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This is to certify that the
dissertation entitled

NEISSERIA GONORRHOEAE REGULATORY NETWORKS
INVOLVED IN THE RESPONSE TO HOST CELL CONTACT

presented by

YING DU

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NEISSERIA GONORRHOEAE REGULATORY NETWORKS
INVOLVED IN THE RESPONSE TO HOST CELL CONTACT

By

YING DU

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Molecular Genetics

2006

ABSTRACT

NEISSERM GONORRHOEAE REGULATORY NETWORKS INVOLVED IN THE
RESPONSE TO HOST CELL CONTACT
By

Ying Du

Neisseria gonorrhoeae is a Gram-negative diplococcus, which causes gonorrhea
in humans and poses a significant public health threat worldwide. The adhesion of
gonococci to host cells is the critical first step of a gonococcal infection. However, our
understanding of the interaction between this pathogen and the host is very limited.

In this dissertation, DNA microarrays, transposon mutagenesis, real time PCR and
a cell culture model of infection are employed to study the regulatory networks of N.
gonorrhoeae upon host cell contact. The underlying theme of this dissertation is the
hypothesis that the initial attachment to the host would transmit signals into the
bacterium, resulting the modulation of gene expression required for its subsequent
survival and amplification in the host.

In this dissertation, we show that gonococcal adherence to host cells indeed
results in changes of gene expression in the bacterium and this response is in part
mediated by the transcriptional regulator, RpoH, whose induction is necessary for the
subsequent invasion step. Furthermore, three of genes induced upon adherence were
identified as RpoH-dependent, as depletion of RpoH from the bacteria abolished their

induction. In particular, knocking out one of these genes, N003 76, encoding a putative

rotarnase in N. gonorrhoeae, compromised their ability to invade human epithelial cells
in culture. This result indicates that signaling is warning across the bacterial membrane.

In addition, many of genes induced upon host contact are independent of RpoH
regulation. One of those genes, NGU340, encoding cysteine synthetase, was found to be
important in both adherence and invasion. Overall, the identification of RpoH-
independent genes indicates a complex regulatory network of N. gonorrhoeae is involved
in this response. Hence, characterization of this regulatory network will be essential to
our understanding of gonococci-host interaction.

In addition to the characterization of the regulatory network in N. gonorrhoeae,
this dissertation describes the identification of a cell division gene, zipA, whose gene

product essentially requires the Signal Recognition Particle system for transport to the

cytoplasmic membrane.

To my grandmother, Xiangzhen Hu

To my dear husband, Qianchuan He

ACKNOWLEDGMENTS

I would like to express my sincere thanks to my mentor, Dr. Cindy Arvidson. I
wish to thank her for letting me work on this wonderful and challenging project. I feel
very grateful and honored to be her student. Without her insightful guidance, continuous
encouragement and infinite patience, I would not be able to accomplish this dissertation
successfully. She was always there to share with me the difficulties or excitements of my
research and I cherish each single moment of them. Her invaluable scientific contribution
and experience has greatly inspired me to be an independent scientific researcher.

I am deeply grateful to my committee members, Dr. Michael Bagdasarian, Dr.
Martha Mulks, Dr. Vincent Young, and Dr. Shengyang He for serving their precious time
on my committee. I wish to thank them for their invaluable suggestions and comments on
my research and help me to think through questions and think big.

I also owe lots of thanks to the current and former members of the Arvidson
laboratory for their help and support. It has always been fun and pleasure to work with all
of them in this wonderful laboratory. I wish to thank Roxie Mason and Cody Frasz for
their warmhearted help in the early years of my research. I wish to thank Johnanson
Lenz, Audrey Butcher and James Tumbrink for their excellent technical assistance in my
research. I also wish to thank Kevin Smith, Jason Jens and Mary Fantacone for their
thoughtful discussion of science and critical reading of my writings. I also wish to thank
Dr. Dennis Arvidson for his advice and help in my research.

I would like to thank Dr. Robert Britton for his invaluable advice in the analysis

of the array data and the use of the array scanner and software in his laboratory. I wish to

thank the Research Technology Support Facility at Michigan State University for their
excellent technical assistances and services.

I would like to thank the department of Microbiology and Molecular Genetics, the
BioSci program and the graduate school for their financial support. I also wish to thank
my friends at Michigan State University for their help in many ways.

Finally, I wish to thank my husband and my family for their unwavering support
and love throughout these years. I am deeply indebted to my parents. They raised me
with endless love, patience and care and it is for this I am sincerely grateful. A special
thanks goes to my dear sister, who is always my good listener and supporter. I also wish
to express my sincere gratitude to my grandmother, who has always been my emotional
support and from whom I have learned to be grateful and joyful in everyday life. Last, but
not the least, I wish to thank my wonderful husband, Qianchuan He, for his deep love,
care, understanding and support. Without him by my side in this long journey, I would

NOT be WHO I AM today.

-vi-

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. viii

LIST OF FIGURES .................................................................................. ix

Chapter 1. Introduction to Neisseria gonorrhoeae ............................................... 1
Neisseria gonorrhoea: the causative agent of gonorrhea .............................. 2
Epidemiology of N. gonorrhoeae: prevention and control ............................. 3
Virulence factors Of N. gonorrhoeae ...................................................... 5
N. gonorrhoeae-host interaction ......................................................... 17
Regulatory networks of N. gonorrhoeae ................................................ 2O
Signal transduction and the Signal Recognition Particle (SRP) system in bacteria.
................................................................................................ 25
Scope of dissertation ....................................................................... 27

Chapter 2. Identification of ZipA, a signal recognition particle-dependent protein from

Neisseria gonorrhoeae ................................................................. 32
Abstract ...................................................................................... 33
Introduction ................................................................................. 34
Materials and Methods ..................................................................... 36
Results ........................................................................................ 40
Discussion ................................................................................... 60

Chapter 3. Expression capable library for studies of Neisseria gonorrhoeae, version 1.0

................................................................................................ 63
Author’s contributions ..................................................................... 64
Abstract ...................................................................................... 65
Introduction ................................................................................. 67
Methods ...................................................................................... 69
Results ....................................................................................... 74
Discussion ................................................................................... 83

Chapter 4. Global gene expression and the role of sigma factors in Neisseria gonorrhoeae

in interactions with epithelial cells .................................................. 88
Abstract ...................................................................................... 89
Introduction ................................................................................. 90
Materials and Methods ..................................................................... 93
Results ....................................................................................... 99
Discussion ................................................................................. 1 18

-vii-

Chapter 5. RpoH mediates the expression of some, but not all genes induced in Neisseria

gonorrhoeae adherent to epithelial cells ........................................... 125

Abstract .................................................................................... 126
Introduction ................................................................................ 127
Materials and Methods ................................................................... 129
Results ...................................................................................... 133
Discussion ................................................................................. 148
Chapter 6. Summary and future work ........................................................... 156
Appendix A ......................................................................................... 163
Abract ....................................................................................... 164
Introduction ................................................................................ 165
Materials and Methods ................................................................... 166
Results and Discussion ................................................................... 168
References ........................................................................................ 172

- viii -

LIST OF TABLES

Table 1-1. Transcriptional regulators in Neisseria gonorrhoeae F A1090 and in related

Neisseria meningitidis serogroup A Z2491 (NMA) and serogroup B MC58

(NMB) ................................................................................... 31
Table 2-1. Sequence analysis of severe SLO clones ............................................ 43
Table 2-2. SLO screen of pSLO7::TnErmUP insertion mutants .............................. 47
Table 2-3. Transformation of M81] with pSLO7::TnErmUP insertion mutants ........... 55
Table 4-1. Genes up-regulated in gonococci adherent to A431 cells ....................... 105
Table 4-2. Genes down-regulated in gonococci adherent to A431 cells ................... 106

Table 5-1. Expression ratios of host cell contact-induced genes in the presence or absence

of RpoH ................................................................................ 142
Table 5—2. Mutants constructed in host cell contact induced genes ......................... 152
Table A-1. Genes expressed higher in P+ (MSl 1A) than F (MSl 1-307) .................. 178
Table A-2. Genes expressed higher in P' (MSl 1-307) than P+ (MSl 1A) .................. 179

-ix-

LIST OF FIGURES

Figure 2-1. Schematic diagram of the pSLO7 insert and the TnErmUP insertions. . . . . ...46
Figure 2-2. Effect of SRP depletion on localization of SLO7ORF3'-His6 in E. coli ....... 49
Figure 2-3. Alignment of the amino acid sequence of putative gonococcal ZipA homolog
(N. g.) with the E. coli (E. c. )and H. influenzae (H. i.) ZipA sequences ......... 53
Figure 2-4. In vivo complementation of E. coli ZipA with N. gonorrhoeae SLO7ORF3..57
Figure 2-5. N. gonorrhoeae zipA partially alleviates filamentation phenotype of E. coli
depleted for ZipA ...................................................................... 59
Figure 3-1. SDS-PAGE analysis of NG ORF-GST fusions .................................. 84
Figure 4-1. RT-PCR analysis of genes induced in gonococci adherent to human epithelial
cells ..................................................................................... 111
Figure 4-2. Adherence to and invasion of A431 cells is not affected in a gonococcal rpoE
mutant .................................................................................. 112
Figure 4-3. RT-PCR of rpoH and groES in an N. gonorrhoeae strain conditionally
expressing rpoH ....................................................................... 117
Figure 4-4. N. gonorrhoeae depleted for RpoH are hyper-sensitive to heat shock ....... 121
Figure 4-5. Depletion of RpoH in N. gonorrhoeae affects invasion of, but not adherence
to A431 cells .......................................................................... 122
Figure 5—1. Real-time quantitative PCR analysis of expression ofN01684, N003 72,
N003 76, groES and rpoH in the conditional rpoH strain MPD288 grown with

and without inducer (IPTG) ......................................................... 143

Figure 5-2. Real-time quantitative PCR analysis of expression ofN01684, N003 76 and
groES in MPD288 depleted for RpoH and adherent to A431 cells ......... 146

Figure 5-3. Interaction of N. gonorrhoeae mutants with A431 cells ....................... 151

-xi-

Chapter 1

Introduction to Neisseria gonorrhoeae

Neisseria gonorrhoeae: the causative agent of gonorrhea

Neisseria gonorrhoeae (GC, gonococcus) is a Gram-negative diplococcus,
causing one of the oldest documented venereal diseases, gonorrhea. Although
descriptions of this disease had appeared in writings years earlier, it was the Greek
physician, Galen, who first named it gonorrhea (latin for “flow of seeds”). In 1879, N.
gonorrhoeae was first described as the causative agent of gonorrhea by a German
physician, Albert Neisser (183). The genus Neisseria includes 12 species and biovars
(193) and only two species, N. gonorrhoeae and N. meningitidis, are important human
pathogens. The latter is significant as a causative agent of septicemia and meningitis in
humans.

N. gonorrhoeae is an obligate human pathogen and only transmitted by intimate
contacts with an infected person. This pathogen usually colonizes the mucous membrane
of the urethra in males and the endocervix in females. However it can also colonize the
mucous membranes of the eyes, throat, and rectum. Symptoms of gonoccocal infections
differ by gender and the site of infection. Men are more likely to develop acute symptoms
that are typically seen as purulent discharges and dysuria; while many infected women
remain asymptomatic. The acute symptoms of endocervical infection are vaginal
discharge and dysuria. Infections in the rectum and pharynx are rarely symptomatic. In
women, gonoccoci may ascend into the fallopian tubes resulting in salpingitis, which may
lead to the development of pelvic inflammatory disease (PID), a major problem that can
lead to infertility or ectopic pregnancy (253). During vaginal birth, newborns can get
gonococcal infections of the conjunctiva from an infected mother and this can lead to

blindness (203, 307). Although most of the time gonococci cause localized inflammation,

the bacteria can occasionally spread through the blood stream to other organs, causing
disseminated gonococcal infections (DGI), which can result in purulent arthritis,
endocarditis and meningitis (347, 417).

Accurate detection of N. gonorrhoeae in patients is key to treatment and requires
highly sensitive and specific diagnostic methods. Traditionally, culturing N. gonorrhoeae
on selective media has been the gold standard, because of its high sensitivity and
specificity (193). Moreover this culture-based method also allows for the subsequent
determination of antibiotic susceptibilities. This is essential in treatment, considering that
N. gonorrhoeae rapidly develops resistance to antibiotics (235, 337, 389). In order to
distinguish N. gonorrhoeae from other related Neisseria spp, biochemical tests are used
to test their ability to ferment carbohydrates, reduce nitrate, or produce polysaccharide
from sucrose, etc. (192-194, 365). Nonculture-based diagnostic techniques have recently
been developed to improve the accuracy of detection, such as the antibody-based enzyme
immunoassays (EIA), the hybridization-based nucleic acid hybridization test and the

DNA/RNA-based nucleic acid amplification test (N AAT ) (196).

Epidemiology of N. gonorrhoeae: prevention and control

According to the World Health Organization (WHO), more than 62 million cases
of gonorrhea occur each year, posing a huge threat to public health worldwide (6).
However, this number is likely underestimated, since the diagnosis and reporting of
gonococcal infections are often incomplete due to asymptomatic carriage among patients.
Upwards of sixty percent of infected women and twenty percent of infected men are

asymptomatic carriers, composing a large reservoir of gonococci in the population (389).

There are more incidents of gonococcal infection in developing countries with South
Asia, South-East Asia, Africa and South America reporting the highest rates every year
(5).

In the United States, N. gonorrhoeae is a leading cause of bacterial sexually
transmitted disease, second only to Chlamydia trachomatis. Although the incidence of
disease has significantly declined since the 19805, this trend has reversed since 1998 with
more than 300,000 cases reported in the United States in 2003 (1). As gonorrhea can
significantly enhance the transmission of the human immunodeficiency virus (HIV) (58,
202), the importance of gonorrhea as a human pathogen has increased along with the
prevalence of HIV worldwide.

Due to the poor immunogenicity or the antigenic and phase diversities of its
surface proteins, an effective vaccine against N. gonorrhoeae is still beyond reach (54,
150, 308). Today, antibiotics are the only effective choice for treating gonococcal
infections. Historically, penicillins were the most widely used antibiotics to treat
gonorrhea; however, penicillin-resistant gonococcal strains quickly appeared globally. In
1998, the proportion of penicillin-resistant gonococci reached more than 60% in South
and South-East Asia (4, 405) and in some Asian countries, resistance to penicillin can be
as high as 96.8% (316). Because of the high prevalence of the penicillin—resistant strains,
penicillins are now only used where they are still effective (4). Tetracyclines and newer
macrolides such as azithromycin are not recommended for treatment, due to the high
spread of the resistant strains or Side-effects (389). Spectinomycin and quinolones have
been withdrawn in many parts of the world because of resistance or side-effects (4, 389,

405). The third generation cephalosporins (cefiriaxone and cefixime) are currently the

preferred choice of treatment for gonorrhea (4), but there has been a decline in
susceptibility to these drugs in recent years (5).

At present, the most effective way to control the transmission of gonorrhea is
education, aggressive detection, and follow-up screening of possible carriers (27, 386)
and the key elements of disease control are “prevention through promotion of safer

sexual practices and the availability of health care services ”(4).

Virulence factors of N. gonorrhoeae

N. gonorrhoeae attaches to and passes through the mucosal epithelial cells into
the subepithelial space, causing a localized infection, although occasionally it can spread
to other tissues and organs. To better prevent and control gonococcal disease, it is
essential to understand the biology of this pathogen. Over decades, extensive studies have
been done towards this aim and as a result, a variety of virulence factors have been
discovered and characterized, most of which are located in the outer membrane or
secreted to the extracellular space of N. gonorrhoeae.

Pili The N-methylphenylalanine (Type IV) pili on the surface of N.
gonorrhoeae are one of the most important virulence factors in a gonococcal infection
(187, 380, 382). Pili are involved in a broad range of functions including adherence to
host cells (291, 324, 380, 400), DNA transformation (42, 354), and twitching motility
(250, 436, 437). Non-piliated gonococci are non-adhesive to host epithelial cells and lose
their ability to establish infection in the host (380). Gonococcal isolates are usually
divided into 4 phenotypic groups, type I-IV, which is partially based on the presence of

pili on their surfaces. Gonococci recovered from patients are usually of type I or II, which

are highly piliated, adhesive and virulent (48, 187). When these piliated clinical isolates
are maintained on laboratory media, they gradually lose their pili after several serial
passages (186, 187).

Gonococcal pili are helical hair like structures composed of one major structural
subunit, pilin, a 17-21 kDa protein encoded by the gene piIE (251). In N. gonorrhoeae
strain M81 1, there are two identical fimctional pilE genes, while there is only one
functional pilE gene in most clinical isolates (251, 349). Stabler and colleagues recently
showed that the piIE gene is only present in the pathogenic Neisseria species, N.
gonorrhoeae and N. meningitidis (372). The amino acid sequence of pilin can be divided
into three regions, a conserved region (C), a semi-variable region (SV) and a highly
variable region containing two conserved cysteine residues (HV) (251, 252, 350). Using
site-directed antibody probes and immunoassays, researchers have shown that the C
region is essential in pilus assembly and the other two regions contribute to the diversities
of pilin structure (99, 288).

Besides the pilE expression locus, there are also several pilS loci scattered on the
gonococcal chromosome (251, 372). These loci have sequences homologous to the pilE
gene, in particular, the SV and HV regions but lacking important 5’ prrnoter region and
the start codon required for the initation of transcription and translation. These pilS loci
form a sequence reservoir allowing the transfer of varied sequences into the pilE gene
through homologous recombination (136, 137, 252, 349). N. gonorrhoeae can obtain the
piIS sequences either from the same strain (intragenic) or from the lysed gonococci
within a population (intergenic) (354). It is likely that the latter might be more common

in vivo, since gonococcus is spontaneously lysable, releasing its DNA into the

surrounding environment (149) and it is also naturally transformable, taking up
exogenous DNA at high fiequencies (369). This leads to extensive antigenic variation,
due to expression of varing pilin proteins on the bacteria in a population. In fact, it is
because of the highly diverse pilin structure that the vaccine against pilin is not practical
in use, since antibodies against pilin from one strain lack broad cross reactivity to other
gonococcal strains (41). Additionally, gonococcal pili are subject to phase variation,
resulting in the “on” or “off” of pilin expression. Pilin phase and antigenic variation are
closely related as a result of RecA-dependent homologous recombination (137, 349, 350).
Recombination between pilS and pilE loci results in the expression of a mosaic pilE gene
leading to production of antigenically variable pilins. Recombination leading to a deletion
in the pilE gene, causing a frame-shift mutation and abortion of translation, results in
phase variation. Additionally, the expression of the pilin gene may also be controlled at
the transcriptional level, as some non-piliated strains still contain the functional pilE gene
and the intact promoter region (349).

Pilus biogenesis has been extensively studied. Although pili are composed of one
major subunit, pilin, several proteins are engaged in the assembly of the pilus, including
PilD-PilF-PilG-PilT and PilQ-PilP (81, 104, 158, 206, 397). It has recently been shown
that these proteins have high homologies to those components involved in the Type II
secretion system of Gram-negative bacteria, raising an interesting question that pilus
assembly might be closely related to the protein secretion system (284, 309).

The gonococcal pilus assembly process is mainly composed of three steps: the
assembly of the fiber in the periplasm, the translocation of the fiber onto the cell surface

and the final stablization of the pili structure on the surface (438). At each step, different

proteins are required. For instance, the prepilin peptidase PilD cleaves the leader peptide
from prepilins so that the pilins can later form the fiber processed by PilF and PilG (104,
397). Then the assembled fiber passes through the pore formed by PilQ subunits to the
cell surface (63, 80, 81, 438), where they continue to form dynamic and stable fibers.
During this whole process, the pilus retraction mediated by PilT, an ATPase, is required
to balance fiber growth (436, 437). A recent report showed that the PilT-mediated pilus
retraction is in fact regulated by PilC protein in N. gonorrhoeae (259).

Besides the essential role as a primary adhesin, pili are also critical in DNA
transformation (106, 436), allowing gonococci to obtain foreign genetic material and
retain genetic diversity (330, 357, 438). In non-piliated gonococci, DNA transformation
efficiency decreases dramatically, which is 10,000 times lower than piliated gonococci
(355, 369). Although the DNA transformation process in N. gonorrhoeae is still not
completely understood, a recent paper reports that the functional pilus assembly
apparatus is required for DNA uptake (223).

Pili also play an important role in signaling pathways in infected epithelial cells.
Purified gonococcal pili are shown to cause Ca2+ influx in epithelial cells and result in
rearrangement of the cytoskeleton in the cell membrane (22, 180). Additionally, part of
these signaling pathways are caused by the twitching motility powered by the pilus
retraction, as it can create tension on the cell surface, which can be sensed by host cells
(250) . Indeed, it has been shown that the mechanical force generated by the pilus
retraction can promote the arrangement of the cytoskeleton in the cell membrane,
influxes of Ca2+ into the cytosol, and the formation of the cortical plaques that are

enriched in proteins necessary for the subsequent cellular responses in host cells (246).

Furthermore, pilus retraction is required for the activation of PI-3 kinase/Akt (PKB)
pathway (210) and changes of expression of genes necessary for the subsequent cellular
responses in host cells (165). Although pili are the primary adhesins of N. gonorrhoeae,
our knowledge about their host receptor is still elusive. CD46, a host cell surface protein,
has been hypothesized as a receptor for pili (182), however, several studies indicate the
existance of other different, yet currently unknown receptor(s), as gonococcal pili can
interact with epithelial cells in a CD46-independent manner (190, 249, 395). Recently,
Edwards and colleagues identified I-domain-containing (IDC) integrins as receptors for
gonococcal pili on urethral epithelial cells (89).

Opa Opa (Opacity associated proteins, Protein II, PII) are a group of major
outer membrane proteins of N. gonorrhoeae that confer an opaque quality to colonies
grown on agar plates and visualized under a microscope (157, 379). Because of their
ability to interact with polymorphonuclear leukocytes (PMNs), they were once named
Leukocyte Association Proteins (LAPs) (382). Opa proteins play critical roles in
gonococcal pathogenesis, facilitating adherence to and invasion of epithelial cells and
inducing the expression of the pro-inflammatory cytokines/chemokines (114, 125, 231,
419)

Like the diverse gonococcal pili, Opa proteins are also subject to antigenic and
phase variation. However, the mechanism is different from that of pilin antigenic
variation. N. gonorrhoeae has as many as 11 opa genes scattered on its chromosome,
named as opaA-K (31, 200). Each opa gene is constitutively expressed and encodes a
different Opa protein differing in the variable domains (31). Opa genes also undergo

extensive “on” and “off” phase variation, the frequency of which is as high as 10'2 to 10'3

per colony-forming unit per generation (237). This opa phase variation is caused by the
slipped-strand mispairing (SSM) mechanism independent of RecA-mediated homologous
recombination (268). The SSM mechanism is associated with short DNA coding repeats
(CR) CTCTT present in the 5’ coding region of opa genes. During DNA replication and
repair, the number of these CR repeats can be easily changed, leading to frame-shift
mutations and the “on” or “off” of the expression of opa genes (268, 373). As a result,
one single N. gonorrhoeae strain is able to express varying combinations of serval Opa
variants scattered on its surface at the same time (237) .

Opa proteins are important in mediating the tighter association between gonococci
and host cells (14, 232). Opa+ gonococcal strains have been shown to bind to and invade
human epithelial cells (363, 419, 426, 427). In clinical observations, the strains recovered
from patients are usually Opa+ (168, 171, 414).

The identification of host Opa receptors come from studies using Chinese
Hamster Ovary (CHO) cells or other animal cells, which were transfected with genes
encoding different human cell surface molecules and were then analyzed for adhesion
and invasion of Opa+ gonococci (61, 415, 416). As a result, two major groups of Opa
receptors were discovered, although different Opa variants have distinct affinities for
these receptors (61, 200, 415, 416). The first group of Opa receptors is Heparan Sulfate
ProteoGlycan (HSPG), whose discovery was based on the speculation that the interaction
of Opa proteins and its receptor might involve electrostatic interactions, as adding of
negative charged DNA or sulfated polysaccharide can Significantly affect gonococcal
adhesion (379). Proteoglycans are an important family of macromolecules of human

epithelial cells and are composed of a protein core covalently attached by one or more

10

glycosaminoglycan side chains. These molecules can be found in the cytosol, on the
surface or in the extracellular matrix of the cell. The syndecan group is a major group of
HSPGs, which contain negative-charged heparansulphates and chondroitin sulphates in
the side chains and are located on the cell membrane (323). Only syndecan-1 and
syndecan-4 seem to interact with Opa proteins and mediate the entry of N. gonorrhoeae
into epithelial cells (103). However, not every Opa variant can interact with HSPG, with
OpaA strongly binding to this receptor followed by OpaC and OpaH (411, 412). HSPGS
are important molecules on the human cell membrane, as they can interact with many
growth factors and ligands and serve as growth factor receptors involved many cell
Signaling pathways (72).

The second type of Opa receptor belongs to the CD66 family, also known as
carcinoembryonic antigen (CEA). This receptor family has immunoglobulin-like domains
(415) and many Opa variants can bind to the N-domain of CD66 molecules, although the
affinity may vary (416). CD66 proteins are broadly expressed in various cells, including
leukocytes, endothelial cells, and epithelial cells. Studies have shown that Opa+
gonococci can specifically bind to CD66 proteins on these cells (116, 124, 270, 363, 415,
422)

Porin Porin (Protein 1, PI) proteins are the most abundant proteins on the outer
membrane of gonococci, and are encoded by the par gene (121). Por is essential in N.
gonorrhoeae, as it has not been possible to construct a null mutant thus far. In N.
gonorrhoeae, there are two par alleles, porIA and porIB, encoding PIA and PB, and
only one of them is expressed in a single gonococcal strain (121, 176). In several studies

of tracking the transmission of gonorrhea, gonococcal strains bearing PIA have shown a

11

strong correlation with the occurrence of DGI (121, 176, 194, 305). It has also been
shown that certain mutations in the porin proteins make the bacterium more resistant to
antibiotics (52, 162, 285, 319).

PIA is a 34 kDa protein, slightly smaller than PIB (35.5 kDa), however, the two
proteins share over 80% similarity in their amino acid sequences (176). The major
difference between PIA and PIB proteins resides in the regions exposed on the cell
surface, in which PIA only has a small portion exposed outside and is therefore less
susceptible to exogenous protease cleavage (176, 410).

Gonococcal porin proteins are homologs of E. coli OmpC, OmpF and PhoE
porins ( 121, 258). The porin proteins can form voltage-gated hydrophilic channels
through the outer membrane, which might allow the nutrients to enter the bacterium (121 ,
410). These channels can also be easily translocated into the membranes of cultured
human cells (121). During a gonococcal infection, gonococcal porins have been observed
to be released from the bacterial membrane and translocated into the host cell membrane,
resulting in the influx of Ca2+ into the cytoplasm and apoptosis in the target (HeLa) cells
(266). A recent study showed that gonococcal porins can be specifically translocated into
the mitochondrial membranes and induces apoptosis in host cells (265, 267). However,
this porin-induced apoptosis might be tissue specific, as in other cells, such as
transformed urethral epithelial cells or primary cells derived from the male urethra,
porins were shown to repress apoptosis (34). In addition, the porin-induced Ca2+ influx is
necessary for the pili-induced Ca2+ influx (22).

Besides their roles in gonococcal pathogenesis, porins are also potential

candidates for vaccines. Porins are conserved outer membrane proteins on the gonococcal

12

surface and don’t undergo the extensive variation observed for Opa and pilin.
Additionally, porins are highly immunogenic and can induce a strong immune response
in the host (55, 445). However, the interaction of porin proteins, LOS and Protein 111
weakens this immune response by significantly masking the porin binding sites from host
bactericidal antibodies (3 7).

Protein 111 Protein III (PIII, or Reduction modifiable proteins, Rmp) is located
on the outer membrane and tightly associated with porins (PI) on the gonococcal surface
(238). The gene encoding P111 is exclusively present in the pathogenic Neisseriae species,
N. gonorrhoeae and N. menigitidis (435). P111 is 236 amino acids in length, with a
cleavable signal peptide of 22 amino acids and the primary structure of this protein is
very similar to that of OmpA in E. coli (119, 120), a protein tightly associated with LPS
and important in conjugation (345, 346). When P111 is subject to sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE), the migrating band of this protein
under reducing conditions is 8,000 kDa larger than the size predicted from its sequence
(238). Because of its unusual migration pattern on SDS-PAGE, P111 is also called
Reduction modifiable protein (Rmp). Blake and colleagues showed that this unusual
migration pattern is due to the two pairs of disulfide bonds formed in this protein. In their
experiments, PIII was first chemically cleaved into two fragments so that the disulfide
bonds could not be formed. These two fragments were then subject to gel electrophoresis.
As a result, no difference in migration was observed in the presence or absence of the
reducing agent (38). Proteolytic analyses showed that P111 is resistant to protease
digestion in intact gonococci, indicating that most of the protein is not exposed to the

outside of the cell (36). Indeed, DNA sequence analysis and surface peptide mapping

13

experiments showed that only a very small portion of P111 was exposed to the surface
(119, 120, 177, 229).

Compared to other gonococcal surface molecules, which undergo extensive phase
and antigenic variation, PHI is highly conserved in N. gonorrhoeae and has very little
variability in its structure. This unique characteristic had once triggered interests of
exploring of this protein as a potential gonococcal vaccine (177, 228). In 1986, Rice and
colleagues found that the antibodies directly against PIII were able to block the killing of
gonococcus from immune serum. This fact was further substantiated by an experiment in
which anti-PIII antibodies can directly affect the efficiency of insertion of the
complement membrane attack complex (318). The anti-PIII antibodies block the binding
of antibody complexes to the gonococcal surface, in particular to the binding sites of
porin proteins by occupying those sites. Therefore, anti-PIII antibodies allow gonococci
to evade the host immune response, instead of activating it. This was supported by the
observation of the declining efficiency of the PI vaccine contaminated with P111 in human
volunteers (175, 299).

LOS Lipopolysaccharide (LPS) has long been known as a major virulence
factor in many bacterial pathogens (152, 217, 282). This is also true in a gonococcal
infection, as it triggers an intense inflammatory response in the subepithelial space of the
mucous membrane including the activation of the complement complex and attraction
and lysis of phagocytes (234). Gonococcal LPS has also been shown to play a role in
gonococcal adhesion and invasion of epithelial cells (90, 368).

Compared to the LPS molecules found in most Gram-negative bacteria, the

gonococcal LOS (lipooligosaccharide) lacks the typical O-antigen. Instead, three

14

branches of oligosaccharides are attached into the Lipid A backbone and the length and
sugar composition of these oligosaccharide branches differs from strain to strain (112,
128). In N. gonorrhoeae, a single strain can display several distinct LOS structures on its
surface (16, 233). In 1994, Gotschlich first identified a locus involved in LOS
biosynthesis on the gonococcal genome, which includes the five genes, lgtA-E, encoding
enzymes responsible for adding different sugars to the end of the LOS structure. Three of
these genes have poly-G repeats in their coding regions, which indicate the potential for
phase variation by the slipped-strand mispairing mechanism (118). This phase variation
was confirmed in later studies (68, 441). In addition to the lgt locus, other genes involved
in LOS biosynthesis were identified and characterized in the following years (24, 82,
451), and many of these genes also bear poly-nucleotide repeats in their coding regions,
indicating that they are prone to phase variation by slipped-strand misrepair.

Gonococcal LOS can be further modified by sialylation. The bacteria themselves
do not provide the source for sialylation. Instead, they use host CMP-N-acetylneuraminic
acid (CMP-NANA) as a donor to add a sialic acid to the LOS moiety (177, 234). The
enzyme responsible for sialylation, a 0t-2,3-sialyl-transferase, is subject to extensive
phase variation, which leads to the “on” and “off” of sialylation of LOS in N.
gonorrhoeae (289). The sialylated LOS is important in the pathogenesis, as it helps
gonococci to resist serum killing as a result of mimicking the human cell membrane
glycosphingolipids (15, 234). However, some studies show that the sialylated LOS can
reduce gonococcal ability to invade epithelial cells (343). In fact, it has been
hypothesized that during different infection stages, gonococcus can control LOS

sialylation accordingly. For instance, in the early infection stages, the bacteria might need

15

the unsialylated LOS to facilitate the bacterial entry into the host; while during the later
phase of infection, the sialylated LOS might be required to evade the immune response
from the host (74, 368).

IgAl Protease IgAl protease has been identified as a virulence factor in several
pathogens, including Haemophilus influenzae, N. gonorrhoeae, N meningitidis and
Streptococcus sanguis ( 189, 263, 264, 297, 298). It has been shown that the gene
encoding this enzyme exists exclusively in pathogenic, but not commensal species of
Neisseria (263, 435). IgAl protease is a secreted enzyme, which can cleave human
immunologlobulin A1 (IgAl), the main immunologlobulin secreted on mucosal surfaces.
N. gonorrhoeae possesses two distinct types of IgAl protease: type I and II, and each of
them cleaves a specific site of human IgAl molecule (262).

In N. gonorrhoeae, the secretion of lgAl protease may be the simplest, as the
protein itself is enough for its secretion into the extracellular space (141), also referred to
as “ autotransporter”. The mature IgAl protease is derived from a 169 kDa precursor,
which is composed of five distinct domains: the N-terminal signal peptide, the central
“protease” domain, the C-terminal “helper” domain and two domains or and y (301).
During the export process, the signal peptide at the N-terminus will first guide the protein
to pass through the inner membrane through the SecYEG translocon. (301). Cleavage of
this signal peptide by a leader peptidase releases the protein to the periplasm. The C-
terminal “helper” domain then associates with the outer membrane and forms a pore to
allow the remainder of the protein to pass through the outer membrane. Enzyme activity
analysis shows that the IgAI protease activity can detected at this step. Autocleavage will

then occur to release the protease domain into the outer space. As a result, the “helper”

16

domain will be retained in the outer membrane. Then, the peptides in the outer space will
continue to develop the active form and will be further cleaved to yield the active
protease and two peptides. In the end, the mature active secreted protease has a molecular
mass of 106 kDa (301).

The human IgAl molecule is the main substrate for IgAl protease. However, the
presence of consensus sequence of the specific cleavage sites in relevant human proteins
indicate that gonococcal IgAl protease may have other potential substrates (302). This
was later confirmed by the finding that the type II IgAl protease could cleave lysosomal
associated membrane protein 1 (Lampl), a major integral membrane glycoprotein of
lysosomes in epithelial cells (20, 145, 163, 216). As a result, the cleavage of Lampl can
significantly facilitate gonococci to enter epithelial cells and promote their survival in
those cells (216). IgAl protease was also found to cleave the tumor necrosis factor alpha
(TNFct) receptor 11 to inhibit the TNFot-mediated apoptosis in human monocytic cells

(26).

N. gonorrhoeae - host interaction

N. gonorrhoeae is an obligate human pathogen with no intermediate animal
reservoirs. There are very few animal models available for gonococcal studies, although a
mouse model was recently developed and is yielding interesting data (172). Our
knowledge of the N. gonorrhoeae - host interaction is primarily from studies using tissue
culture and organ culture models derived from tissues normally infected by gonococci
(48, 144, 240, 241, 244, 360, 363, 419). Currently, there are several cell lines used in

studies of gonococcal infection. HEC-l-B, a human epithelial line derived from a cervical

l7

carcinoma has been used to study adhesion and invasion by N. gonorrhoeae and N.
meningitidis (60, 363). A431, an epithelial cell line derived from an epidermal carcinoma
(110), is particularly useful in that they are easy to grow and handle in the laboratory and
excellent for observation under the microscope. ME180 cells and T84 cell are two other
epithelial cell lines used in gonococcal adhesion and invasion studies (179, 247, 422).

N. gonorrhoeae initiates infection via the primary adhesin, type IV pili and forms
microcolonies on the surface of the host cell. Following the initial attachment, Opa
proteins and other outer membrane proteins mediate an intimate association between the
gonococcus and host cells. In fact, this association is so close that some studies suggest
that the gonococcal outer membranes are actually fused with the host cell membranes
(14, 261, 360). It is also at this step that the microcolonies start to disperse and the
bacteria start to lose pili on their surfaces. Losing pili is a beneficial step, as pili can
inhibit the subsequent invasion step (232, 360). Furthermore, the association between
gonococci and host cells results in the formation of cortical plaques underneath the
contact site, site of entry into the cells, and the loss of microvilli from the surface of
nonciliated cells (123, 111). The invasion step is mediated by actin-dependent
endocytosis, as this step can be disrupted by the anti-actin drug cytochalasin D (CCD)
(248, 261, 320, 360). Using Hec-l-B cells as a culture model, Shaw and coworkers
showed that the number of invaded gonococci usually increased after 4-hour of
incubation (360). Once inside the epithelial cells, gonococci are usually seen in vacuoles,
however, other studies have also found gonococci in the cytosol of the epithelial cells

(14, 240, 360, 422). Gonococci then exit from the basal side of the cell by exocytosis into

18

the subepithelial space of the mucosa, where an inflammatory response is elicited (240,
261)

Overall, the interaction between N. gonorrhoeae and the host is a dynamic
process involving multiple factors from both sides. Gonococcal adhesion transmits
signals to the host cells, which leads to cytoskeletal rearrangements, uptake of the
pathogen, and production and release of inflammatory cytokines from the host mucosal
epithelial cells (123, 248, 277, 278, 430). Extensive studies on gonococcal infection over
the past two decades have greatly increased our understanding of signaling pathways
happening inside host cells, in particular, the pilus and Opa-mediated signaling pathways.

Pilus-mediated attachment is the essential first step in gonococcal infection. The
binding of gonococcal pili to host epithelial cells results in the influx of Ca2+ within the
target cells (20, 180). This Ca2+ influx happens very rapidly in host cells and can be
detected within 10 minutes after the initial adhesion (261). Consequently, the transient
increase of Ca2+ in the cytosol of the target cell results in the subsequent sigraling
cascades, as Ca2+ is an important signal in the target cells (181, 248). Additionally, the
Ca2+ influx can increase the distribution of the Lampl molecules on the host cell
membrane by triggering the lysosome exocytosis so that more Lampl can be cleaved by
the secreted gonococcal IgAl protease thus promoting the survival of gonococci in
epithelial cells (20). Recent experiments show that the porin-mediated Ca2+ influx might
be required for the pilus-mediated Ca2+ influx (22).

Opa-mediated signaling pathways are mediated by two distinct groups of Opa
receptors on host cell membranes, HSPG and CD66 (discussed in the previous section).

The HSPG-Opa interaction results in distinct signaling cascades in different cell lines and

19

leads to the activation of the phosphatidylcholine (PO-dependent phospholipase C
(PLC), acidic sphingomyelinase (ASM) and protein kinase C (86, 122, 114). In contrast,
Opa-CD66 interaction results in the activation of the Src family kinases, which can in
turn activate the Rho-dependent GTPase Racl (147). As a result, the activation of these
kinases can eventually induce the expression of the transcription factor nuclear factor
kappa B (NF -KB) and the dimeric sequence-specific transcription factor activator protein
1 (AP-1), which subsequently up-regulate the expression of several pro-inflammatory
cytokines, IL-8, IL-6 and TNF-or through the activation of the JNK kinase pathway (277,
278). The induction of NF-KB and AP-l happens very quickly, as the NF-KB complex
could be transported into the nucleus within 10 minutes after infection (240).

Compared to our understanding of the signaling pathways within host cells, little
is known about the signaling pathways happening inside the gonococcus during this
interaction. Does the attachment to host cells transmit signals to the bacterium? If so,
what are the regulatory networks involved? Once attached to host cells, gonococci
undergo several changes including the formation and dispersal of microcolonies,
disappearance of pili fiom the surface and the translocation of porin into the target cell
membrane. These changes are highly organized and coordinated, indicating the
involvement of regulatory networks. Thus identifying and dissecting these networks is

very important for us to understand gonococcal pathogenesis.

Regulatory networks of 1V. gonorrhoeae

When a pathogen enters a new host it faces a variety of new challenges, involving

acquisition of nutrients, colonization, competing for space with existing flora and

20

circumventing the immune response of the host. In order to adapt to the new
environment, pathogens have developed many strategies, including modulating gene
expression at the transcriptional, translational, or post-translational levels (66, 67, 151,
154, 317, 336, 340). Rapidly turning genes on or off as required may be the most efficient
way to do this as it controls gene expression at the starting point. In fact, many pathogens
regulate virulence genes to achieve a selective advantage by enabling them to rapidly
adapt to changing environments. For example, Yersinia pestis up-regulates the expression
of a set of genes encoding type-III-secreted virulence factors upon host contact; the
products of these genes are then injected into the cytoplasm of the host cell, causing
cytoskeletal changes and interfering with phagocytosis (398). In Helicobacter pylori,
nearly 10% of its open reading frames (ORFs) are differentially regulated when the
pathogen is transferred to an acidic environment reflecting a complex, coordinated
regulatory network involved in adaptation to the harsh environment in the stomach (13).
The availability of completed N. gonorrhoeae genome sequence provides us
opportunities to study its regulatory systems (http://www.uohsc.edu). In the annotated
gonococcal genome (43), there are about 50 putative transcription factors (Table 1-1).
Compared to about 150 transcription factors identified in E. coli (396), N. gonorrhoeae
apparently has far fewer transcription regulators, which might be because N. gonorrhoeae
has very limited niches to reside, unlike E. coli, which can inhabit a broad range of hosts
and environments. Thus, some of transcription factors encoded in the gonococcal genome
are likely involved in the ability of the pathogen to survive and multiply in the host and

are interesting to study.

21

Based on amino acid sequence homologies to well-characterized regulators in
other bacteria, gonococcal regulators can be grouped into several families, including the
AraC, DeoR, GntR, LysR, and MarR regulatory families (Table 1-1). Most of these
families contain the helix-tum-helix (HTH) motif and can directly bind to DNA and
quickly regulate gene expression in response to differing environmental conditions (108,
338). In N. gonorrhoeae, regulatory systems involving regulation of iron acquisition, the
redox response, and control of antimicrobial efflux have been described (29, 138, 159,
188, 207, 224, 348, 402).

Iron is essential for bacterial growth, as iron is an important cofactor for many
enzymes such as catalase, peroxidase, cytochromes and ribonucleotide reductase (221).
N. gonorrhoeae encodes several proteins that can scavenge iron from the environment
and the Fur (Ferric gptake regulation) protein is a global regulator that regulates the
expression of most of iron-uptake systems (29, 220, 348, 393). The gene encoding Fur
was first detected in N. gonorrhoeae by Southern blot using an E. coli fur DNA fragment
as probe to hybridize gonococcal genomic DNA. This gene was then cloned and shown
to complement an E. coli fur mutant, confirming that the gonococcal fur gene encodes a
functional Fur protein (29). In 1996, Thomas et al directly characterized the function of
the gonococcal Fur regulator in N. gonorrhoeae (393), demonstrating that the Fur
regulator regulates several iron-uptake genes, frpB, tpr and tpr. Two-dimensional gel
electrophoresis and DNA binding experiments later indicated that the gonococcal fur
regulon may includes proteins of a broad range, which are not only involved in iron-
uptake, but also involved in protein secretion and DNA recombination (348, 393). In

addition to Fur, a MerR-like regulator was recently identified in microarray analysis as a

22

regulator of iron-uptake systems, suggesting that the regulation of this system is
controlled by several regulators (85).

N. gonorrhoeae lives in an environment which is rich in antimicrobial fatty acids
(FAs), bile salts, hormones and antimicrobial peptides that are toxic to the bacteria,
however, gonococci have developed several mechanisms to resist these antibacterial
molecules. This makes the current antibiotic treatments challenging. Mtr (Multiple
transferable resistance) and Far (Fatty acid resistance) efflux pumps are two such systems
that help N. gonorrhoeae to resist antimicrobial agents. The Mtr system is composed of
three cell envelope proteins (MtrC, MtrD and MtrE) that confer the resistance to
hydrophobic agents (HAs), such as erythromycin and Triton X-100 (138). The Far system
is composed of a membrane protein FarA and cytoplasmic transporter protein FarB,
which mediate the resistance to long-chained fatty acids and depend on MtrE to export
these agents. However, these two systems act independently to mediate the resistance to
host-derived agaents (208). The regulation of these two systems is mediated by the same
transcription repressor protein, MtrR (Multiple rransferable resistance Regulator) (138,
139, 207, 224). MtrR binds the DNA sequences between mtrR and mtrC and represses
the expression of the mtrCDE operon (138, 159, 224). Mutations in mtrR can lead to an
increase in the resistance of gonococci to HAS including Triton X-100, erythromycin and
penicillin (359, 413, 446). MtrR can also activate expression of the farAB encoded efflux
pump by repressing the expression of the farAB repressor, FarR (207). In addition to the
two repressors, MtrR and FarR, MtrA (Multiple rransferable resistance Activator) was
recently discovered as an activator of the mtrCDE encoded efflux pump, indicating that

the regulation of gonococcal resistance to antimicrobial molecules is controlled by both

23

positive and negative regulatory mechanisms (327). Recently, another efflux system
(MacAB) was identified in N. gonorrhoeae, although its function remains to be
determined (326).

During a gonococcal infection, reactive oxygen species (ROS) are an important
weapon of phagocytes of the host against gonococci. The ROS include hydrogen
peroxide (H202), hydroxyl radical (HO‘), and superoxide anion (02"), which are
generated in the lysosomes of the phagocytic cells of the host. They are highly toxic and
can damage macromolecules in the bacterium such as DNA, lipids and proteins.
Gonococci are always associated with activated polymorphonuclear leukocytes on the
inflamed mucosal membrane. To manage the ROS encountered, gonococci possess
catalase and peroxidase, which can convert the encountered ROS into harmless products
(02 and H20) (17, 174, 449). Recent studies show that the regulation of this ROS
response system in N. gonorrhoeae is mediated by two important regulators, OxyR and
leR. OxyR represses expression of the gene encoding catalase (kat), while leR can
up-regulate the expression of several genes (adhC, copA and tpr) encoding
dehydrogenases and reductases (188, 402).

There are four pairs of putative two-component regulatory systems in the
annotated N. gonorrhoeae genome (43). Two-component regulatory systems are
composed of a “sensor kinase/His-kinase” located in the cell membrane and a “response
regulator” in the cytoplasm (5 7, 92). Two-component regulatory systems are important in
bacterial pathogens, because the sensor kinase is able to sense an external environmental
stimulus or signal and then transmit it to the “response regulator” in the cytoplasm via a

phospho relay (92, 375, 376). As a result, the corresponding response regulator can

24

directly bind to target genes to regulate gene expression or cell behaviors (25, 28). In the
E. coli K—12 genome, genes encoding more than 30 pairs of two-component systems have
been identified (257, 286). These regulatory systems are involved in a wide range of
cellular functions, including responses to the osmotic pressure, nitrogen availability,
glutamate metabolism and pathogenesis (KEGG database) (95, 129, 236, 312). Our
understanding of two-component systems of N. gonorrhoeae is comparatively limited.
The only characterized two-component system of N. gonorrhoeae is the NarQ-NarP
system, which is involved in nitrite reduction under oxygen limiting conditions (219,
432). Studies have shown that NarQ-NarP is able to initiate transcription of aniA, a gene
encoding the nitrite reductase AniA, which is essential for anaerobic respiration in
gonococci (219, 243, 432).

Many bacteria use multiple sigma factors to control gene expression in response
to a variety of environmental stimuli. The sigma factor is the subunit of the RNA
polymerase and confers promoter specificity. There are at least seven sigma factors in E.
coli (51, 222). However, N. gonorrhoeae appears to possess relative few sigma factors.
Western blot using antibodies against E. coli or Salmonella enterica serovar
Typhimurium sigma factors first indicated the presence of multiple sigma factors in N.
gonorrhoeae, which are later identified as (:70 (RpoD), O'E (RpoE), and 0'32 (RpoH) (43,
191, 204). (:70 (RpoD) is the primary “house-keeping” sigma factor, highly conserved
among Gram-negative bacteria and responsible for the expression of most housekeeping
genes. In other bacteria, O'E (RpoE), 0'32 (RpoH), and 0'54 (RpoN) have been shown to be
important in regulating a variety of genes including heat shock, nitrogen assimilation, and

bacterial pathogenesis (45, 283, 392, 423). The sigma factor 054 (RpoN) regulates

25

expression of a variety of genes involved in nitrogen assimilation in E. coli (43, 184,
399). Although there is an moN homolog in N. gonorrhoeae, it has been shown to be
non-functional (assessed by the transcriptional and translational analysis of rpoN and its
gene product RpoN) (204). This is likely due to a deletion in the rpoN gene, which
removes the region encoding the essential HTH DNA binding motif (204). In E. coli, the
extreme heat shock sigma factor, O'E (encoded by moE) responds to misfolded extra-
cytoplasmic proteins (135), which is important for the maintenance of membrane and
periplasm homeostasis. Additionally, in S. typhimurium, GE also regulates the expression
of virulence genes (166). 0’32 (encoded by rpoH) is another heat shock sigma factor,
which is partially regulated by CE in E. coli. Although N. gonorrhoeae has homologs of
both moE and rpoH on its chromosome, their functions remain to be identified.
Interestingly, recent work in our lab shows that the gonococcal rpoE homolog may be
different from that in other bacteria, as it does not regulate the gene expression of rpoH in
the standard laboratory media or upon host cell contact (84). Moreover, our study also
shows that the gonococcal O32 (RpoH) is involved in gonococcal infection, as several

genes induced upon host cells contact are regulated by 0'32 (RpoH) (Details in Chapter 4

and 5).

Signal transduction and the Signal Recognition Particle (SRP) system in bacteria
Bacteria, particularly pathogens, have evolved many ways to modulate the

production of proteins in order to adapt to changing environments. This modulation

occurs at many levels, including transcription, translation, protein folding and protein

localization. In N. gonorrhoeae, many of the important proteins identified so far are

26

involved in interaction with host cells and are found in the outer membrane (OM) such as
pili (380), Opa (379) and porin (176). However, our knowledge about cytoplasmic
membrane (CM) proteins is very limited, although this class of proteins may also play
critical roles in pathogenesis. CM proteins include many proteins with a variety of
functions, such as transporting nutrients, excluding antimicrobial agents and some basic
housekeeping functions, such as energy generation, cell division and protein
translocation. Moreover, CM proteins also contain proteins important in signal
transduction, which is necessary for the organism to sense its environment and respond to
signals.

In bacteria, there are at least two conserved pathways to transport proteins to the
cytoplasmic membrane: the general secretory pathway (GSP) and the signal recognition
particle (SRP) pathway (303, 309). The major functional difference between these two
systems is that the GSP is a posttranslational targeting system that transports fully
synthesized proteins to and across the cytoplasmic membrane and the SRP functions
cotranslationally to target a subset of cytoplasmic membrane proteins to the membrane.

The SRP system was first identified in eukaryotes and later identified in
prokaryotes, and is highly conserved across species. The well-characterized eukaryotic
SRP system has six proteins (SRP 9, 14, 19, 54, 68 and 72) and one RNA molecule (78
RNA) (226, 433). By contrast, the bacterial SRP system is much simpler with two
proteins called th (“Fifty-four homolog” of eukaryotic SRP54), and FtsY, the SRP
receptor (SR) and a 4.58 RNA, all of which are essential for cell grth (49, 113, 294).

In the model for SRP targeting in Gram-negative bacteria, the SRP complex

(Fm/4.58 RNA) first binds to the newly synthesized signal peptide as it emerges from the

27

ribosome. This nascent peptide/SRP/ribosome complex is then targeted to the
cytoplasmic membrane through the interaction between the SRP complex and SR. The
role of 4.5S RNA in the SRP system is not only to increase the interaction between th
and the signal sequence of the nascent polypeptide, but also to increase the interaction
between F111 and FtsY (450). The binding and hydrolysis of GTP by th and/or FtsY
results in the dissociation of SRP complex from the Ribosome-nascent-chain complex
(RNC) and transfer the RNC to the translocon (SecYEG) located in the cytoplasmic
membrane, where translation resumes and the newly synthesized peptide crosses through
the cytoplasmic membrane cotranslationally (292, 306). The SRP system is beneficial to
the bacterium, likely because this system primarily targets CM proteins, thus preventing
the aggregation of these hydrophobic proteins in the cytoplasm (73, 230, 358, 407).
Orthologs of SRP components have been identified in N. gonorrhoeae (19, 102).
The gonococcal SRP system has some unique characteristics that are being studied in our
laboratory. For instance, gonococcal 4.58 RNA can directly stimulate the GTPase activity
of gonococcal FtsY (PilA), but not that of gonococcal F fl’l. Moreover, the promoter
sequences of genes encoding several SRP-dependent proteins can specifically interact
with gonococcal FtsY (PilA) and stimulate its GTPase activity (102). The role of DNA

binding in SRP funtion in N. gonorrhoeae is still to be determined.

Scope of dissertation
N. gonorrhoeae is a public health problem worldwide. To better prevent and
control diseases caused by this organism, we must improve our understanding of

gonococcal biology. Towards this goal, this dissertation focuses on the study and

28

characterization of regulatory networks of N. gonorrhoeae. DNA microarrays and
transposon mutagenesis were developed and used to examine gene expression in N.
gonorrhoeae in a tissue culture model of infection. In addition, this dissertation also
describes the identification and characterization of an essential Signal Recognition
Particle (SRP) targeted protein in N. gonorrhoeae.

Chapter two of this dissertation describes the characterization of a gonococcal
gene whose product requires the SRP system for transporting to the cytoplasmic
membrane. This gene was initially identified in a genetic screen designed to isolate
potential cytoplasmic membrane proteins, based on the fact that overproduction of any of
three SRP components would be lethal to the bacterium. This gene was later shown to
encode a firnctional cell division protein, ZipA, in N. gonorrhOeae, as that can
complement an E. coli conditional zipA mutant. This observation is significant in that it is
the first ZipA homolog identified in a non-rod-shaped organism and its essential role in
cell division may in part explain why SRP components are essential in bacteria.

Chapter three describes the development and construction of a gonococcal
expression-capable clone set and an associated DNA microarray that were critical for the
later studies. The constructed expression-capable clone set represents 83% (1672 out of
2250) protein-coding ORFs of the gonococcal genome including 42 ORFS of the
gonococcal genetical island of N. gonorrhoeae strain MSll (78). The construction of this
clone set was based on a lambda phage-based site-specific recombination system, which
provides multiple choices for expression and analysis of genes of interest (420). PCR
products used to construct the clone set were spotted on glass slides to generate an N.

gonorrhoeae DNA microarray. This microarray represents 2035 ORFS of N. gonorrhoeae

29

genome. The availability of this array allows us to examine the transcriptional profiles of
N. gonorrhoeae at the genome wide level in a single experiment.

In chapter four, we discuss the identification of genes differentially regulated in
gonococci upon host cell contact using the DNA microarray and a cell culture model of
infection. The findings show that host cell contact results in changes of gene expression
and this response is in part mediated by a heat shock sigma factor, RpoH. Studies also
show that while induction of rpoH expression is not necessary for adherence of
gonococci to epithelial cells, it is important for the subsequent invasion step, as
gonococci depleted for rpoH invade cells less efficiently than a wild-type strain.

Chapter five describes the identification of additional genes regulated by RpoH.
Several different approaches were used here, including DNA arrays, real-time PCR,
transposon mutagenesis and a cell culture model of infection. In this work three genes
induced upon adherence were identified as RpoH-dependent for this induction, as the
expression of these genes was no longer induced upon host cell contact when RpoH was
depleted. Additionally, one RpoH-independent gene was identified as important in both
adhesion and invasion steps. Furthermore, the observation that only a few of genes
differentially regulated upon host cell contact are regulated by RpoH indicates that more
than one regulator is involved in the response to host cell contact.

In chapter six, the dissertation is summarized and suggestions for future research
to expand upon the current knowledge of gonococcal regulatory networks during

infection are discussed.

30

Table 1-1. Transcriptional regulators in Neisseria gonorrhoeae FA1090 and in
related Neisseria meningitidis serogroup A Z2491 (NMA) and serogroup B MC58

 

 

(N MB)

GC ID NMA ID NMB ID Name Gene product

Transcription: Transcription factors

NG0199 NMAO825 NMBO617 rho Transcription termination factor rho

NG0256 NMA0885 NMB0683 nusB NusB protein (transcription termination
factor)

NG0262 None NMBO689 greB Transcription elongation factor

NG0288 NMA0917 NMB0712 7720}! RNA polymerase sigma-32 factor

NG0899 NMA1643 NMB 1430 greA Transcription elongation factor (GreA)

NG0999 NMA1737 NMBlS38 rpoD RNA polymerase sigma factor RpoD

NGl766 NMA0049 NMB0217 rpoN Sigma factor 54

NG1856 NMA0147 NMB0126 nusG Transcription antiterrnination protein

NG1944 NMA0230 NMB2144 moE ECF-family RNA polymerase sigma factor

Regulatory functions: No subcategory

NGOOZS NMA0578 NMBl878 Possible AraC family transcription regulator

NGOOSS NMA0613 NMB l 843 MarR family transcriptional regulator

NG0128 NMA0701 None wrbA Trp repressor binding protein, WrbA

NG0152 NMA1632 NMB 1420 fis Factor for inversion stimulation protein, F is

NG0267 NMA0897 NMB0694 folI FolC transcriptional regulator

NGO393 NMAlOZO NMBOSIO TetR-family transcriptional regulator

N00602 NMA1517 NMB 1303 nmIR MerR-farnily transcriptional regulator Nran

NG0671 NMA1559 NMB 1 347 suhB Extragenic suppressor protein (inositol
monophosphatase)

NG0692 NMA1437 NMBIO49 LysR-family transcriptional regulator

NG0718 NMA1605 NMBl389 hexR RpiR/HexR family transcriptional regulator

NG0797 NMA1375 NMBlZO4 Transcriptional regulator

NGO99O NMA1729 NMB1529 araC AraC-family transcriptional regulator

NG1053 NMA1689 NMB 1479 oraA recX RecX-family transcriptional regulator

NG1064 NMA1698 NMB 1493 cstA Carbon starvation protein (CstA)

N01 1 16 None NMB0910 prtR Transcription regulatory protein, PrtR

NG1185 None None asrR ArsR-farnily transcriptional regulator

NG1219 NMA1749 NMBlS61 deoR ngR DeoR-family transcriptional regulator;
glycerol-3-phosphate regulon repressor

NG1221 NMA1751 NMB1563 gntR mdcY GntR-family transcriptional regulator

NG1244 NMA1774 NMB l 585 MarR-family transcriptional regulator

NG1250 NMA1783 NMB1591 mtrA Transcriptional regulator MtrA

NG1294 NMA1905 NMB 1650 lrp Leucine-responsive regulatory protein (Lrp
family)

NG1360 NMA1965 NMBl7ll GntR-family transcriptional regulator

NG1366 NMA1971 NMBl7l7 ach mtrR MtrCDE transcriptional regulator, repressor

NGl40l None None nosR Regulatory protein NosR

NO 1402 NMA0761 NMB0577 nosR Regulatory protein NosR

NG1407 NMA0756 NMBOS 73 aan Asn C-family transcriptional regulator

NG1427 NMA0738 NMBOSS6 Transcriptional regulator, repressor

NG1474 NMA0601 NMB 1 856 crgA LysR-family transcriptional regulator

NG1562 NMA2087 NMBO398 arsR ArsR-family transcriptional regulator

NO 1 578 NMA2 1 06 NMB038 l cysB Cysteine transcriptional regulatory protein,

31

NG1579
NG1630
NGl652

NG1706
NG1779
NG1813
NG2027

NG2102
NG2130

NG2131

NG2161

NMA2 107
None
NMAl 544

NMA2 197
NMA0064
NMA0098
NMA03 8 1

NMA0463
NMA0498

NMA0499

NMA0529

NMBO38O
None
NMB0900

NMBOZ90
NMB0205
NMB0173
NMB2055

NMBl981
NMB1953

NMB1952

NMB 1924

lysR

fur

oxyR
metR

luxS

regF sspA

regG sspB

Regulatory functions: Two component system

NG0111

NG0112

NG0176

NG0177

NG0314

NG03 l 3

NG0752

NG0753

NG1866

NG1867

NMA0798

NMA0797

NMA0798

NMA0797

NMA0947

NMA0946

NMA1419

NMA1418

NMA0159

NMA0160

NMB0595

NMBOS94

NMB0595

NMBOS94

NMBO737

NMB0736

NMB1250

NMB1249

NMBOIlS

NMB0114

basR
basS

resE, phoP,
MisS

ompR , pth,

MisR
Her

narP

NarQ

12de ntrX

AtoC

atoS 1:de

activator (CysB)

Transcriptional regulator, Anr/Fnr family
Lambda repressor-like protein cI
Probable antirepressor protein - phage
associated

LysR-family transcriptional regulator
Ferric uptake regulation protein Fur
LysR-farnily transcriptional regulator
Methionine biosynthesis transcriptional
regulator (MetR)

LUXS protein -- autoinducer A12 synthesis
Regulator of piIE expression; stringent
starvation protein A

Regulator of pilE expression (RegG);
stringent starvation protein B

Possible extragenic suppressor; inositol
monophosphatase family protein

DNA-binding component of two-component
regulatory system

Sensor component of two-component
regulatory system, BasS

Sensor histidine kinase of two-component
system

Transcriptional response regulator of two-
component system

Hpr kinase/phosphatase (two component
system)

Putative regulatory protein, probable PTS
system, nitrogen regulatory IIA protein
NarP (nitrite/nitrate two-component response
transcriptional regulator )

Nitrate/nitrite two-component system sensor
protein NarQ

Probable nitrogen assimilation regulatory
protein

Probable two-component system sensor
kinase

 

(Table 1-1 cont’d)

32

Chapter 2

Identification of ZipA, an SRP-dependent protein from Neisseria gonorrhoeae

This manuscript has been previously published in Journal of Bacteriology:

Du, Ying and Arvidson, Cindy Grove 2003 Identification of ZipA, a Signal Recognition
Particle-Dependent Protein from Neisseria gonorrhoeae, J.Bacteriol. 185:2122-2130

33

ABSTRACT

A genetic screen designed to identify proteins that utilize the signal recognition
particle (SRP) for targeting in Escherichia coli was used to screen a Neisseria
gonorrhoeae plasmid library. Six plasmids were identified in this screen and each is
predicted to encode one or more putative cytoplasmic membrane (CM) proteins. One of
these, pSLO7, has three open reading frames (ORFS), two of which have no similarity to
known proteins in GenBank other than sequences from the closely related N.
meningitidis. Further analyses showed that one of these, SLO7ORF3, encodes a protein
that is dependent on the SRP for localization. This gene also appears to be essential in N.
gonorrhoeae as it was not possible to generate null mutations in the gene. While
appearing unique to Neisseria at the DNA sequence level, SLO7ORF3 was found to
share some features with the cell division gene zipA of E. coli. These features included
similar chromosomal locations (with respect to linked genes) as well as similarities in the
predicted protein domain structures. Here, we Show that SLO7ORF 3 can complement an
E. coli conditional zipA mutant, and therefore encodes a functional ZipA homolog in N.
gonorrhoeae. This observation is significant in that it is the first ZipA homolog identified
in a non-rod-shaped organism. Also interesting is that this is the fourth cell division
protein (the others are FtsE, FtsX, and FtsQ) shown to utilize the SRP for localization,
which may in part explain why the genes encoding the three SRP components are

essential in bacteria.

34

INTRODUCTION

The prokaryotic signal recognition particle (SRP) is a complex of two proteins,
FtsY and Fill, and a 4.5S RNA that targets a subset of proteins to the cytoplasmic
membrane (CM) co-translationally (73, 230, 279, 358, 407). The use of a co-translational
targeting system by this subset of proteins is thought to be advantageous in that it avoids
their aggregation in the cytoplasm, which might occur if they were targeted post-
translationally. All of the SRP components are essential for viability in Escherichia coli
(49, 113, 294) suggesting that this targeting system is critical for the proper localization
of proteins involved in essential cell processes.

This is certainly true for the sexually transmitted pathogen, Neisseria
gonorrhoeae. The gonococcal FtsY ortholog PilA was shown to be essential to the
gonococcus (387) before PilA was shown to be part of the SRP (19). Since N.
gonorrhoeae is an important human pathogen, the most intensively studied proteins of
this bacterium are proteins involved in interactions with host cells. These virulence
factors are mostly outer membrane (OM) components such as pili (380), P11 or Opa
(200), P1 or porin (176), LOS (341) and iron utilization proteins (35, 59, 64). A few, such
as the IgAl protease, are secreted (364). Relatively little is known, however, of other
membrane-associated proteins in Neisseria or their functions, especially those of the CM.
CM proteins include many transporters for nutrients as well as enzymes involved in the
maturation of outer membrane components. Also included are efflux pumps, which
prevent otherwise harmful materials from accumulating within the cytoplasm. This class

of proteins can be very important for pathogens as a mechanism to exclude harmful

35

antimicrobial agents and are important targets for vaccine and drug development. Other
CM proteins include those involved in energy generation and conservation, respiration,
cell division, and protein translocation. The CM also contains proteins involved in signal
transduction, which is necessary for the organism to sense its environment, and
components necessary to respond to such signals. One of the goals of our research has
been to identify and characterize putative CM proteins of N. gonorrhoeae, with the
ultimate goal of identifying unique proteins that might be useful as targets for drug
development.

Using a screening approach that takes advantage of the fact that the relative levels
of each of the components of the prokaryotic SRP are critical for function and survival of
the organism (407), we have identified several genes of N. gonorrhoeae that encode
proteins that utilize the SRP for localization. Sequence analysis of these genes revealed
one that is apparently unique to Neisseria, having no close matches in the GenBank
database. Further examination of this gene, however, suggested that it might be

structurally and functionally related to the cell division protein, ZipA, of E. coli.

36

MATERIALS AND METHODS

DNA manipulations. E. coli recombinant DNA manipulations were performed as
described (333). Cloning vectors used were pET24a (Novagen, Madison, WI),
pACYC 184, pHSS6 (356), pWSK129 (424) and pWSKlacIOPEl. pWSKlacIOPEl was
constructed by ligating a 2.8 kb lacIOPErm fragment of pHSXErm (351) into the NotI
site of pWSK29 (424). Restriction enzymes and T4 DNA ligase (New England Biolabs,
Beverly, MA) were used according to the manufacturer’s recommendations. Polymerase
chain reaction (PCR) was done using a GeneAmp 9700 therrnocycler (Applied
Biosystems, Foster City, CA) and Taq DNA polymerase (Roche Molecular
Biochemicals, Indianapolis, IN). Oligonucleotide primers were purchased from Sigrna-
Genosys (The Woodlands, TX) or the Michigan State University Macromolecular
Structure, Sequencing and Synthesis Facility and the sequences are available on request.
The QIA quick PCR Purification kit (Qiagen, Valencia, CA) was used to purify PCR
products. DNA sequence determination of pSLO plasmids was done by the Core Facility
of the Department of Molecular Microbiology and Immunology at Oregon Health
Sciences University. All other plasmids were sequenced by the Michigan State University
Macromolecular Structure, Sequencing and Synthesis Facility. DNA sequences were
analyzed using the Oxford Molecular Group DNA analysis programs MacVector and

Omiga (Accelrys, San Diego, CA).

Transposon mutagenesis. Transposon mutagenesis of plasmid DNA was performed by

in vitro transposition using EZ::‘I'N'M transposase (Epicentre Technologies, Madison,

WI) and the modified transposon TnErmUP contained on the plasmid pMODErmUP (H.

37

S. Seifert, unpublished). The TnErmUP transposon was prepared by PCR amplification
from pMODErmUP using Oligonucleotide primers FP-l and RP-l (Epicentre). Following
the transposition reaction, plasmid DNA was transformed into E. coli strain DH5a, and
transforrnants selected for EmR. The position of the transposon insertion was determined
by PCR using Oligonucleotide primers homologous to the ends of the transposon (SqFP
and SqRP, Epicentre) and to the ends of the pSLO7 insert, followed by restriction

analysis.

Southern blot analysis. Chromosomal DNA fi'om N. gonorrhoeae transforrnants was
isolated and digested with ClaI. Fragments were separated on 1% agarose gels and DNA
blotted to nylon membranes as described (19). DNA probes for hybridization were
generated by the random priming method with the digoxygenin (DIG) DNA-labelling and

detection kit (Roche).

Growth of bacterial strains. E. coli strains used were DH50L, BL21XDE3 (378), S17-
lkpir, HDB29 (214), N4156::pAra14-FtsY (227), CH3pCH32 and CH5pCH32 (140). E.
coli were routinely grown in Luria broth supplemented as necessary with ampicillin (Ap)
at 100 mg/l, chloramphenicol (Cm) at 20 mg/l, kanamycin (Kn) at 50 mg/l,
Spectinomycin (Sp) or streptomycin (St) at 25 mg/l, or erythromycin (Em) at 300 mg/l. N.
gonorrhoeae strain MSl 1A [P+tr;](349) was maintained in a humidified 5% C02
atmosphere on GC agar (Difco Laboratories, Sparks, MD) with supplements (187). N.
gonorrhoeae transformation was performed as described (355). Em was used at 3 mg/l

for N. gonorrhoeae.

38

Protein isolation and analysis. Bacterial cells were fractionated into periplasm,
cytoplasm, cytoplasmic and outer membranes as described (19) and protein
concentrations determined by the Bradford method (BioRad Laboratories, Richmond,
CA). Proteins were separated on 15% polyacrylamide gels and transferred to
nitrocellulose membranes electrophoretically. Detection of Hisé-tagged proteins was done
using Ni-NTA-HRP (horseradish peroxidase) conjugate (Qiagen) in TBS (Tris-buffered-
saline) buffer. Membranes were incubated in 10% (w/v) nonfat dry milk in TBS to block
and then with Ni-NTA-HRP in 2% milk. HRP was detected with the chemiluminescent
detection agent SuperSignal (Pierce, Rockford, IL) used according to the manufacturers’

directions.

Construction of the SL0 screen strain. E. coli fih was amplified from genomic DNA
by PCR and cloned into pBluescript II SK- to create pBluFflr. pBluFflr was then digested
with HpaI to remove an internal 777 bp fragment which was replaced with a 1222 bp
fragment encoding resistance to erythromycin (401). This is the same deletion as was
used for construction of the flh::kan-1 allele (294). The entire fihxerm fiagment was then
cloned into the suicide plasmid pKAS32 (367), which contains the n-dependent R6K
origin of replication (255), an RP4 origin of transfer for conjugation, and the rpsL gene,
which renders the host strain (Sl7-17tpir) sensitive to streptomycin (Sm) which is
dominant to SrnR alleles. The resulting strain Sl7-1Xpir/pSFfl1::erm was mated with
HDB29 (SmR) and transconjugants selected for EmR and SmR. pSFflizzerm cannot
replicate in HDB29, forcing the flhxerm allele to recombine with the flh::kan-1 allele on

the chromosome of HDB29. Selection for SmR insures that a double crossover event

39

(replacement offlh::kan-1 with flh: :erm ) and not a single crossover event (integration of
pSFfli::erm into the chromosome) occurs. HDB29 also contains an flh gene under the
control of Pm on a separate replicon, although if the flh: :erm allele were to recombine at
this locus, there would be no intact flh gene and these recombinants would not survive,
since flh is essential to E. coli (294). Multiple EmR transconjugants were obtained and
screened for KnS and Cbs, to insure that the fih::kan-1 allele was no longer present and
that the entire plasmid, pSth::erm had not integrated into the chromosome. The resulting

strain was called CGA29 (flhxerm 7tD69 HinDIII::lacIQ P-mfjh).

40

RESULTS

SLO screen of a gonococcal gene bank. Previous studies have shown that multicopy
plasmids expressing SRP-dependent E. coli proteins confer a lethal phenotype in strains
producing limiting amounts of th (407). In E. coli strain CGA29,fi’h is under the control
of the IPTG-inducible trc promoter. LacI repression offlh is not complete in this strain,
and a small amount of F 111 is made in the absence of IPTG. Ulbrandt et al. (407) showed
that this small amount of F 111 is sufficient for survival of the bacterium under normal
conditions. However, if a multicopy plasmid expressing a protein that utilizes the SRP is
present in this strain, it can no longer survive unless the level of F fh in the cell is
increased by the addition of IPTG to the growth medium. They termed this effect SLO,
for §ynthetic lethality upon gverproduction. Since the SRP proteins from E. coli and
Neisseria are similar, and we have Shown that N. gonorrhoeae PilA can functionally
replace FtsY in E. coli (19), we reasoned that gonococcal proteins that utilize the SRP
might also confer a SLO phenotype to the conditional Fflr strain in this system.

The N. gonorrhoeae strain MSl 1A gene library pCBB (19) was introduced into
CGA29 and transforrnants selected on plates containing 10 uM IPTG. Transformants
were next cultured on duplicate plates with and without IPTG, and examined to identify
those with little or no grth under Fflr-limiting conditions. In a screen of 1008
CGA29pCBB transformants, 14 were found to be sensitive to growth under F ill-limiting
conditions. These were divided into two groups based on the severity of their growth
defect. Five isolates were moderately affected, with colonies noticably smaller than a
vector-only control on plates lacking IPTG. Nine isolates were severely affected, with

little or no grth in the absence of IPTG.

4l

Plasmid DNA was isolated from each strain and retransforrned into CGA29 to
confirm that the SL0 phenotype was plasmid-linked. Of the 14 original isolates, 4 were
eliminated because the inserts were unstable. The SLO phenotypes of the remaining 10
were the same as the original isolates (6 severe and 4 moderate) and the DNA sequences
of each of the plasmids were determined. First, the sequences were scanned for the
presence of open reading frames (ORFS) using the DNA analysis programs MacVector
and Omiga. The deduced protein sequence for each ORF was then analyzed using
PSORT (272), which predicts the cellular localization of a protein. The protein sequences
were also used to search the GenBank database using BLAST (10). And finally, the
sequences of each insert were used as query in a BLAST search of the annotated N.
gonorrhoeae strain F A1090 database (http://www.stdgen.lanl. gov).

A summary of the sequence analyses of the 6 severe SLO clones is shown in
Table 2-1. These results showed that each of the plasmids encoded at least one putative
inner/cytoplasmic membrane protein. Since the bacterial SRP targets a subset of proteins
whose final destination is the CM (73, 279, 332, 358, 407), these results indicate that this
heterologous screen does identify proteins from a Neisseria gene bank that interact with
the E. coli SRP. Of the 4 plasmids conferring a moderate SLO phenotype, 3 encoded at
least one putative CM protein and only 1 appeared to be a false positive, encoding 2
putative cytoplasmic proteins (data not shown). The frequency obtained in this
preliminary screen is 0.9% (9 of 1008), similar to the 1-2% estimated by Ulbrandt and
co-workers for SRP-dependent proteins in E. coli (407). Of the 6 severe SLO clones, one,
SLO7, appeared to encode a putative CM protein that was not similar to any sequences in

the GenBank database, suggesting it was unique to Neisseria.

42

Table 2-1. Sequence analysis of severe SLO clones.

 

 

 

 

 

 

 

SLO clone Insert size BLAST results PSORT location
7 2121 bp AmpD cytoplasm
Conserved hypothetical protein periplasm
(5’ 914/945”)
Unknownb ORF (5’ 642/1284“) CMc
16 4307 bp cytosine permease CM
(5’ 883/1224 bp)
TldD (CsrA supressor) cytoplasm
histidine permease CM
glutamine permease CM
24 2953 bp protein disulfide isomerase CM or OMd
(3’ 312/492 bp)
ubiquinone oxidoreductase CM
ABC-type exporter periplasm
48 2273 bp phosphoglucomutase cytoplasm
(5’ 463/1383 bp)
proline cis/trans isomerase cytoplasm
Na‘L/H+ antiporter CM
53 2366 bp ABC transporter permease CM
(5’ 694/1932 bp)
Dst (protein disulfide periplasm
isomerase)
DNA replication protein cytoplasm
(5’ 658/2199 bp)
87 2161 bp Hypothetical protein CM or OM
glutamate permease CM
(5’ 618/1213 bp)

 

 

a partial sequence bnot in GenBank; °cytoplasmic membrane; douter membrane

43

Analysis of the ORFS of pSLO7. Sequence analysis of the pSLO7 insert showed it
contains 3 putative ORFS which we designated ORFl, ORF2, and ORF3 (Figure 2-1).
Only the 5’ 914 bp of the 945 bp ORFl are on pSLO7, and this ORF encodes a
hypothetical protein with no similarities in the GenBank database, other than a conserved
hypothetical protein from N. meningitidis. PSORT analysis of the predicted protein
sequence of ORFl suggests it is localizes to the periplasm. ORF2 encodes a protein with
49% identity and 61% similarity to AmpD of E. coli. AmpD is thought to be a regulator
of B-lactamase induction (403) and is probably involved in the recycling of products of
peptidoglycan turnover (160). PSORT analysis of the putative AmpD homolog predicts it
to localize to the cytoplasm. ORF3, like ORFl, is only partly on pSLO7 (5’ 642 bp of
1284 bp). Also like ORF], ORF3 appears to encode a protein unique to Neisseria, and
encodes a protein with no similarities in the GenBank database, aside from hypothetical
proteins in N. meningitidis. PSORT analysis of the predicted protein encoded by ORF3

suggests that it localizes to the cytoplasmic membrane.

Transposon mutagenesis of pSLO7. In order to determine which of the ORFS of pSLO7
was responsible for the SL0 phenotype, each ORF was disrupted by in vitro transposition
(117) using the transposon TnErmUP contained on the plasmid pMODErmUP (kindly
provided by H. Seifert). pMODErmUP is a modification of pMODm-2<MCS>
(Epicentre) in which the ermC gene (418) and a Neisseria uptake sequence (115) have
been inserted between the mosaic ends (ME) recognized by the EZ::TNT" transposase
(Figure 2-1B). Following transformation of the transposition mix, KnREmR transforrnants

were selected and the positions of the TnErmUP insertions determined by PCR and

44

restriction analysis (Figure 2-1). Each of these plasmids was then transformed into
CGA29 and transforrnants scored for growth on plates with and without IPTG (Table 2-
2). Plasmids with insertions in ORFl (pSLO7::TnEm #11) or ORF2 (pSLO7::TnEm #3)
only grew in the presence of IPTG, i.e., confer a SLO phenotype. However, CGA29
harboring either of the ORF3 insertion plasmids (pSLO7::TnEm #10 or #26) grew well in
the presence and absence of IPTG, no longer conferring a SLO phenotype. This indicates
that the remaining ORFS on this plasmid, ORFl and ORF2, do not encode SRP-
dependent proteins, and suggests that ORF3 is responsible for the SL0 phenotype of
pSLO7. To confirm this, since pSLO7 contains a truncated ORF3 gene, the entire
predicted ORF 3 gene along with associated upstream expression sequences was PCR
amplified from MSllA genomic DNA and cloned into pHSS6 (356). The resulting
plasmid, pSLO7ORF 3, conferred a SLO phenotype when transformed into CGA29 and
grown without IPTG (Table 2-2), confirming that ORF3 is responsible for the SL0

phenotype.

Effects of SRP-depletion on cellular localization of SLO7ORF3. In order to detect
small amounts of the SLO7ORF3 protein in cellular protein extracts, a plasmid was
constructed such that an epitope tag (Hi86) was added to the C-terminus of the full-length
ORF3 protein. We were unable to detect a firll length SLO7ORF3-His6 when
overexpressed (data not shown), therefore a truncated version was constructed. The 5’
840 bp of the SLO7ORF3 coding region was PCR amplified from N. gonorrhoeae strain
MSl 1A genomic DNA and ligated into pET24a, placing a truncated (280 amino acids)

ORF3 with a C-terminal His6 tag under the control of a T7 promoter.

45

A. pSLO'I insert

 

 

 

 

 

 

 

11E SE 105 265
V V V
l l E——'1 1 l
‘— ORF1 ORF2 —’ ORF-'3' —’
B. TnErmUP
ME ME

Figure 2-1. Schematic diagram of the pSLO7 insert and the TnErmUP insertions. A.
pSLO7 insert. Arrows of ORFS indicate direction of transcription. V indicates position of
the TnErmUP (3E, 10E, 11E, 26E) insertions. B. TnErmUP transposon. Small arrows
indicate Oligonucleotide primers used to prepare the transposon for in vitro transposition

(FP-l and RP-l) and to map transposon insertions (SqRP and SqFP).

46

Table 2-2. SLO screen of pSLO7::TnErmUP insertion mutants.

 

 

Plasmid Genotype Growth + IPTG Growth - IPTG
pHSS7 Vector control + +
pSLO7 Wild-type insert + -

+
I

pSLO7::TnErmUP #3 orf1+orj2'orj3+

pSLO7::TnErmUP #11 om°orj2+orf3+ + _

pSLO7::TnErmUP #10 orj7+orj2+opj3' + +
pSLO7::TnErmUP #26 0rf1+opj2+o;j3‘ + +
pSLO7ORF3 043+ + _

 

 

The resulting plasmid, pSLO7ORF3’-His(,, was transformed into E. coli strain
BL217tDE3 (378) and induced with IPTG. Cells were then fractionated into periplasmic,
cytoplasmic, and membrane fractions as described (19). The membrane fraction was
extracted with 0.2 % Sarkosyl to separate soluble (CM) and insoluble (OM) proteins
(140). Samples were separated by SDS-PAGE and transferred to nitrocellulose, and the
ORF3’-Hi56 protein was detected using an Ni-NTA-HRP conjugate. ORF3’-His6 was
found in both membrane fiactions, but not the cytoplasm or periplasm, suggesting that
SLO7ORF 3 localizes to the cytoplasmic membrane.

To determine whether the localization of the ORF3’-Hi56 protein was dependent
on the SRP in E. coli, the pSLO7ORF3’-His(, construct was subcloned onto a derivative
of pWSK129 (424) placing an IPTG-inducible lac-promoter upstream of ORF3. This

construct, pWSKORF3’-Hi56, was transformed into the conditional SRP E. coli strain,

47

N4156::pAra14-FtsY (227), in which the production of the SRP receptor, FtsY, is
controlled by the araBAD promoter. This strain absolutely requires L-arabinose for
growth, and culturing in the absence of this sugar results in the depletion of FtsY and
eventually kills the cell. However, before the cells die, the depletion of FtsY causes an
accumulation in the cytoplasm of proteins that are dependent on the SRP.
N4156::pAra14-FtsY/pWSKORF3’-His6 was grown overnight in medium containing
0.2% L-arabinose. The culture was washed with medium lacking arabinose, and
subcultured to medium containing 0.2% glucose with or without arabinose at a starting
concentration of ~107 CFU/ml. After growth for 7 hr at 37°C, the cultures were harvested
and cells fractionated as described above. ORF3’-His6 protein was detected with Ni-
NTA-HRP and the results showed that under conditions of FtsY-depletion (medium
lacking L-arabinose) ORF3’-His6 accumulates in the cytoplasm (Figure 2-2), indicating
that under these conditions ORF3’-Hi36 is not targeting to the membrane as it does under
FtsY-replete (w/arabinose) conditions. We conclude from this result that SLO7ORF3 is

directly or indirectly dependent on the SRP for CM localization.

ORF3 is essential for N. gonorrohoeae. To determine whether any of the genes on
pSLO7 were essential to N. gonorrhoeae, plasmids containing transposon insertions in
each of the putative ORFS were used to transform N. gonorrhoeae strain MSl 1A (349).
Neisseria are naturally competent for transformation and will take up DNA at high

fi‘equencies as long as the DNA contains a Neisseria uptake sequence (NUS) (115).

48

cytoplasm CM

I-lm- IW”MI fl Fl 9” I l . kl I. In I" h ”C L ‘5 “f P‘V ..- l. I . A ll . 9 I I. I ‘ I" V' r I ‘l ‘4
I
I t ; 3| ’ a h . ti
‘ 0
.

 

4. . , .
1‘..- “P _

 

arabinose -— + _. +

Figure 2-2. Effect of SRP depletion on localization of SLO7ORF3'-His6 in E. coli.
N4156::pAra14-FtsY transformed with pSLO7ORF3'-His6 was grown for 7 h with (FtsY
replete) or without (FtsY depleted) arabinose. The cells were fractionated, and 100 ug of
cytoplasm or Sarkosyl-extracted membrane (CM) proteins were separated by SDS-PAGE
and transferred to a nitrocellulose membrane. SLO7ORF3'-His6 was detected with Ni-

NTA-HRP, followed by chemiluminescent detection of HRP.

49

Since there are two NUS in pSLO7 (both in ORF 2) as well as one on TnErmUP, it was
predicted that the transposon insertion mutant plasmids would be taken up efficiently by
MS] 1A. MSl 1A was transformed with linearized pSLO7::TnErmUP plasmid DNA and
selected for EmR. If a gene is essential to the gonococcus, it would be predicted that no
EmR transformants would be obtained in such an experiment. Table 2-3 summarizes the

results of 3 independent transformation experiments. Experiments 1 and 3 utilized 0.2 pg

of DNA, and experiment 2 was done using 0.6 ug of plasmid DNA. In each experiment,
high frequencies of transformation were observed for plasmids with insertions in ORFl
and ORF2 (2.1 x 10'5 to 4.6 x 10‘1 EmR/total CFU), but very few EmR transformants were
obtained with plasmids containing insertions in ORF3. This strongly suggested that
ORF3 is essential to the gonococcus. pSLO7::TnErmUP#10, which contains an insertion
just after the predicted start codon of ORF 3, yielded a total of six EmR transformants, two
in experiment #2, and four in experiment #3. In order to determine whether these
transformants had a disruption of the ORF 3 gene, Southern blot analyses were performed
on DNA from 2 of the ORF3 EmR transformants and one each of the ORFl and ORF2
EmR transformants (data not shown). When the TnErmUP transposon was used as a
probe, a single band was observed for each of the mutants, as expected. When pSLO7
was used as a probe, two bands were observed in the putative ORF3 mutants, whereas
only a single band (the same mobility as that observed with the transposon probe) was
observed for the ORF 1 and ORF2 mutants. The mobility of this band was Slightly slower
than the band observed in MS] 1A (wild-type) consistent with an insertion of 1.3 kb, the
size of the transposon. The two bands observed for the putative ORF 3 mutants suggested

a duplication event, such that these isolates are heterodiploids containing a mutated as

50

well as a wild-type copy of ORF3. This supports the conclusion that ORF 3 is essential to

the gonococcus.

Analysis of the putative ORF3 gene product. The ORF3 sequence on the N.
gonorrhoeae strain FA1090 chromosome is located between ligA, the gene for DNA
ligase (211) and ampD. Comparison to the ligA region of the E. coli K-12 genome (39)
revealed that the cell division gene, zipA (140), is found in the same position with respect
to ligA as ORF3 in Neisseria.

The predicted SLO7ORF3 polypeptide (Figure 2-3) is 428 amino acid residues
with a calculated molecular weight of 47.5 kDa, somewhat larger than E. coli ZipA at
328 residues and 36.4 kDa. E. coli ZipA has an hydrophobic N-terrninal domain (residues
1-21) with no apparent signal peptidase cleavage site, followed by a stretch of basic
residues, which reportedly prevents membrane translocation (12). This is followed by a
large apparently cytoplasmic domain with no hydrophobic stretches that are long enough
to span the membrane. It has been suggested that ZipA is anchored in the cytoplasmic
membrane with the remainder of the protein in the cytoplasm (140). Comparison of the
predicted SLO7ORF 3 protein sequence and E. coli ZipA showed no significant
similarities in the primary sequence (using pairwise BLAST). Interestingly, however,
these two proteins do appear to share features of their predicted secondary protein
structure, including the N-terminal hydrophobic region (residues 1-21), the following
basic region (residues 26-50, net charge +6) and the remaining cytoplasmic regions. An
interesting feature of E. coli ZipA is the unusually high number of proline (31%) and

glutamine (23%) residues, which are thought to form a rigid linker that holds the C-

51

terminal domain in place extended from the membrane anchored domain (21).
SLO7ORF3 also has an atypically high number of proline (13%) and glutamine (8%)
residues in a similar region of the predicted protein sequence (Figure 2-3). Furthermore, a
ClustalW alignment of the amino acid sequences of the C-terminal region of E. coli ZipA
with the similar region of ORF3 and the putative ZipA of H. influenzae (Shown in Figure
2-3) shows that there is Significant similiarity in this region, leading us to hypothesize

that SLO7ORF3 might encode an ortholog of ZipA.

Complementation of an E. coli conditional zipA mutant. The entire SLO7ORF3 region
from N. gonorrhoeae strain MSl 1A was ligated into pET24a placing a full length ORF3
under the control of the T7 promoter. The resulting plasmid, pET-ORF3, was
transformed into E. coli strain BL217tDE3 (378) and analyzed for protein production
upon induction with IPTG. SDS-PAGE analysis showed the induction of a protein of ~50
kDa, similar to the predicted 47.5 kDa size of the ORF protein (data not shown).

In order to determine whether ORF3 could complement a conditional zipA mutant
strain of E. coli, we obtained two strains, CH3(recA::Tn10, zipAU and CH5(recA::TnlO,
zipAzzaph), both harboring the plasmid pCH32(repA‘szipA+flsZ+) from Piet deBoer (140).
The zipA allele on the chromosome of CH5 has been insertionally inactivated [zipAzzaph],
such that this strain can only grow when harboring pCH32 and at the permissive
temperature (30-32°C), since zipA is an essential gene. CH3 is a wild-type (zipA+) control
that grows well at the permissive (30-32°C) as well as non-perrnissive (37-42°C)

temperature, even while harboring pCH32.

52

Figure 2-3. Alignment of the amino acid sequence of putative gonococcal ZipA
homolog (N.g.) with the E. coli (E.c.) (21) and H. influenzae (Hi) (14) ZipA
sequences. The entire coding region of SLO7ORF3 is annotated in the STDGEN
database (http://www.stdgen.lanl.gov/) as NG0236. ClustalW alignment was done using
the DNA analysis program Omiga. Numbering is sequential for N. g. The amino-terminal
hydrophobic residues thought to anchor the protein in the CM are double underlined. The
positively charged region (residues 26-50) believed to prevent further export of the
protein through the CM is indicated by bold underline. Proline (P) and glutamine (Q)
residues in a region of high concentration of these residues are indicated in black boxes.
Gray boxes indicate residues in the most conserved C-terminal region that are identical or
functionally similar in at least two of the proteins indicated, and shows the high degreee

of Similarity between SLO7ORF 3 and these other two ZipA homologs.

53

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296 366
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367 423
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" Dionkmgomomtmmw

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54

Table 2-3. Transformation of M81] with pSLO7::TnErmUP insertion mutants.

 

 

 

Plasmid Transformation reguency‘
(genotype)

Exp #1 Exp #2 Exp #3
pSLO7::TnErmUP#3 4.6 x 10“ 6.3 x 10'5 7.0 x 10'5
(or/2‘)
pSLO7::TnErmUP#11 3.6 x 10‘5 5.1 x 10'5 2.1 x 10'5
(orfI')
pSLO7::TnErmUP#lO <1b x 10'7 2.4 x 10'7 3.9 x 10'7
(or/3‘)
pSLO7::TnErmUP#26 <1 x 10‘7 <1 x 10'7 <1 x 10'7

(0'13")

 

 

aFrequencies are reported as erythromycin resistant (EmR) transformants per total number

of CFU.

bThe limit of detection in this assay is 1.0 x 107; a value of <1 x 10'7 indicates that no

transformants were obtained.

55

To construct appropriate plasmids to test whether SLO7ORF 3 could complement
ZipA in E. coli, ORF3 was subcloned from pET—ORF3 to a plasmid containing a lac
promoter and the lac]Q gene, pWSKlacIOPEl (see Materials and Methods). The entire
lacIQ-Plac-SLO7ORF3 construct was then ligated into pACYC184. The resulting
plasmid, pACYCLacORF3, was used to transform CH3pCH32 and CH5pCH32 (along
with pACYC184 as a control). Transfonnants were then streaked on LBCm plates in
duplicate and incubated at 30°C or 37°C (Figure 2-4). At 30°C, all four strains grew well,
as expected. At 37°C the wild-type strain CH3pCH32 harboring pACYC184 or
pACYCLacORF3 grew well, but CH5pCH32 harboring pACYC184 did not, which was
also expected, since at the non-permissive temperature ZipA would be depleted in this
strain. However, CH5pCH32 harboring pACYCLacORF3 did grow well at 37°C, the
non-permissive temperature for replication of the ZipA+ plasmid, demonstrating that
SLO7ORF3 can complement a zipA null mutation in E. coli.

We next asked whether SLO7ORF3 could alleviate the filamentous phenotype of
ZipA-depleted cells previously reported (140). Strains CH3pCH32pACYCLacORF3,
CH3pCH32pACYC184, CH5pCH32pACYCLacORF3, and CH5pCH32pACYC184
were grown in liquid culture overnight at 30°C, then diluted 1:500 and shifted to 37°C
and grown until an optical density (OD) at 600 nm of ~0.2-4 was reached. All strains
except CH5pCH32pACYC184 reached this density in about 2 hr. After 6 hr of growth
CH5pCH32pACYC184 reached a maximum OD600 of just under 0.2, consistent with the
inability of this strain to grow at the non-permissive temperature. Samples of each culture

were spotted onto glass microscope slides and gram-stained (Figure 2-5).

56

 

ORF3" 0RF3"

 

ORF3‘ ORF3'

CH5 c113
(Zip-4") (2594*)

 

Figure 2-4. In vivo complementation of E. coli zipA with N. gonorrhoeae SLO7ORF3.
E. coli strains CH3(zipA*)pCH32(repA‘szsz*ftsZ*) and CH5
(zipA::aph)pCH32(repA“zipA+ftsZ+) transformed with pACYC184 (ORF3') or
pACYCLacORF3 (ORF3+) were streaked on LB szo plates and grown at the permissive
(30°C) or non-permissive (37°C) temperature. Plates were photographed using a UVP

BioDoc-It system with back lighting.

57

As expected, the zipA+ control strain CH3pCH32 showed normal individual cells, with
the occasional pair in the process of dividing. This was observed whether pACYC184 or
pACYCLacORF3 was present, suggesting this level of ORF3 expression did not affect
cell division in the presence of E. coli zipA. CH5pCH32 harboring pACYC184, however,
was extremely filamentous. Few, if any, individual cells were observed in any slide (4
slides prepared from 2 different cultures were examined). Interestingly, CH5pCH32
harboring pACYCLacORF3 showed an intermediate phenotype. Some filaments were
observed, although none were as long as those of CH5pCH32pACYC184. Numerous
individual as well as short filaments of 2-5 cells were visible, indicating that septation did
occur, but not as efficiently as in the wild-type. This suggests that while SLO7ORF 3 can
complement the growth defect caused by ZipA-depletion in E. coli, it only partially
alleviates the defect in cell division. This may explain our inability to cure these strains of
pCH32 (data not shown).

The growth of E. coli strain DHSct containing pSLO7ORF3 (ORF3 expressed
from its own promoter) had a slightly reduced growth rate, which could be explained by a
defect in cell divison. Examination of these cultures by light microscopy showed
numerous filamentous cells, suggesting that overexpression of N. gonorrhoeae
SLO7ORF3 blocks cell division (data not shown) consistent with the phenomenon
observed upon overexpression of zipA in E. coli (140). However, when SLO7ORF 3 was
overexpressed from an IPTG-inducible promoter (pACYCLacORF3) and pCH32
(zip/17132" ) was also present, this effect on growth was alleviated, presumably due to the

excess FtsZ produced fiom this plasmid.

58

Figure 2-5. N. gonorrhoeae zipA partially alleviates filamentation phenotype of E.
coli depleted for ZipA. E. coli strains harboring various zipA plasmids were cultured
overnight at 30°C, then diluted 1:500 and shified to 37°C and incubated for 2 hr (A-C) or
6 hr (D). Cultures were harvested and concentration adjusted to ~2 x 109 CFU/ml. 10 pl
was Spotted onto a microscope slide and gram-stained.

A: CH3(zipA*)pCH32(repA“zipA+fisz*) pACYC-ORF3;

B: CH3(zipA+)pCH32(repAtszipA+ftsZ+)pACYC184;

C: CH5(zipA::aph)pCH32(repA”zip/1732i)pACYC-ORF3;

D: CH5(zipA::aph)pCH32(repA“zsz+flsT)pACYC l 84.

Photomicrographs (courtesy of Matti Kiupel) were taken using an Olympus Carnedia E-
10 digital camera attached to an Olympus BX40 microscope. Images were viewed under

oil immersion at a magnification of 1000X.

59

 

 

 

 

 

 

 

 

 

 

 

60

DISCUSSION

The prokaryotic SRP is a ubiquitous protein targeting system that targets a subset
of proteins to the cytoplasmic membrane co-translationally (73, 230, 279, 358, 407). All
of the components of the SRP appear to be essential in bacteria (49, 113, 161, 273, 294,
434) suggesting that at least one of the proteins dependent on this system for localization
is involved in an essential cell process. In this work, we set out to identify proteins from
N. gonorrhoeae that utilize the SRP in an effort to identify essential CM proteins from
this organism. Using a heterologous screening approach in E. coli, we identified several
genes encoding proteins that appeared to utilize the SRP. Of these, one (SLO7ORF 3) was
determined to be a functional homolog of ZipA, an essential cell division protein in E.
coli.

In E. coli, there are at least ten components (FtsA, -I, -K, -L, -N, -Q, -W, -Z,
ngQ, and ZipA) involved in the assembly of the septal ring, a membrane-associated
cytoskeletal element that directs the formation of the division septum (50), reviewed in
(325). In the initial stages of cell division FtsZ self-associates to accumulate at the
prospective division site on the inner side of the CM, forming a structure called the Z ring
(31), which then acts as a scaffold to which the other cell division proteins are recruited.
ZipA is an essential protein that interacts with F tsZ (140), which while not required for Z
ring formation, is required for recruitment of additional proteins to the Z ring (20, 29).
Although ZipA is essential in E. coli, it appears to be less conserved in Gram-negative
bacteria, and is the least conserved cell division proteins (325). Putative homologs of
ZipA have been identified in several gram-negative bacteria (140, 325) mostly based on

sequence homologies between the N-terminal and C-terminal domains of the protein,

61

both of which are reported to be important for ZipA function (22, 29). Predicted proteins
with significant homology to ZipA have not been identified in genome sequences of
grarn-positive bacteria, archaea, and some gram-negative bacteria, leading to the
conclusion that it has either divergently evolved or other proteins serve its function in the
cell (325).

While the similarity of N. gonorrhoeae SLO7ORF3 to E. coli ZipA is low at the
amino acid sequence level, there are significant similarities in key domains (Figure 2-3).
These include: an N-terrninal hydrophobic region (with no signal peptidase cleavage site)
which is followed by a basic region (net positive charge of 5-8) which likely functions to
anchor the protein in the CM; and central region rich in proline and glutamine residues
(140). A ClustalW alignment of this fragment with the N. gonorrhoeae ZipA shows 15%
identity and 49% similarity at the amino acid level in this region (Figure 2-3). Taken
together, these observations and our data indicate that SLO7ORF 3 does indeed encode
the gonococcal cell division protein, ZipA. To the best of our knowledge, this is the first
ZipA homolog identified in a non-rod-shaped bacterium. Thus, it will be interesting to
more closely examine the role of ZipA in cell division in N. gonorrhoeae.

Of the ten proteins shown to be involved in formation of the division septum in E.
coli, genes encoding seven of these have been identified in N. gonorrhoeae, ftsZ, ftsA,
ftsQ, and ftsI (101, 332) and ftsK, ftsW and yng (http://www.stdgen.lanl.gov). In
addition to these, the min genes, which encode three proteins, MinC, MinD, and MinE,
which are involved in positioning of the division septum have also been identified and
characterized in N. gonorrhoeae (380). Studies of the function of these proteins in N.

gonorrhoeae by Jo-Anne Dillon and coworkers indicates that they play similar roles in

62

cell division as in E. coli (315, 385). Genes encoding FtsE and FtsX have also been
identified in N. gonorrhoeae (30), although it is not clear if they are involved in cell
division in Neisseria. FtsL and FtsN are the only key cell division proteins remaining to
be identified in Neisseria.

Numerous proteins have now been identified that utilize the SRP for targeting in
bacteria (73, 230, 279, 358, 407, 408). These include an efflux pump (Ach), several
transport systems (LctP, LacY, thP, MalF, MtlA, ProW), and some membrane-
associated enzymes (PgsA, MdoH, CdsA), none of which are essential for cell viability.
In addition to these, however, are several proteins that may be involved in cell division.
These include FtsQ (394, 409), which is essential in formation of the division septum (7,
109, 227, 429), and FtsE and FtsX (407). While most SRP-dependent proteins are
polytopic membrane proteins, two SRP-dependent cell division proteins (F tsQ and FtsE)
are bitopic, having a single membrane-spanning domain, similar to ZipA.

There are several links between cell division and the SRP. Depletion of F III, the
signal sequence binding SRP component, or cells expressing a mutated jfh gene results in
defects in cell division in E. coli (294, 335). A point mutation in fife, which encodes the
4.58 SRP RNA, of Caulobacter crescentus confers a temperature-sensitive defect in cell
division (434). And finally, fts Y, which encodes the SRP docking protein, FtsY, in E. coli
was initially identified as part of an Operon (ftsYEX) containing genes which are
temperature-sensitive for filamentation (fts; (113), and FtsY-depleted cells are
filamentous, apparently defective in completion of septation during cell division (227).
All of these observations can be explained if one or more essential cell division proteins

is dependent on the SRP for targeting. Since all of the other cell divison proteins, with the

63

exception of FtsZ and FtsA, are reported to localize to the CM, it will be interesting to

determine whether any of these utilize the SRP.

64

Chapter 3

Expression capable library for studies of Neisseria gonorrhoeae, version 1.0

This manuscript has been previously published in BMC Microbiology:

Thomas Brettin, Michael R. Altherr, Ying Du, Roxie M. Mason, Alexandra Friedrich,
Laura Potter, Chris Langford, Thomas J. Keller, Jason Jens, Heather Howie, Nathan J.
Weyand, Susan Clary, Kimberly Prichard, Susi Wachocki, Erica Sodergren, Joseph P.
Dillard, George Weinstock, Magdalene So, and Cindy Grove Arvidson, 2005, Expression
capable library for studies of Neisseria gonorrhoeae, version 1.0, BMC microbol. 5:50

65

Authors’ contributions

Thomas Brettin: design of clone set construction, primer design

Michael R. Altherr: design of clone set construction, design and supervision of high-
throughput PCR amplification of ORFS and recombination cloning into entry vectors
Ying Du: construction and tests of DNA microarrays, amplification and cloning of
several ORFS not obtained in the initial batch cloning

Roxie M. Mason: cataloging of all cloning reactions processed at MSU (Arvidson lab),
screening of transformants and preparation of clones for sequencing

Alexandra Friedrich: recombination of selected clones into expression vectors and
analysis of proteins produced (Figure 3-1)

Laura Potter: initial project design, screening and cataloging of all cloning reactions
processed at OHSU (So lab), screening of transformants and preparation of clones for
sequencing

Chris Langford: batch BLAST analysis of sequencing results

Thomas J. Keller: batch BLAST analysis of sequencing results, wrote new program to
faciliate analysis of sequence data

Jason Jens: processing of sequence data, batch program design for analysis of subset of
clones from Gonococcal Genetic Island

Heather Howie: screening of transformants and preparation of clones for sequencing
Nathan J. Weyand: screening of transformants and preparation of clones for sequencing
Susan Clary: screening of transformants and preparation of clones for sequencing,
amplification and cloning of several ORFS not obtained in the initial batch cloning
Kimberly Prichard: primer design

Susi Wachocki: high-throughput PCR amplification of ORFS and cloning into entry
vectors

Erica Sodergren: sequencing of clone set

Joseph P. Dillard: contributed data of the Gonococcal Genetic Island prior to
publication, participated in project design

George Weinstock: initial project design

Magdalene 80: initial project design, supervison of all work performed at OHSU,
substantial writing of manuscript

Cindy Grove Arvidson: initial project design, supervison of all work performed at MSU,
screening of transformants from LANL, cloning of several ORFS not cloned in initial
high-throughput cloning, collated and organized all sequence data, primary writing of
manuscript, corresponding author.

66

ABSTRACT

The sexually transmitted disease, gonorrhea, is a serious health problem in
developed as well as in developing countries, for which treatment continues to be a
challenge. The recent completion of the genome sequence of the causative agent,
Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this
organism and the diseases it causes. Here, we describe the initial phases of the
construction of an expression-capable clone set representing the protein-coding ORFS of
the gonococcal genome using a recombination-based cloning system.

The clone set thus far includes 1672 of the 2250 predicted ORFS of the N.
gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set
are 48 of the 61 ORFS of the gonococcal genetic island of strain M811, not present in the
sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase
(GST)-fusions were constructed from random clones and each was shown to express a
fusion protein of the predicted size following induction, demonstrating the use of the
recombination cloning system. PCR amplicons of each ORF used in the cloning reactions
were spotted onto glass slides to produce DNA microarrays representing 2035 genes of
the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the
analysis of global gene expression in gonococci.

This archived set of Gateway® entry clones will facilitate high-throughput
genomic and proteomic studies of gonococcal genes using a variety of expression and
analysis systems. In addition, the DNA arrays produced will allow us to generate gene

expression profiles of gonococci grown in a wide variety of conditions. Together, the

67

resources produced in this work will facilitate experiments to dissect the molecular
mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the

determination of the functions of unknown genes in the genome.

68

INTRODUCTION

Neisseria gonorrhoeae (gonococcus), a Gram-negative diplococcus, is one of two
pathogenicmembers of the Neisseriaceae family of bacteria. N. gonorrhoeae is the
causative agent of the sexually transmitted disease, gonorrhea, one of the oldest
documented infectious diseases. Gonorrheal disease has significant morbidity both in the
U8 and worldwide. According to the Centers for Disease Control (6), >350,000 cases of
gonorrhea were reported in the United States in 2002. The World Health Organization
(www.who.org) estimates that over 19 million cases occur annually in the African
continent alone. Treatment of gonorrhea is increasingly problematic due to the high
frequency of acquisition of resistance to multiple antibiotics (100, 361) and to the
observation that gonococcal infection does not elicit protective immunity (342).
Gonorrheal infections, though not usually life-threatening, also enhance the transmission
of HIV (202).

N. gonorrhoeae is strictly a human pathogen, with no known animal reservoir.
The bacterium has no environmental niche, and cannot survive outside the human host. In
adults, N. gonorrhoeae is acquired primarily through sexual contact. However, the eyes
of newborn infants may be infected by passing through an infected birth canal, resulting
in the condition, ophthalmia neonatorum, which can lead to blindness. In most cases,
gonococcal infections are limited to the urogenital tract, causing urethritis in men and
cervicitis in women. Occasionally, gonococci cross the epithelial barrier to enter the
bloodstream causing septicemia, and transit to the joints resulting in arthritis. In women,

ascending infections from the endocervix can result in pelvic inflammatory disease,

69

salpingitis, tubal blockage and infertility. N. gonorrhoeae can also establish a carrier state
in which apparently healthy individuals harbor culturable and infectious bacteria (98).
Carriers are thought be important for disease dissemination. A recent study revealed that
the gonococcal caniage rate in women was 6.7% in a major metropolitan area (404).

Due to the importance of N gonorrhoeae to human health, much research effort
has focussed on identifying virulence factors and elucidating the biochemical interactions
of these factors with the host cell (146, 249, 310), with the goal of developing vaccines
and alternative treatments. It is clear, however, that in order to fully understand the
capabilities of this organism to cause disease and elude eradication, it will be necessary to
ultimately determine the functions of a great deal more of the gene products encoded by
the gonococcal genome. The recent genome sequencing makes possible a variety of
genomic and proteomic studies of N. gonorrhoeae. To facilitate such studies, we have
cloned into a bacteriophage lambda-based recombination cloning system (Gateway®
(420), Invitrogen, Carlsbad, CA) 1624 of the 2189 predicted ORFs from the genome of
N. gonorrhoeae strain FA1090 (43), and 48 of the 61 ORFS of the gonococcal genetic
island (GGI) of strain M811 (78, 142). This clone-set allows the generation of
transcriptional and translational fusions without the necessity of additional cloning and
sequencing. Coupled to the construction of this clone set, DNA microarrays were
generated by spotting the insert DNA onto glass slides. Preliminary experiments with the
clone set and DNA arrays indicate that this system is suitable for studies of expression of
genes from N. gonorrhoeae in heterologous systems as well as for the study of global

gene expression in this organism.

70

METHODS

Gene predictions and initial sequence preparation. All ORF IDs for strain FA1090
reference records can be found at the STDGEN Neisseria gonorrhoeae annotated genome
sequence database (43). All gene sequences were prepared to include the natural start and
stop codons. NG0540 and N00634 have internal stop codons in the sequence database,
and nothing was done to correct for this. Sequences of the ORFS of the M811 GGI (142)

have been deposited in the GenBank database with the accession number AY803022.

Signal peptide identification. In order to determine whether the ORFS contained signal
sequences, the programs PSORT (272) and SignalP (272) were employed. If both
programs predicted a cleavable signal peptide for a given ORF sequence, that constituted
“high support”. The result of the signal peptide analyses showed 149 sequences with high
support for a signal peptide. For those gene nucleotide sequences with a high support
signal peptide, the nucleotide sequence representing the signal peptide was removed prior
to cloning primer design. When both programs predicted different cleavage sites, the
cleavage site that represented the shorter signal peptide was chosen. The rationale for this
choice was that it would be better to include a bit of the signal peptide in the PCR product

than to exclude a bit of the mature protein in the PCR product.

Primer Design. In the first cycle, the forward and reverse primers were fixed at the same
length. This was due to the case at which Primer3 (329) could be used. The input file was
all gene sequences for which no signal peptide sequence was detected (see above). For

the second cycle, the prim.aux file was manually inspected. This file contained

71

information about successful primer picks where only a left or right primer could be
picked. The strategy was to combine primers of different length. The potential for primer-

dimer formation using this strategy was also assessed.

N.g. ORF cloning. Primers for each of the genes were purchased from Illumina, Inc (San
Diego, CA) and were designed to add sequences corresponding to part of the attB site
necessary for recombination into the Gateway® entry vector, pDONR221 (Invitrogen).
Primary amplification was done using genomic DNA at a concentration of ~10
ng/reaction and gene specific primers at 2.5 pM. Reaction mix contained dNTPs, reaction
buffer, Mng and Taq polymerase as recommended by the manufacturer (Roche).
Reactions conditions for primary PCR were as follows: denaturation at 94°C for 10 min;
10 cycles of 94°C 30 sec, 50°C 1 min, 74°C 1-5 min (depending on length of predicted
product); 20 cycles of 94°C 30 sec, 55°C 1 min, 74°C 1-5 min (depending on length of
predicted product); and a final extension at 74°C for 10 min. Following the primary
amplification with the primer set, products were diluted 1:100 and 1 pl used as template
for a secondary amplification using a pair of primers corresponding to the partial attB site
common to all of the amplicons, and including additional sequences to generate a
complete attB site for the cloning reaction. Reactions conditions for secondary PCR were
as follows: denaturation at 94°C for 1 min; 5 cycles of 94°C 15 sec, 45°C 30 sec, 68°C 2
min; 15 cycles of 94°C 15 sec, 55°C 30 sec, 68°C 2 min. 5 pl of the 50 pl PCR was
removed and used for cloning, and the remainder used for agarose gel analysis and
printing of DNA arrays (N.g. array version 1.0, see below). Cloning reactions were

performed according to the manufacturer’s instructions (Invitrogen), transformed into E.

72

coli strain DH50t, and transformants selected for kanamycin resistance. Individual
transformants were picked into wells of 96-well plates containing 100 pL broth
containing kanamycin (50 mg/l) and the same toothpick then used to place a small
amount of bacteria directly into another plate containing a PCR cocktail. PCR was done
using the M13 universal primers, which flank the att sites of the entry vector,
pDONR221. A product of 350 bp was observed if no insert was present, providing an
internal control for the PCR reactions. Individual clones were identified, stocked in

duplicate, and grown for DNA isolation for sequencing.

Sequencing. Sequencing runs from each end of the insert of each of the clones was
determined to verify the ORF inserted. Complete sequence verification, (ie. both strands
completely across the insert) was not done as it was determined to be impractical. DNA
sequencing reactions were performed at the Baylor College of Medicine HGSC.
Additional File 1 is a zipped file containing each of the sequence reads as .exp files
generated using PREGAP4, and can be opened and read using word processing software.
The file is separated into folders labelled Sequate #, which refers to sequencing plate
number (1-21), and corresponds to the SP# designation in the list of clones in Additional
File 2. 8Pl8 sequencing reactions were done twice (Sequate 18-1, Sequate 18-2) as a
sequencing reaction control, and the reactions of SP5 were analyzed three times
(Sequate 5-1, Sequate 5-2, Sequate 5-3) as controls. SP20 does not exist, SP22 was not
sent for sequencing, and the quality of SP17 sequence was too poor to be readable. The
naming of the individual read files is as follows: BGACA(project code) # (1, 2, or 3 =

reaction) D or F (primer D = forward, F = reverse) # (box, m1 same as SP) # (01-96;

73

well, 1 = A1, 2 = B1, 9 = A2, and so on) A or B (run). For example: BGACA3D1701A
(found in Sequate 1 file) corresponds to SP1 well Al sequenced with the forward primer

(5’ end of ORF), result from the third reaction and the first gel.

Gene expression analysis. pDONR221 derivatives chosen from the clone set were
recombined with the destination vector, pDEST15 (Invitrogen), an N-terminal GST
fusion vector. Plasmid DNA from the pDONR221 derivatives was isolated and incubated
with pDEST15 in a LR recombination reaction performed according to the
manufacturer’s instructions. The recombination mixes were transformed into E. coli
DH50t and transformants were selected on LB plates containing 50 mg/l Carbenicillin
(Cbso). To test the possibility of false positive clones, the overexpression clones were
tested for growth on chloramphenicol (20 mg/l), on which expression recombinants
should not grow since the chloramphenicol resistance gene of pDEST15 is replaced by
the insert. The resulting plasmids were purified and transformed into E. coli BL2l-AI
(Invitrogen), which expresses T7 RNA polymerase from the araBAD promotor, and
transformants were selected on LB CbsO plates. BL21-AI strains containing the pDEST15
derivatives were grown overnight and used to inoculate fresh LB medium containing
Cb50 to an OD600 of 0.05. Expression of the GST-fusion proteins in E. coli BL2l-AI was
induced at an OD600 of 0.5 by addition of L-arabinose to a final concentration of 0.2%.
Aliquots were removed after 2 hr and total proteins electropheresed on 10%

polyacrylamide SDS gels (201).

74

DNA microarray construction. PCR amplicons remaining from the cloning reactions
(described above) were concentrated and then spotted in duplicate onto TeleChem
SuperAmine glass slides (TeleChem International, Inc., Sunnyvale, CA) using a
GeneMachines Omnigrid 100 (GeneMachines, Inc., San Carlos, CA) with 16 TeleChem
Chipmaker 3 pins at the Genome Technology Support Facility (GTSF) at Michigan State
University. Preparation of probes and hybridization conditions were as described (84).
Hybridized microarray slides were scanned using a GenePix 4000B scanner (Axon
Instruments, Union City, CA) and images were processed and analyzed using GenePix

version 4.1 software.

Availability of clone set. The master library was used to generate a limited set of stock
plates, which are stored at various locations as reference stocks. An additional set is
stored at Michigan State University and is regarded as the working copy, which is the
sole source of material for distribution. Interested parties should contact Cindy Arvidson
(corresponding author) to arrange for a copy of the clone set. The clones will only be

available as an intact set, individual clones or groups of clones are not available.

Availability of microarrays. DNA arrays will be generated by re-amplification of the
amplicons used for the recombination-cloning reactions using primers recognizing the
common sequences at the ends of the amplicons at the MSU GTSF. Interested parties
should contact Cindy Arvidson (corresponding author) to request slides, which will be

produced upon request.

75

RESULTS

Design of Oligonucleotide primers. The goal of this project was to create a plasmid
library representing the annotated ORFS of N. gonorrhoeae. The Gateway® Cloning
System from Invitrogen (420) was selected for several reasons. First, Gateway® uses a
recombination-based cloning method which has the added benefit that once an archival
clone is sequence-validated, subsequent recombinants (ie. into expression vectors) do not
need to be sequenced. Second, the initial clones lack transcriptional machinery such that
the cloned ORFS are not expressed, thus avoiding problems from lethality due to
troublesome gene products. Third, there are several expression and epitope-tagging
vector options for the subsequent study of proteins encoded by the cloned ORFS,
allowing a variety of approaches to study their functions. The high efficiency also lends
itself to high throughput approaches that are suitable for automation.

A total of 2071 unique primer pairs were successfully designed for the 2189
annotated ORFS of the FA1090 genome (43) and 61 ORFS of the G01 (78, 142). These
primers were gene specific, and their termini contain sequences for recombination
cloning into the entry vector, pDONR221 (Invitrogen). All primers were designed such
that the final recombination product yielded the native start codon at the 5’ end of the
gene, including the 206 of the 229 predicted ORFS with alternative (non-ATG) start
codons. Since nearly half of the genes of this group (110/229) are annotated (that is
encode putative proteins with significant similarity to proteins of known function), it is
very possible that they are functional genes in the gonococcus and were thus included in
the clone set design. Whether or not they encode functional proteins will ultimately

depend on the results of future expression and mutation studies. The 23 ORFS of this

76

subset not included were less than 400 bp in length and considered too small for the
Gateway® system (see below). For the 149 ORFs encoding predicted proteins with an
amino-terminal signal sequence (as identified by PSORT (272) and SignalP (281)),
sequences encoding the signal sequence were removed and an ATG start codon placed at
the 5’ end of the remaining coding sequence. This was done to reduce problems of
expression of hydrophobic signal sequences and to facilitate future expression and
targeting studies for such recombinant proteins.

The primer design strategy was iterative, starting with an annealing temperature
range of 62°-72°C and primer length set to 18 nt. All ORFS were included in the first
iteration, and those ORFS for which a primer pair was not selected were subjected to
subsequent iterations. In each subsequent iteration the primer length parameter was
increased by one, up to a maximum primer length of 34 nt. Primers larger than this were
not designed in part due to cost and convenience of oligo synthesis in our 96-well format.
Next, the annealing temperature range was expanded 5°C in both the positive and
negative directions and each primer size from 18 to 34 nt was tried again. No further
iterations were attempted after the annealing temperature range exceeded 47°-87°C.
Failing to meet either of these criteria resulted in a primer pair not being designed for the
ORF. There were a total of 177 ORFS for which no primers were designed (see
Additional file 2), 174 from FA1090 and 3 from the GGI. Most of these ORFS (165/177
= 93%) were less than 500 bp in length, and were not included since the Gateway®
system is reportedly less efficient for cloning fragments of this small size.

To each of the gene specific primer sequences for the 5’ ends of the ORFS was

added a 21 nt sequence including a consensus ribosome binding site (Shine-Dalgamo

77

sequence). To each of the gene specific primer sequences for the 3’ ends of the ORFS was
added 20 nt corresponding to the 3’ end of the attBZ site necessary for recombination into
pDONR221. These were the primary PCR primers, the sequences of which are available
on request. A single pair of primers was then designed for a secondary amplification to
generate gene specific products containing the attB sites for the recombinatory cloning
step. The 5’ secondary primer contained the 24 nt attB] tail and the 21 nt sequence
(Shine-Dalgarno) common to all of the primary 5’ primer sequences. The 3’ secondary
primer contained the remaining 10 nt of the attBZ site and the 20 nt attBZ sequence
common to all of the primary 3’ primer sequences. The attB sequences were as

recommended in the Gateway® manual.

PCR amplification of ORFS. The first round of PCR, using gene specific primers,
included a total of 2071 different primer pairs. Plates 1 and 2 were organized as pilot
reactions and contained primers designed to amplify products ranging from 143 bp to
3485 bp in length. Genomic DNA from N. gonorrhoeae strain FA1090 was used as a
template for the reactions. Agarose gel analysis of the amplicons showed that 173 of the
192 reactions yielded a product of the expected size, an efficiency of 90%. Plates 3-21
were then arranged with increasing size of expected product, with plate 3 containing the
smallest products and plate 21 the largest. Several primer pairs (from plates 1 and 2) were
included in plates 3-21 as internal controls. Plate 22 was an additional control plate, with
one half of the plate (rows A-D) duplicated on the other half (rows E-H). Plate 23
corresponded to the genes of the GGI present in strain M811 (78, 142), and M811

genomic DNA was used as the template for this plate of reactions. 5 pl of each reaction

78

from the first round of PCR of each plate were run on agarose gels and scored for
production of a product and whether it was of the expected size. The results showed 89%
of the reactions to produce a product of the correct size.

Following the primary amplification with gene specific primers, all products (in
the original 96-well format) were diluted 1:100 and an aliquot subjected to a secondary
amplification using a pair of primers corresponding to the sequences common to all of the
amplicons, and including additional sequences to generate a complete attB site for the
cloning reaction. Aliquots of the secondary amplification were then used directly in the

recombination reaction to generate entry clones.

Construction of the library. Secondary amplicons were inserted into pDONR221 by in
vitro recombination between the attB sites introduced at the ends of the amplicons and
the attP sequences of the vector, maintaining the 96-well format arrangement. Cloning
reactions were then transformed into E. coli strain DH50t and a portion of the
transformation mix plated on LB plates containing kanamycin. Individual transformants
were screened by PCR to determine the presence of and size of the insert. For the first
round of screening, four independent transformants from each reaction were screened,
maintaining the original 96-well format. A product of 350 bp was observed if no insert
was present, a positive clone was identified as having a product 350 bp larger than the
size of the corresponding primary PCR product.

For the initial set of 2147 transformation reactions, clones corresponding to 1165
genes were identified in the first four transformants screened, an efficiency of 54%. This

efficiency varied greatly with the predicted size of insert, the smallest inserts (plate 3)

79

were 83% positive in the first four screened and the next largest inserts (plate 20) had
17% positive in the first four screened. Plate 21, which had the largest inserts, only
yielded 3 transformants as positive after several rounds of screening. Additional
transformants for those clones not identified in the first round of screening were
individually cultured, screened by PCR, and positive clones frozen down as they were
identified. This approach yielded an additional 354 clones.

Following the initial rounds of screening, a list of missing clones was generated
and the amplification and cloning steps repeated, optimizing several parameters and
analyzing on an individual basis. This approach yielded an additional 283 clones, for a
total of 1802 which were subsequently sequenced. Arrangement of the clones in plates
for the master set and for sequencing was on an “as identified” basis, such that they are
not arranged as in the original 96-well format. Each of the 21 plates contain viable clones
in up to 95 of the 96 available positions, with position H12 (and additional wells on some

plates) left empty for controls, providing a unique identity for several of the plates.

Sequence verification of the clones. Transforrnants identified as having an insert of the
predicted size were grown in 96-well plates and DNA isolated for sequence analysis.
DNA isolation and sequencing was done by at the Human Genome Sequencing Center at
Baylor College of Medicine (HGSC) using the same set of primers used to screen
transformants for insert size. Sequence reads were posted onto an HGSC website and
subsequently downloaded by FTP. DNA reads were processed initially using the
STADEN DNA analysis software package (3). Binary files were converted into .exp files

using PREGAP4 to generate text files for each individual sequence read, with sequence

80

quality cutoffs. These data are provided in Additional File 1 (AF 1 exp sequence files.zip),
and a description of the labelling scheme for the files is in the Methods section.

The data were next analyzed by BLAST (11) against the N. gonorrhoeae genome
sequence database (43). Clones expected to contain inserts from the G01, not present in
FA1090, were analyzed by BLAST 2 (390) using sequence of the individual GGI ORFS
((142), GenBank accession number AY803022). BLAST results were then manually
tabulated in a file containing the expected gene for each archived clone. Of the 1802
sequenced clones, 58 were expected to be duplicates, leaving 1744 unique clones
expected in the clone set. 1550 of the sequences were readable and corresponded to a
predicted ORF from N. gonorrhoeae, 1399 of them unique, corresponding to 151
duplicates. Some duplicates were expected, and the remainder likely due to cross
contamination from neighboring wells. Of those sequence validated, 55 were not in
positions predicted. Most of these were due to human error, such as obvious well
transpositions and numbering transpositions. 26 of these, however, had inserts in a
backwards orientation and were incomplete. These clones will not be usable in
subsequent recombination reactions using the Gateway® system. Together, these data
indicate that at present we have a collection of 1672 individual clones from N.
gonorrhoeae, 48 of which are from the GGI, and 83% of which have been sequence
validated. A list of genes in the clone set with sequencing result information can be found
in Additional File 2 (AF 2 NG clone set seq status.xls).

Overexpression of randomly chosen ORFS. In order to examine the flexibility of using
the clone set to construct various derivatives for which the Gateway® system was

designed, three randomly selected pDONR221 derivatives were used to create inducible

81

glutathione-S-transferase (GST) fusions. Plasmids containing ORFS N01490 (aspS,
encodes aspartyl-tRNA synthetase), N01561 (xthA, encodes exodeoxyribonuclease III),
and N01641 (pivN0, encodes a pilin gene inverting homolog, PivNG) are predicted to
encode native proteins of 9.6, 29.0, and 36.2 kDa respectively. Plasmid DNA of
pDONR221 derivatives containing these three genes were recombined with the
destination vector pDEST15, an N-terrninal GST fusion vector. The resulting
recombinants were then transformed into E. coli BL21-AI for expression analysis. SDS-
PAGE analysis of the proteins after a 2 hr induction with 0.2% L-arabinose is shown in
Figure 3-1. The results show high levels of induction for each of the fusion proteins, with

the sizes as predicted (GST adds 29 kDa).

DNA microarray production. Since a very small portion of the secondary amplicons
were used for the cloning reactions (5 pl of a 50 pl reaction), the remaining products
were used to produce a set of DNA arrays for gene expression analysis. Gel
electrophoresis (data not shown) indicated that the efficiency after the secondary
amplification was 81%, representing 1681 ORFS of a total of 2071. To generate a more
complete DNA array, a second set of primers (360) were designed to amplify internal
portions of those ORFS not visible following the secondary PCR. Primers to amplify
sequences of two small RNAs were also designed: N00892.1, fls, encodes the 4.58 RNA
component of the gonococcal signal recognition particle (102); and N00880.1, tmRNA,
encodes an RNA that tags abnormal proteins in the cell arising from stalled ribosomes
and targets them for proteolysis (133). Gel analysis of the amplicons produced from the

internal PCR primers showed 294 of the 362 to produce products of the expected size, for

82

an efficiency of 82%. Together, the cloning amplicons (1681) and internal ORF and RNA
amplicons (294) represent a minimum of 1975 ORFS of the N. gonorrhoeae genome.
This is a minimal estimate since all products (regardles of gel result) were to be spotted,
and some products might be present, but at amounts too low to be visualized. DNA
samples were processed and spotted onto glass slides as described in Methods. As a first
test, the arrays were hybridized with a Cy3-labelled random nonamer Oligonucleotide
(Qiagen). A scan of the slide at 532 nm showed spots at the appropriate positions where
DNA had been spotted, and blank spots at the buffer control spots. The next test was to
hybridize the arrays with labelled genomic DNA. Total DNA from N. gonorrhoeae strain
M811 was digested with RsaI and labelled by including Cy3-dCTP in a random-primed
Klenow DNA polymerase reaction (Roche Applied Science, Indianapolis, IN). DNA
arrays were hybridized and then scanned at 532 nm. Valid hybridization signals (at least
one standard deviation above background) were detected for 98% of the spots expected to
contain DNA, with those of the M811-specific island comparable in intensity to those of
FA1090 amplified DNA. Overall, the results showed valid signals for 2035 individual
genes, with several in duplicate. The fact that this number is higher than expected based
on agarose gel analysis of the amplicons before spotting (see above) indicates that some
of the reactions produced a product, but at amounts too low to be visualized by ethidiurn
bromide (EtBr) staining. Thus, these arrays represent 90% (2035/2250) of the predicted
ORFS of N. gonorrhoeae (including 58 ORFS of the GGI; (78), (142)) and 98% of those

for which primers were designed.

83

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Figure 3-1. SDS-PAGE analysis of NG ORF-GST fusions. Equivalent amounts of total
protern was boiled in sample buffer and electrophoresed on 10% polyacrylamide gels
Gels were stained with Coomassie Blue. Lane 1: MW markers; lane 2: NG1490(AspS)

GST, uninduced; lane 3: NG1490 induced (38.6 kDa); lane 4: NG1561(XthA)-GST
uninduced;

lane 5: NG1561 induced (58.0 kDa); lane 6: NG1641(PivNG)-GST

uninduced; lane 7: NG1641 induced (65.2 kDa).

84

DISCUSSION

Despite the advent of antibiotics in the 1940's, disease due to infection with IV.
gonorrhoeae remains a major health problem worldwide. The reasons for this are multi-
fold. First, resistance to antibiotics by N. gonorrhoeae continues to rise (6, 100, 361). In
addition, treatment with high levels of broad spectrum antibiotics (which is fiequently
done since patients often do not return for follow up treatment) kills many bacteria of the
(ofien beneficial) normal flora as well as the disease-causing microbe. Second, there is an
incredibly high frequency of asymptomatic gonococcal infection, occurring in 5-10% of
infected men and up to 50% of infected women. This represents a major reservoir for
transmission of the infection. Furthermore, undiagnosed and untreated gonococcal
salpingitis can lead to fallopian tube blockage. Partial blockage can result in ectopic
pregnancy, which can be life threatening, and complete blockage of the fallopian tubes
ofien leads to infertility. Third, development of a vaccine to protect against gonorrhea has
been seriously hampered by the observation that gonococcal infection does not elicit
protective immunity (342). Patients can be reinfected following treatment, and can even
be infected by multiple strains at a given time. Thus, alternative treatments and
preventative strategies for gonococcal infection are sorely needed.

As a first step in the identification of such alternative treatments and
preventatives, it will be necessary to more thoroughly understand the biology of the
gonococcus and the molecular mechanisms involved in its interactions with the host
environment. Much of the studies to date have focused primarily on identifying the
molecules on the surface of the bacterium that directly interact with the host, and the

toxic moieties involved in damage to host cells. Many of the molecules identified are

85

outer membrane (OM) components (97, 311, 331, 380, 411), and iron utilization proteins
(reviewed in (344)). A few are secreted (364), or shed in blebs (126, 128, 245). There has
also been significant work in the identification of eukaryotic host cell receptors for
gonococcal surface proteins (reviewed in (250)).

Interactions between gonococci and epithelial cells are beginning to be unraveled.
Chen and Clark showed that contact with Hec-l-B human endocervical epithelial cells
increases gonococcal infectiveness and that the process involves, in part, de novo protein
synthesis by the bacterium (249). The gonococcal type IV pilus (Tip) and Opa proteins
promote attachment, invasion and trans-epithelial trafficking. The mechanisms
underlying Tfp- and Opa-mediated virulence are not yet understood, but these surface
structures modulate a series of events in the infected epithelial cell, among them Ca2+
fluxes (20, 180), cortical rearrangements (246), and receptor phosphorylation (62, 124,
209). Tfp retraction enhances the activation of stress-responsive “kinases and the
transcription of cytoprotective genes in the infected cell (165), and triggers the infected
cell to produce a molecule that alters bacterial motility behavior (210). Finally, binding of
gonococci to primary urethral cells up-regulates anti-apoptotic factors (33). These and
other observations indicate that gonococcal infection requires the active participation of
both the bacterium and the host cell.

The recent completion of the annotated genome sequence of N. gonorrhoeae (43,
321), coupled with the development of high throughput methods for the analysis of gene
expression and function, provide an opportunity to significantly advance the study of
gonococcal biology and pathogenesis. Like many sequenced genomes, nearly half (44%)

of the genes of the annotated gonococcal genome encode hypothetical proteins of

86

unknown function. Furthermore, many of the annotations are based on homologies at the
nucleotide and/or amino acid level, and the actual function of the gonococcal proteins
have not been demonstrated. In order to realize the full potential of information gleaned
from the genome sequence of this (and any) organism, it will be necessary to assign
functions to all of the genes of the genome. The newly emerging fields of functional
genomics and proteomics offer much promise towards achieving the goal of eventual
assignment of fiinctions for each and every gene in a given organism.

In this work, we describe the initial phases of the construction of an expression-
capable clone set representing the annotated ORFS of the gonococcal genome using a
recombination-based cloning system. The advantages of the system used for this set are
numerous. 1) The original sequences in the clone set contain only the ORFS, not the gene
expression sequences, thus avoiding the issue of expression-related lethality of the
recombinants. 2) The clones can be transferred to a number of expression systems
(prokaryotic and eukaryotic), allowing the regulation of genes for overproduction of
proteins, or the production of proteins out of the context of the particular environments so
as to study their functions. 3) The clones can also be transferred to vectors that result in
epitope fusions, such as hexa—histidine, GST, green fluorescent protein (GFP), Lumio'm,
etc., to the proteins of interest. Protein fusions are usefi11 in localizing proteins (within the
bacterium or infected cell), in determining protein-interacting partners, in allowing
smaller step purification protocols for structural and activity studies, and for antibody
production. This ability has been demonstrated by constructing IPTG-inducible GST-
fusions from three random clones from this set (Figure 3-1). 4) The clone set is also

catalogued in such a way that individual clones of interest are easily identified and

87

recovered from the clone bank (see Additional file 2). 5) Entry clones can also be used to
create knockouts by in vitro transposition (83, 84) or shuttle mutagenesis (355) followed
by transformation into naturally competent gonococci (115). This system is also
ammenable to automation, thus increasing the potential output and consistency in the data
obtained.

The N. gonorrhoeae clone set thus far includes 1672 of the 2250 predicted ORFS
of the genome (43), of which 83% are sequence-validated. Included in this set are 48 of
the 61 ORFS of the MS11 GGI (78, 142). While this clone set is not yet complete, we
believe these initial efforts have resulted in generating a valuable resource for the
Neisseria research community. It is hoped that others in the community will share
compatible reagents and add to the clone set, making it more comprehensive over time.

Coupled to the clone set construction, a PCR-amplicon based DNA microarray
was generated. DNA microarrays are a powerful tool that allow one to measure relative
transcript levels for essentially each gene of the genome simultaneously. These DNA
arrays represent 2035 ORFS of the N. gonorrhoeae genome: 1977 from strain FA1090
(77) and 58 from the MS11 GGI (78), comprising 90% of the genes of the genome.
Preliminary studies show that these arrays are suitable for examining global gene
expression in N. gonorrhoeae.

Many bacterial pathogens are known to respond to changes in their physical
environment, often integrating responses to several environmental signals via complex
regulatory networks to control expression of a variety of genes (65, 79, 225, 293, 366).
The examples of regulatory systems characterized in gonococci are few, with the best

characterized being the response to iron availability (348, 431) and antimicrobial

88

compounds (139, 359). The advent of microarray technologies has opened avenues of
research on global gene expression in both prokaryotes and eukaryotes, providing
opportunities for studying a variety of organisms, including such genetically intractable
microbes as T repanema pallidum (242) and Chlamydia trachomatis (280). Thus, the use
of DNA arrays will allow us to more fully explore the response of N. gonorrhoeae to
environmental signals at the gene expression level. Since genes are typically only
transcribed when the gene product function is required, expression profiles and cluster
analyses will allow us to begin to determine the functions of unknown genes in the
genome. Thus far, use of these DNA arrays has led to the identification of a regulator
involved in the modulation of gonococcal gene expression upon adherence to epithelial
cells in (84).

In summary, the tools described in this work represent a resource which will
facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis
on a global scale. Combining these tools with the gonococcal infection models [tissue
culture (360), organ culture (239-241), and the mouse model (170)], will allow us to
make significant advances in the study of this important pathogen, thus providing us with
the knowledge necessary to design therapeutics with which to treat and prevent

gonococcal disease.

89

Chapter 4

Global gene expression and the role of sigma factors in Neisseria gonorrhoeae in

interactions with epithelial cells

This manuscript has been previously published in Infection and Immunity.
Du,Y. Lenz, J. and CG. Arvidson. 2005 Global Gene Expression and the Role of Sigma

Factors in Neisseria gonorrhoeae in Interactions with Epithelial Cells. Infect. Immun.
73:4834-4845

Arvidson, CG performed the Southern blot experiment on MSll genomic DNA.
Lenz, J, performed the heat shock experiment.

90

ABSTRACT

Like many bacterial pathogens, Neisseria gonorrhoeae must adapt to
environmental changes in order to successfully colonize and proliferate in a new host.
Modulation of gene expression in response to environmental signals is an efficient
mechanism used by bacteria to achieve this goal. Using DNA microarrays and a tissue
culture model for gonococcal infection, we examined global changes in gene expression
in N. gonorrhoeae in response to adherence to host cells. Among those genes induced
upon adherence to human epithelial cells in culture was rpoH, which encodes a homolog
of the heat shock sigma factor, 032 (RpoH), as well as genes of the RpoH regulon, groEL
and groES. Attempts to construct an moH null mutant in N. gonorrhoeae were
unsuccessful, suggesting that RpoH is essential for viability of N. gonorrhoeae. The
extra-cytoplasmic sigma factor, RpoE (GE), while known to regulate rpoH in other
bacteria, was found not to be necessary for the up-regulation of rpoH in gonococci upon
adherence to host cells. To examine the role of RpoH in host cell interactions, a N.
gonorrhoeae strain conditionally expressing rpoH was constructed. The results of our
experiments showed that while induction of rpoH expression is not necessary for
adherence of gonococci to epithelial cells, it is important for the subsequent invasion
step, as gonococci depleted for moH invade cells 23 fold less efficiently than a wild-
type strain. Taken together, these results indicate that 032, but not 65, is important for the

response of gonococci in the initial steps of an infection.

91

INTRODUCTION

Neisseria gonorrhoeae (gonococcus, GC), an obligate human pathogen, is the
causative agent of the sexually transmitted disease (STD) gonorrhea. Gonococcal disease
is prevalent world-wide, with ~500,000 reported cases each year in the US alone. It is
estimated that at least as many additional cases go unreported. While aggressive safe sex
practice campaigns significantly reduced the incidence of several STDs in the 1980’s and
1990’s, the last five years have shown a gradual increase in gonorrhea as well as another
bacterial STD, syphilis (Centers for Disease Control and Prevention,
http://www.cdc.gov/std/stats/toc2002.htm). There are several challenges in the treatment
of gonococcal disease, including continuing acquisition of antibiotic resistance (2, 100,
361), a high incidence of asymptomatic infection (especially in women), and the
observation that gonococcal infection does not elicit protective immunity (343). Patients
can be reinfected following treatment, and can even be infected by multiple strains at a
given time. Thus, understanding the biology of this organism and how it senses and
responds to its environment will be key in the development of alternative treatments and
preventative strategies for gonococcal infection.

Gonorrhea is typically a disease of the urogenital tract, although the bacterium
can also infect tissues of the pharynx, rectum, and the conjunctiva (which can lead to
blindness in newborns who are infected during birth). The initial step of a gonococcal
infection is colonization of mucosal tissues. Following adherence to mucosal epithelial
cells, the bacteria then enter the cells and traverse across them to exit to the subepithelial

space where they elicit an acute inflammatory response, resulting in the symptoms

92

characteristic of gonorrhea. In some cases, the organism will ascend from the site of
infection into the fallopian tubes resulting in gonococcal salpingitis, a leading cause of
infertility in women. It will also occasionally spread to other tissues in both men and
women resulting in a full-blown bacteremia, termed disseminated gonococcal infection
(DGI), a major sequelae of which is arthritis.

The colonization of mucosal tissues by N. gonorrhoeae is a critical first step in a
gonococcal infection. Adherence to cells of the mucosal epithelia is essential to prevent
the bacterium from being washed away by urine (in the male urethra) or vaginal fluid (in
the female genital tract). Colonization of the host epithelia by gonococci is a complex
process involving multiple components of both the bacterial and host cell. The incoming
bacterium first binds to epithelial cells via interactions between specific receptors on the
bacterial and host cell surfaces. The primary adhesins on the gonococcal cell surface are
the type IV pili (380, 384), although there are additional surface structures of the
bacterium that can participate in adherence (97, 127, 176, 194, 232, 233, 341, 419, 428).
Binding of gonococcal pili to host cells results in the induction of signal transduction
pathways in the eukaryotic cell that affect multiple cellular processes (20, 181, 209, 276,
278, 296). Adherence of piliated gonococci to epithelial cells also results in a variety of
rearrangements in the components of the cellular cytoskeleton (249)] as well as
significant changes in host cell gene expression (295). However, our understanding of
gonococcal infection with respect to the specific response of N. gonorrhoeae at the level
of gene expression is still incomplete.

Our hypothesis is that the initial contact between gonococci and host epithelial

cells signals the bacterium to modulate the expression of genes that will allow it to

93

colonize and proliferate in the new host. In this work, we sought to identify genes that are
triggered by the adherence of gonococci to host cells and to identify regulatory
phenomena that are involved in this response. Using a tissue culture model of infection
and gonococcal DNA microarrays, we examined changes in patterns of gene expression
in the gonococcus in response to contact with human epithelial cells in culture. Here we
report that rpoH, which encodes RpoH, a homolog of the heat shock sigma factor, 032, as
well as genes expected to be controlled by RpoH, are induced after host cell contact.
Further studies show that moH is essential to N. gonorrhoeae and is important for the

invasion of, but not adherence to human epithelial cells. These data therefore suggest a

role for the heat shock regulon in gonococcal pathogenesis.

94

MATERIALS AND METHODS

Growth and construction of bacterial strains. E. coli strain DHSa was used for all
recombinant DNA manipulations (334) and was grown in Luria broth (LB) supplemented
as necessary with ampicillin (Ap) at 100 mg/l, chloramphenicol (Cm) at 20 mg/l,
kanamycin (Kn) at 50 mg/l, or erythromycin (Em) at 300 mg/l.

N. gonorrhoeae strain MSll (P+ Tr) (349) was grown in a humidified 5% C02
atmosphere in GC medium (Difco Laboratories, Sparks, MD) with supplements (187).
When necessary, antibiotics (Em and Cm) were added at 3 mg/l and 6 mg/l respectively.
N. gonorrhoeae transformation and genomic DNA isolation were performed as described
(349, 355). Gonococcal strains used in these studies were predominantly Opa” and P+ as

determined by colony microscopy.

DNA manipulations. Cloning vectors used were pHSS6 (356), pDONR221 (Invitrogen,
Carlsbad, CA), pET24a (Novagen, Madison, WI) and pKH35 (142). Restriction enzymes,
T4 DNA ligase (New England Biolabs, Beverly, MA) and GatewayTM PCR cloning kits
(Invitrogen) were used according to the manufacturer’s recommendations. Polymerase
chain reaction (PCR) was done with Taq DNA polymerase (91) in a GeneAmp 9700
thermocycler (Applied Biosystems, Foster City, CA). When necessary, amplification
products were purified using the QIAquick PCR Purification kit (Qiagen, Valencia, CA).
Oligonucleotide primers were synthesized at the Michigan State University
Macromolecular Structure, Sequencing and Synthesis Facility (MSU GTSF, sequences
available on request). DNA sequence determination was done by the MSU GTSF and

analyzed using the Oxford Molecular Group DNA analysis program Omiga (Accelrys,

95

San Diego, CA). Transposon mutagenesis was performed by in vitro transposition using
EZ::TNTM transposase (Epicentre Technologies, Madison, WI) and the modified
transposon TnErmUP as described (83). The position of transposon insertions was
determined by PCR using primers homologous to the ends of transposon (SqFP and
SqRP; Epicentre) and to the ends of the gene in question and corroborated by restriction

analysis.

Cell culture. Human epithelial cell line A431 (ATCC CRL 1555) was grown at 37°C in
a humidified 5% C02 atmosphere in Dulbecco modified Eagle medium (DMEM,
Gibco/Invitrogen) supplemented with 5% fetal calf serum (FCS, Gibco). Adhesion and
invasion assays were performed as described (19, 419). Briefly, gonococci grown on GC
agar (16-20 h cultures) were swabbed into GC broth and concentration determined
spectrophotometrically. Bacteria were diluted in DMEM/5% F CS to appropriate
concentrations, and the bacterial suspension added to A431 cells grown to 50-90%
confluency in 24-well or 100 mM tissue culture plates as indicated in the text. Serial
dilutions of each bacterial inoculurn were diluted and plated on GC agar plates to
determine the actual number colony forming units (CFU) added. Infected cells were
incubated at 37°C w/5% CO; for 3 h (for adherence assays) or 7 h (for invasion assays).
Following incubation, the samples were divided into two sets (for adherence assays) or
three sets (for invasion assays). For the first set (total CFU), bacteria in the supernatant
were transferred to a sterile tube. Cells (with adherent bacteria) were lifted with saponin
(1% in GCB) and this suspension added to the bacteria from the supernatant, and serial

dilutions were plated onto GC agar. This represents the total number of CFU in the well

96

at the end of the experiment, a necessary control since gonococci multiply in the cell
culture media thoughout the experiment, and adherence is measured as a function of total
number of bacteria at the end of the experiment. For the second set (cell-associated CFU),
infected cells were washed with PBS to remove non-adherent bacteria and cells (with
adherent bacteria) lifted with 1% saponin in GC broth. Serial dilutions were plated to
determine cell associated CFU. The ratio of cell-associated CFU to total CFU at the end
of the experiment was defined as the adhesion frequency. For invasion assays, a third set
of wells were washed to remove non-adherent bacteria (as above) and fresh DMEM/5%
FCS containing 50 mg/l gentamicin (Gmso) added to each well to kill extracellular
bacteria. Following incubation at 37°C w/5% C02 for 1 h, Gm-containing media was
removed, cells were washed with PBS, and cells lifted with 1% saponin in GC broth.
Serial dilutions were plated to determine intracellular CFU. The ratio of GmR CFU to

cell-associated CFU at the end of the experiment was defined as the invasion frequency.

RNA isolation. RNA was isolated from adherent bacteria or bacteria grown in the
absence of cells using Trizol reagent (Invitrogen). RNA was quantified

spectrophotometrically, and quality assessed by agarose gel electrophoresis.

Construction of GC DNA arrays. DNA microarrays were made by amplification of
2043 ORFs of the N. gonorrhoeae genome, 1985 from strain FA1090 (43, 77) and 58
from the gonococcal genetic island (GGI) of N. gonorrhoeae strain MS11 (78, 142).
Sizes of the amplicons spotted ranged from 143 bp to 3485 bp in length, and

corresponded to the predicted ORF of each gene. An additional 362 amplicons

97

corresponding to internal ORF sequences were generated for genes with insufficient
DNA observed upon agarose gel electrophoresis of the initial PCR. PCR amplicons were
spotted in duplicate onto SuperAmine glass slides (TeleChem International Inc.,
Sunnyvale, CA) at the MSU GTSF. The presence of DNA on the arrays was validated by
hybridization with a Cy3-labelled random nonamer Oligonucleotide (Qiagen). A scan of
the slide at 532 nm showed spots at the appropriate positions where DNA had been
spotted, and blank spots at the buffer control spots (data not shown). DNA hybridization
of the arrays with MS11 genomic DNA showed valid hybridization signals (above
background) for 98% of the genes spotted on the slides, and indicated that these arrays
would be suitable for the analysis of gene expression in N. gonorrhoeae strain MS] 1. (A
manuscript detailing the construction and use of the arrays has been submitted for

publication.)

Microarray hybridization. 10 pg of total bacterial RNA was labeled using the CyScribe
first-strand labelling kit (Amersham Biosciences, Piscataway, NJ) which employs
random primed reverse transcription in the presence of Cy3 or CyS-dCTP. Cy3 and Cy5
labeled cDNA were purified on QIAquick Cleanup columns (Qiagen) and concentrated
using Microcon YM-30 microcentrifugation units (Millipore, Billerica, MA).

DNA array slides were pre-hybridized at 42°C in prehybridization buffer (5X
SSC, 0.1% SDS, 1% BSA). For hybridization, Cy3- and CyS-labelled probes were mixed
(10 pl each) with 1 pl 1 mg/ml sheared, sonicated herring sperm DNA and denatured by

boiling for 5 min, followed by chilling on ice. 20 p1 4X hybridization buffer (Amersham)

and 40 p1 formamide were added to the denatured DNA and the entire hybridization mix

98

was pipetted onto the prehybridized array slide. Following hybridization overnight at
42°C, the coverslips were removed by gently immersing in the first wash (1X SSC; 0.2%
SDS). Washes were: once in 1X SSC; 0.2% SDS at 42°C, twice in 0.1X SSC; 0.2% SDS
at room temperature, once in 0.1X SSC. Slides were then dried and scanned using a
GenePix 400 scanner (Axon Instruments, Union City, CA) and images were processed

and analyzed using GenePix version 4.1 software.

DNA array data analysis. Data from four independent experiments (biological
replicates) including one in which the dyes were swapped, were normalized to eliminate
labelling artifacts (intensity based normalization) and expression ratios determined. In
these four hybridization experiments, 75-85% of the spots on the slide had at least 40% of
the pixels >1 standard deviation above background in at least one channel (535 or 632 nm
wavelength). Spots not above this cutoff were not included in the normalization or outlier
analyses. Outliers were identified by iterative outlier analysis in which three successive
rounds of analysis are done to determine genes with ratios (log transformed) greater than
2.5 standard deviations from the mean (47). Confidence limits of the data were calculated
including a standard error factor (distance from gene’s average value to the nearest 2.5
standard deviation cutoff) and the number of standard deviations separating the spot from
the 2.5 standard deviation cutoff. This data is not included in the tables, but was taken
into consideration when determining genes for which data from multiple spots or

experiments were not consistent.

99

RT-PCR. Total bacterial RNA was prepared as described above. cDNA was synthesized
from 100 ng RNA with Superscript II RNase H' reverse transcriptase (RT) (Invitrogen)
using random nonamers (Sigma-Aldrich, St. Louis, MO). Gene specific primers for rpoH,
groES and 16S rRNA (internal control) were used in the subsequent PCR, which was
carried out for 30 cycles. Negative controls for each experiment included a no template
control and a no RT control where RNA was added as a template for the final PCR (to
rule out genomic DNA contamination of RNA preparations). Products were analyzed by
agarose gel electrophoresis and stained with EtBr for photographing and/or quantification

using KODAK 1D Image Analysis Soflware (Eastman Kodak, Rochester, NY).

100

RESULTS

Analysis of global gene expression in gonococci adherent to cells in culture. Several
cell lines have been used to characterize interactions between gonococci and host cells
(19, 60, 164, 167, 182, 200, 246, 247, 274, 360, 419). Each cell line has slightly different
features with respect to specifics of interactions with gonococci and handling in the
laboratory. For the experiments described in this work, A431 cells (110), an epithelial
cell line derived from the endocervix (275) were used. Gonococci have been shown to
adhere to and invade A431 cells at a high frequency (19, 164, 209, 246, 371) and there
have been several reports investigating the response of these cells to gonococcal
adherence at the molecular level (20, 21, 40, 209).

Total RNA was isolated from A431 cells alone, bacteria alone (M81 1), and A431
cells infected with MSll for 3 h. A 3 h infection time was chosen since this has been
reported as the minimum time where most gonococci that will adhere to cells have done
so, yet few if any, have invaded (360). cDNA was synthesized from each RNA
incorporating digoxigenin-l l-dUTP (Roche Applied Science, Indianapolis, IN) and used
to probe a Southern blot of ClaI-digested MSll genomic DNA (19). The results of this
experiment (data not shown) showed that cDNA from bacteria alone and cells infected
with bacteria gave a strong signal following chemiluminescent detection, with intense
bands at positions corresponding to the bacterial ribosomal RNAs, and a fainter banding
pattern (upon extended exposure) that is presumably from mRNA. cDNA from
uninfected A431 cells gave no signal at all, even after prolonged exposure. This indicated

that any remaining eukaryotic RNA in the infected cell samples (minimal since the cells

101

are lysed before bacteria are harvested) would not create a background problem in DNA
array hybridizations.

We next analyzed global gene expression in gonococci adherent to A431 cells. N.
gonorrhoeae strain MSll was used to infect A431 cells at a multiplicity of infection
(MOI) of 100. An MOI of 100 was chosen to insure that sufficient quantities RNA would
be isolated for hybridization experiments. At 3 h post-infection (p.i.), the cells were
washed with PBS to remove non-adherent bacteria. Cells (with adherent bacteria) were
lifted with 1% saponin in GC broth and samples diluted for plating to determine cell
associated CFU. Duplicate plates containing media without cells were “infected” as the
bacteria alone sample, and bacteria harvested without washing. Adhesion frequencies
were determined by comparing the number of adherent CFU to the total number of CFU
(CFU in the supernatant plus adherent CFU) at the end of the experiment. The total
number of CFU in wells containing A431 cells was comparable to the number of CFU in
the bacteria alone control, indicating that the grth of gonococci was not appreciably
altered in the presence of epithelial cells. Adhesion frequencies varied from 40-66%,
comparable to previous reports of gonococcal adherence under similar conditions at an
MOI of 10 (19, 164, 246). Bacteria from both samples (adherent bacteria and bacteria
alone) were concentrated by centrifugation and RNA isolated immediately using Trizol
(Invitrogen). Labelled cDNA probes were generated from isolated RNA and hybridized
to DNA arrays as described in Materials and Methods.

Global gene expression in adherent gonococci was compared with that of
gonococci grown in cell culture media in the absence of A431 cells at 3 h pi. Four

independent experiments were performed and outliers were identified by iterative outlier

102

analysis in which three successive rounds of analysis are done to determine genes with
ratios (log; transformed) greater than 2.5 standard deviations from the mean (47).
Averages of ratios (and standard deviations) of genes identified as outliers in at least two
experiments as differentially regulated by host cell contact are shown in Tables 4-1 (up-
regulated) and 4-2 (down-regulated).

Of the 94 genes identified as differentially regulated, several genes (35/94)
encode hypothetical proteins. This was not surprising, as nearly half of the genes in the
annotated genome of N. gonorrhoeae encode putative proteins with no similarities to
proteins of known function (43). The remaining 59 genes identified as differentially
regulated encode proteins with a broad spectrum of functions in N. gonorrhoeae
including amino acid metabolism, protein export, transcription, translation, protein
modification, glycolysis, oxidation-reduction reactions, cell division and replication.

A control microarray experiment was done to determine whether saponin
treatment (used to lift cells with adherent bacteria) affected gonococcal gene expression.
The results of this experiment showed no genes significantly down-regulated, and six
genes (NGO387, NGU652, NGIO55, N01 767, N01 779, and NGZO65) consistently up-
regulated in bacteria treated with saponin for 10 min (data not shown). However, none of
the genes up-regulated by saponin were identified as up-regulated by host cell contact

(Table 4-1).
RpoH is induced upon contact with A431 cells. Among the genes identified as up-

regulated following adherence to epithelial cells is mail, which encodes the putative heat

shock sigma factor, 0’32 (RpoH). In E. coli, RpoH has been shown to regulate the

103

expression of genes encoding a number of molecular chaperones and other proteins that
are important for keeping proteins in the cell folded properly under conditions of stress
such as increased temperature (131). Two major genes known to be transcribed fi'om 032-
dependent promoters in gram-negative bacteria are groEL and groES, which encode such
molecular chaperones. Not surprisingly, our results showed the gonococcal groEL and
groES homologs to be up-regulated in gonococci upon cell contact (Table 4-1). This is
consistent with a previous report showing that transcription of the gonococcal groESL

operon is increased following heat shock (391 ).

RT-PCR analysis of gonococcal genes induced upon host cell contact. In order to
corroborate the DNA microarray results, quantitative RT-PCR was employed to measure
differential expression of rpoH and groES. 16S rRNA expression was used as an internal
control, since its expression was not expected to be influenced by bacteria- host cell
contact. N. gonorrhoeae strain MSll was used to infect A431 cells at an MOI of 10. At 3
h p.i., RNA was isolated from adherent bacteria and from bacteria grown in cell culture
medium in the absence of cells essentially as described for the DNA array experiments.
RNA was quantified and subjected to RT-PCR using primers specific for moH, groES,
and 16S rRNA. Figure 4-1A shows the results of a representative RT-PCR experiment.
These data show that expression of rpoH was greatly increased (4.32-fold) in wild-type
gonococci (MSll) at 3 h p.i. Expression of groES was also significantly increased in
MS11 (1.77—fold), while the expression of 16S rRNA was unchanged. These data are

consistent with the results of the DNA microarray experiments.

104

Table 4-1. Genes up-regulated in gonococci adherent to A431 cells

 

 

 

a expression ratio
ORF ID gene gene product “cells/4:9“) std dev
NG1684 conserved hypothetical protein 3.27 1.33
NG0288 rpoH RpoH, 032 2.61 0.61
NG1363 pitrE MtrE, multidrug efflux pump 2.48 0.88
NG2094 groES GroES, heat shock chaperone 2.39 0.42
NG1989 Neisseria-specific protein, uncharacterized 2.38 0.67
NGO340 cysK cysteine synthase 2.34 0.95
NGOZ38 conserved hypothetical protein 2.34 1.10
NG1210 conserved hypothetical protein 2.24 0.50
NGZOQS groEL GroEL, heat shock chaperone 2.10 0.24
N60634 hypothetical protein 2.03 0.72
NGO372 ABC transporter 1.97 0.41
NG0791 conserved hypothetical protein 1.93 0.71
NG1927 Neisseria-specific protein, uncharacterized 1.91 0.43
N60635 hypothetical protein 1.91 0.51
NG1083 hypothetical protein 1.89 0.50
NGOG33 nifU Fe—S scaffold protein 1.89 0.78
NG1988 conserved hypothetical protein 1.88 0.48
NGOZ34 hemN porphyrin oxidoreductase 1.88 0.63
NGOS76 ppiB peptidyI-prolyl cis-trans isomerase B 1.87 0.86
NGO721 Neisseria-specific protein 1.85 0.38
NG1366 mtrR MtrR. multidrug efflux regulator 1.84 0.65
NG1291 conserved hypothetical protein 1.83 0.25
NGO199 ho transcription termination factor Rho 1.79 0.62
NG1515 ier prephenate dehydrogenase Ter 1.76 0.11
NG1493 rpsT 30$ ribosomal protein 820 1.73 0.36
NG1439 Equ ABC transporter. ATP-binding 1.72 0.34
NG0989 conserved hypothetical protein 1.69 0.44
NG1831 rme 508 ribosomal protein L29 1.68 0.61
NG1841 rspJ 308 ribosomal protein S10 1.68 0.24
NG1668 pgi glucose-G-phosphate isomerase (Gpi) 1.67 0.38
NG0637 conserved hypothetical protein 1.67 0.18
NG1773 sdaA L-serine dehydratase; L-serine deaminase 1.65 0.35
NG2180 conserved hypothetical protein 1.59 0.56
N60962 pncB nicotinate phosphoribosyltransferase 1.59 0.31
NGSO41 yth hypothetical protein. MS11 island 1.46 0.37
NG1587 mafB2 adhesin MafBZ 1.41 0.39
NGOZ76 comA competence protein (ComA) 1.32 0.30

 

aORF IDs and gene designations are fi'om the annotated N. gonorrhoeae genome

sequence (43).

1’Ratio of expression in adherent bacteria to expression in bacteria grown in the absence
of epithelial cells.

105

Table 4-2. Genes down-regulated in gonococci adherent to A431 cells

 

expression ratio std

 

a
ORF ID gene gene product (cells/wells) d ev
NG1024 conserved hypothetical protein 5.44 1.26
NGO186 ald Zn-alcohol dehydrogenase 2.55 1.24
NGO715 g6pd glucose 6-phosphate 1-dehydrogenase 2.54 0.25
NGO186.1 transposase fragment of IS1016 2.54 1.57
N60574 cah carbonic anhydrase 2.52 0.72
NG0771 recD RecD 2.35 0.26
NG1 21 5 conserved hypothetical protein 2.24 0.70
NG1358 gdhA glutamate dehydrogenase 2.04 0.50
NG1473 mdaB drug activity modulator B 1.99 0.26
NG1882 hypothetical protein 1 .96 0.79
NG1600 gInA glutamine synthetase 1.92 0.44
NG1545 conserved hypothetical protein 1.89 0.56
NG1627 hypothetical protein 1.85 0.53
NGO656 oxiT oxalate/formate antiporter 1.84 0.89
NG1630 lambda repressor-like protein cl 1.83 0.85
NG1698 comE competence protein ComE 1.78 0.44
NG1139 Neisseria-specific protein 1.76 0.70
NGO479 phage-related repressor (CI) 1.76 0.26
NGOB49 proB ProB 1 .73 0.57
NG1249 conserved hypothetical protein 1.71 0.45
NG1 1 17 conserved hypothetical protein (phage-like) 1.71 0.68
NG0249 ach acetyI-CoA carboxylase 1.70 0.20
NG1881 pykA pyruvate kinase II 1.70 0.64
NGO177 cpxR CpxR 1 .70 0.62
NG0850 proA ProA 1 .69 0.33
NG1769 yth cytochrome c peroxidase ' 1.68 0.37
NGO788 Neisseria-specific protein, uncharacterize 1.67 0.29
NG1258 pgm phosphoglycerate mutase 1.67 0.20
N60561 conserved hypothetical protein 1.67 0.33
NG1406 gcsT aminomethyltransferase, glycine cleavage system 1.66 0.40
NG1815 minD cell division protein, MinD 1 .66 0.44
NGOQO4 conserved hypothetical protein 1.66 0.40
NG0062 fthS formate-tetrahydrofolate ligase 1.65 0.46
NGOQ59 conserved hypothetical protein 1.65 0.35
NGO719 ngi glucose-6-phosphate isomerase 1.65 0.26
NGO794 bfrA bacterioferritin A 1.64 0.58
NGO410 cspA cold-shock protein 1.64 0.14
NG1 122 hypothetical protein 1.64 0.37
NGO318 recN RecN 1 .64 0.50
NG0449 conserved hypothetical protein 1.63 0.50
NGO717 hka gluookinase 1.63 0.40
NG1577 omp3 Plll 1.63 0.37
NGO732 Neisseria-specific protein, uncharacterized 1.62 0.59
N60626 mItB murein transglycosylase 1.62 0.16
N60692 LysR family transcriptional regulator 1.62 0.34
NGO472 hypothetical protein 1.61 0.42
NG1300 ych adenine specific methylase 1.61 0.29
NGO787 Neisseria-specific protein, uncharacterized 1.59 0.25
NG1374 ccoN cytochrome c oxidase 1.59 0.31
NG1643 conserved hypothetical protein 1.58 0.14
NG1138 conserved hypothetical protein 1.57 0.19
NG0248 trpA tryptophan synthase alpha chain 1.56 0.22
NG0203 gph phosphoglycolate phosphatase 1.55 0.30
NG1110 dnaB DnaB 1.55 0.42
NGO716 6ng 6-phosphogluconolactonase 1 .54 0.37
NG1381 glr2 glutaredoxin 2 1.51 0.41
NG1411 conserved hypothetical protein 1.40 0.66

 

 

106

aORF IDs and gene designations are from the annotated N. gonorrhoeae genome

sequence (43).

bRatio of expression in bacteria grown in the absence of epithelial cells to expression in
adherent bacteria.

107

An additional control was done to rule out effects of saponin (used to lifi cells
with adherent bacteria). Gonococci grown on plates were swabbed into GC broth and
divided into two aliquots: the first into GC broth, the second into GC broth containing 1%
saponin. After 10 min incubation, RNA was isolated from both samples and subjected to
RT-PCR using moH-specific primers. Results of this experiment showed that rpoH

expression was not affected by treatment of the bacteria with saponin (data not shown).

RpoE does m); mediate the induction of rpoH in gonococci upon host cell contact. In
E. coli, the expression of moH is under the control of multiple promoters, three that are
transcribed under most growth conditions via the housekeeping sigma factor, 070 (131),
one of which is subject to catabolite repression (269). The fourth promoter is dependent
on the extreme heat shock sigma factor, (:5 (encoded by 17705) which responds to
misfolded extra-cytoplasmic proteins (328). It is thought that RpoE is important for the
maintenance of membrane and periplasm homeostasis, and has been shown to have a role
in pathogenesis in several gram-negative bacteria (70, 197, 328).

Since N. gonorrhoeae has an annotated gene (NGI944) with high similarity to
rpoE (43), we initially hypothesized that the observed response to host cell contact might
be mediated by RpoE-dependent induction of rpOH, which in turn would lead to RpoH-
dependent induction of groEL/groES. To test this hypothesis, an rpoE mutant of N.
gonorrhoeae strain MS11 was constructed. The gonococcal rpoE gene was PCR
amplified from N. gonorrhoeae genomic DNA and cloned into pDONR221, and the

resulting plasmid (pNG1944) mutagenized by in vitro transposition using the transposon

108

TnErmUP as described (83). Two plasmids containing rpoE with transposon insertions
approximately 320 bp (11F) and 500 bp (9G) from the rpoE start codon were isolated and
used to transform N. gonorrhoeae strain MS11. EmR transformants were obtained at a
high frequency (~1 x 10'5 EmR transforrnants/CFU), indicating that rpoE is not essential
in N. gonorrhoeae. This is inconsistent with observations that rpoE is essential in several
bacteria, including E. coli and Y. enterocolitica (75, 156). To rule out the possibility that
the EmR transformants were rpoE heterodiploids, having a copy of the intact moE gene in
addition to the insertionally inactivated copy, PCR was done using primers specific for
rpoE. Agarose gel analysis of the amplification products showed that the mutants yielded
a band ~1.2 kb larger than the wild-type rpoE, consistent with the insertion of the
TnErmUP transposon, (data not shown). None of the mutants yielded a band
corresponding to the size of the wild-type moE gene. Expression of rpoE in the mutants
was analyzed by RT-PCR, with no amplification products observed except in the wild-
type (M81 1) control. Hence, our results indicate that strains NG1944.11F and
NG1944.9G are indeed rpoE null mutants and that rpoE is not essential to N.
gonorrhoeae.

To examine the role of RpoE in the initial stages of a gonococcal infection,
adhesion and invasion assays were done using A431 cells as described in Materials and
Methods. Adhesion was scored as the number of CFU cell-associated/total CFU at the
end of the experiment, while invasion was scored as the number of bacteria protected
from gentamicin killing (GmR)/number of cell-associated CFU. The results of these
assays are summarized in Figure 4-2. The rpoE mutant (NG1944.11F) adhered to and

invaded A431 cells at essentially the same frequencies as the wild-type parent strain,

109

MS11 (p > 0.05, Student’s t test). These results suggest that RpoE is not necessary for
gonococcal adherence to or invasion of A431 cells.

To determine whether rpoH expression and induction upon adherence to epithelial
cells was dependent on RpoE, RT-PCR was done using RNA isolated from NG1944.11F
grown either alone in cell culture medium, or following adherence to A431 cells for 3 h.
The results of these experiments (shown in Figure 4-1B) showed that rpoH is up-
regulated in the rpoE mutant at 3 h p.i., similar to what was observed in the wild-type
strain, MS11 (Figure 4-1A). Expression of groES followed a similar pattern. These data
suggest that rpoE is not required for the up-regulation of rpoH or groES upon host cell

contact.

110

 

 

 

Figure 4-1. RT -PCR analysis of genes induced in gonococci adherent to human
epithelial cells. RNA isolated from MS11 (wild-type) or NG1944.11F (rpoE::TnEmUP)
adherent to A431 cells (+) or grown in the absence of A431 cells (-) was reverse
transcribed using random primers. The subsequent PCR was performed with primers
specific for rpoH, groES and 16S rRNA, as indicated on the right. Images of EtBr stained
gels were reversed using Adobe Photoshop for clarity and band intensities quantified
using Kodak image analysis software, normalizing to 16S rRNA. Results shown are

representative of three independent determinations.

111

P
m

 

 

a)

d

O

o
1

Percent cell associated
01
O
GmR CFU/cell associated CFU x 10-3
A

o
1

 

 

 

 

WT rpoE WT rpoE

Figure 4-2. Adherence to and invasion of A431 cells is not affected in a gonococcal
rpoE mutant. WT: MS11; rpoE: rp0E22TnEmUP. (A) Adherence to A431 cells.
Adhesion data are presented as a percentage of the wild-type control (M811) and are
averages of five independent experiments performed in triplicate. (B) Invasion of A431
cells. Invasion data are presented as the ratio of Gentamicin-resistant (GmR) bacteria per
number of cell associated bacteria and are averages of two independent experiments

performed in triplicate. Error bars indicate standard deviation.

112

To analyze global gene expression in rpoE mutant gonococci, a DNA microarray
experiment was performed. Labelled cDNA was synthesized from RNA isolated from
NG1944.11F (rpoEzzTnEmUP) following 3 h adherence to A431 cells as described above
for wild-type gonococci. Analysis of the data showed a similar list of genes up-regulated
by host cell contact as was observed for the wild-type parent, MSll, including rpoH,
groEL, and groES (data not shown). Taken together, our results indicate that RpoE is not
involved in regulating rpoH gene expression in gonococci, at least under the conditions

tested.

RpoH is essential to N. gonorrhoeae. In order to examine the role of RpoH in gonococci
and its interaction with host cells, we next attempted to construct an moH null mutant.
The gonococcal moH gene was PCR amplified and cloned into pHSS6, and the resulting
plasmid (pADCl) mutagenized with the transposon TnErmUP as described for moE
above. Three different mutants with transposon insertions approximately 220 bp (1H),
435 bp (5D), and 700 bp (66) from the rpoH start codon were used to transform N.
gonorrhoeae strain MS11. Despite several attempts at transformation with increasing
amounts of input DNA, EmR mutants were never isolated. Positive transformation
controls included plasmid DNA used to construct the rpoE mutants (described above), as
well as genomic DNA from a previously characterized EmR N. gonorrhoeae strain,
MS11-306 (247). The inability to obtain EmR rpoH null mutants strongly suggested that
rpoH is essential in N. gonorrhoeae. An alternative explanation for our result could be a

polar effect on expression of a gene downstream (3’) of rpoH, however this is unlikely as

113

the gene immediately 3’ of rpoH (NG0287) is transcribed in opposite direction (43).

Hence, we concluded from these results that rpoH is essential to gonococci.

Construction of a conditional rpoH mutant. Since we were unable to construct an
rpoH null mutant in N. gonorrhoeae, a strain was constructed in which rpoH expression
was under the transcriptional control of the lactose repressor (LacI) such that rpoH
expression could be controlled by the addition of the LacI inducer, IPTG, to the culture
medium. We utilized a plasmid, pKH35 (142), which contains a segment of the N.
gonorrhoeae chromosome (3.8 kb) in which large sequences can be inserted without
being deleterious to the cell (H. S. Seifert, personal communication). Between the aspC
and lctP genes on the plasmid is a selectable marker (CmR), a lacIQ gene (since N.
gonorrhoeae has no lac operon of its own), two tandem lac promoter-operator sequences
(351), and three Neisseria uptake sequences (NUS) which are necessary for gonococcal
transformation (351). A gene of interest cloned downstream of the lacOPOP and
transformed into N. gonorrhoeae results in recombination between the incoming DNA
and the aspC-lctP region of the chromosome. Since pKH35 can replicate in E. coli, but
not N. gonorrhoeae, the plasmid itself is lost following transformation. What remains on
the chromosome is the selectable marker and the IPTG-inducible construct.

The rpoH gene was PCR amplified fiom MSll genomic DNA and cloned
immediately downstream of lac promoter on pKH35. This plasmid, pNGR18, was then
used to transform MS11, selecting for CmR. A double crossover event following
transformation results in the insertion of the inducible rpoH between aspC and lctP. The

resulting strain, MP288, has two copies of rpoH on the genome, the native copy and the

114

inducible copy. To eliminate the native copy of rpoH (such that rpoH expression is solely
under IPTG control) MP288 was transformed with the rpoH::TnErmUP plasmid,
pNGR20.5D, and transformants selected for EmR on plates containing IPTG, to insure
some rpoH expression in the event the native copy of rpoH was inactivated, as intended.
Since this incoming DNA could recombine with either the native copy of rpoH or the
inducible rpoH locus, EmR transformants were screened by PCR using primers that
differentiated between the native rpoH locus and the inducible construct at the lctP-aspC
locus. A mutant was identified in which only the wild-type copy of rpoH gene was
insertionally inactivated, which was named MPD288 [rpoH::TnEmUP, lacIOP-
rpoHCm], and expresses rpoH only in the presence of IPTG. MPD288 was maintained
on GC agar plates containing 20 pM IPTG, which allowed growth similar to the wild-
type strain. Subsequent passage of this strain onto media lacking IPTG resulted in an
eventual growth defect, but only after several passages. This may be due to a small
amount of RpoH still existing in the cell, and/or leaky expression of rpoH from lacOPOP
promoter.

The expression and inducibility of rpoH in strain MPD288 was examined by RT-
PCR using RNA isolated from bacteria grown with and without IPTG. Figure 4-3 shows
that rpoH is expressed at barely detectable levels in the absence of IPTG in MPD288, but
at nearly wild-type levels in the presence of 20 pM IPTG. Increasing the IPTG resulted in
corresponding increases in rpoH mRNA (data not shown).

Since groES, which is also induced in gonococci upon adherence to A431 cells
(Table 4-1 and Figure 4-1), is expected to be dependent on RpoH, we also analyzed

expression of this gene in the conditional rpoH strain. As predicted, expression of groES,

115

but not 16S rRNA, was reduced in the conditional rpoH strain grown under rpoH-
depletion conditions (no IPTG, Figure 4-3), confirming that groES expression in

gonococci is dependent on RpoH.

116

MS11 MPDZBB
IPTG (pM) o o 20

 

rpoH ~ '- ‘* “
groES It: m ‘1
16S rRNA fl u ~~

Figure 4-3. RT-PCR of rpoH and groES in an N. gonorrhoeae strain conditionally
expressing rpoH. RNA isolated fi'om MS11 (wild-type) or MPD288 (rponzTnEmUP,
lacIOP-rpoHCm) grown overnight on GC agar with or without 20 mePTG (as
indicated) was reverse transcribed using random primers. The subsequent PCR was
performed with primers specific for rpoH, groES and 168 rRNA, as indicated on the left.
Images of EtBr stained gels were reversed using Adobe Photoshop for clarity and band
intensities quantified using Kodak image analysis software, normalizing to 16S rRNA.

Results shown are representative of three independent determinations.

117

Temperature sensitivity of a conditional rpoH strain. In gram—negative bacteria, the
RpoH regulon is induced in response to a sudden increase in temperature, as well as other
stresses. This response is necessary to maintain the proper folding of proteins in the
cytoplasm under such conditions which could otherwise be lethal to the cell. An inability
to respond to heat shock (as is mediated by RpoH) would likely make the bacterium more
sensitive to heat shock. To test this in gonococci, we asked whether depletion of RpoH,
using our conditional rpoH strain, MPD288, would affect the strains’ ability to survive
heat shock. N. gonorrhoeae strains MS11 (wild-type) and MPD288 (inducible rpoH)
grown on GC plates with or without 20 pM IPTG were swabbed into GC broth and
diluted to 107 CFU/ml. The bacterial suspensions were then subjected to heat shock at
42°C for various times over a 1 h period. At 10 min intervals, samples were collected and
plated to determine viable CFU. The data obtained from four independent experiments
was averaged and is plotted in Figure 4-4. Gonococci depleted for RpoH (MPD288
grown without IPTG) were more sensitive to heat shock than the wild-type strain (MS11)
or the conditional rpoH strain grown in the presence of IPTG. This result indicates that

rpoH expression contributes to gonococcal survival in response to heat shock.

Effect of RpoH on interactions between gonococci and A431 cells. DNA array
analysis of gene expression in gonococci adherent to A431 cells showed that rpoH is up-
regulated more than two-fold at 3 h p.i. (Table 4-1). Genes expected to be dependent on
RpoH for expression, such as groES and groEL are also significantly up-regulated under

these conditions. These results imply that RpoH and genes it regulates might be important

118

for subsequent steps in a gonococcal infection. Thus, we next asked whether rpoH
expression is necessary for gonococci to adhere to and invade A431 cells in culture.

Wild-type N. gonorrhoeae strain MS11 and MPD288 were used to infect A431
cells at an MOI of 10. To deplete RpoH in MPD288, this strain was maintained on GC
agar containing 20 pM IPTG, and then grown overnight on medium lacking IPTG prior
to infection of A431 cells. For infection, bacteria were harvested fi'om plates and a
suspension prepared in cell culture medium with or without IPTG. At the end of the
experiment, samples were plated on GC agar containing 20 pM IPTG and CFU
enumerated following incubation at 37°C.

The results of these experiments are summarized in Figure 4-5. Adhesion
frequencies, determined at 3 h p.i., showed no significant differences between MPD288
grown with or without IPTG, or the wild-type parent strain, MS11 (p > 0.05). However,
the subsequent step in infection, invasion of epithelial cells, appeared to be reduced for
the mutant MPD288 depleted for RpoH. The relative invasion frequency at 7 h p.i. for
this strain was significantly less than that observed for the wild-type strain, MSll (p =
0.002). MPD288 grown in conditions which express levels of rpoH comparable to the
wild-type (Figure 4-3) invaded A431 cells at a higher frequency than this strain grown
without IPTG, although the frequency is not as high as observed for the wild-type parent,
and may not be significantly different (p = 0.06). The reduced ability of the rpoH-
expressing MPD288 strain to invade A431 cells compared to MSU may be due to an
inability of this strain to induce rpoH expression upon adherence, since the native
regulatory sequences are not present upstream of the expressed rpoH gene in this strain.

Taken together, these data show that in wild-type gonococci, rpoH expression is turned

119

on in response to initial contact with epithelial cells, and this expression is necessary for
the subsequent invasion of these cells. This demonstrates a mechanism for this bacterium
to sense its environment and adjust gene expression in preparation for the next step of the

infection.

120

 

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10’ 5“
‘\

..l ‘\

g

:>1O6 -

ll.

0

105 ._

104 l i i i l T

010 20 30 4o 50 60 70

Heat Shock Time (min)

Figure 4-4. N. gonorrhoeae depleted for RpoH are hyper-sensitive to heat shock.
Bacteria were grown overnight on GC agar plates with or without 20 pM IPTG (as
indicated) and then swabbed into GC broth at 107 CFU/ml. Bacterial suspensions were
subjected to a heat pulse at 42°C and aliquots removed at 10 min intervals. Serial
dilutions of samples were plated on GC agar plates (+20 pM IPTG as necessary).
Symbols: diamond (O), MSll (wild-type); triangle (A), MDP288 (inducible rpoH) +20
pM IPTG; box (I), MDP288 (inducible rpoH) w/o IPTG. Error bars indicate standard

deviation.

121

.>
on

 

 

 

 

 

 

100 100
u 9
.9 a:
3
.§ 80 E; 80
3 u—
2 60 5 60
8 i
E 40 5 40
6 .3
8 20 E 20
a)
(I
0 O
[IPTG]I - ll0 20 pMI [IPTG]l - II0 20 pMI
MS11 MPD288 MS11 MPD288

Figure 4-5. Depletion of RpoH in N. gonorrhoeae affects invasion of, but not
adherence to A431 cells. MS11: wild-type; MDP288: rpoH::TnEmUP, lacIOP-
rpoHCm. (A) Adherence to A431 cells. Adhesion data are presented as a percentage of
the wild-type control (MS11) and are averages of four independent experiments
performed in triplicate. (B) Invasion of A431 cells. Invasion data are presented as relative
invasion frequency, normalized to MS] 1. These data are averages of five independent

experiments performed in triplicate. Error bars indicate standard deviation.

122

DISCUSSION

The interaction between a host and a pathogen is a dynamic process, involving
responses from both the pathogen and the host. Several bacterial pathogens respond to
changes in their environment by modulating gene expression, providing a selective
advantage to organisms that are, at least initially, outnumbered, and must deal with
differences in nutrient availability as well as with the (often hostile) response of the host.
Often, these regulatory systems result in the integration of responses to several signals via
complex networks to modulate the expression of a variety of genes that are important
during an infection (79, 130, 225, 254, 339, 366). Some bacteria, such as Yersinia
pseudotuberculosis (293) and uropathogenic E. coli (448) regulate gene expression
specifically in response to contact with host cells. Since one of the first events to occur
when the N. gonorrhoeae enters a new host is adherence to the mucosal epithelia, we
hypothesized that this might serve as a signal for the bacterium to modulate the
expression of genes that will allow it to colonize and proliferate in the new host. It has
been shown that adherence of gonococci to epithelial cells in culture enhances their
invasiveness (60), and it is likely that this increased invasiveness results from changes in
gene expression that occur in response to adherence to cells. Thus, in this work, we
sought to identify the genes that are differentially expressed in gonococci in response to
attachment to epithelial cells and to identify the regulatory networks involved.

A PCR-amplicon based genome microarray was used to examine changes in gene
expression in N. gonorrhoeae upon adherence to human epithelial cells. The results of
these experiments showed 37 genes up-regulated (Table 4—1) and 57 genes down-

regulated (Table 4-2) in adherent gonococci relative to bacteria grown in the absence of

123

host cells. These genes fall into several categories including transcriptional regulation,
bacterial metabolism, molecular transport and (as expected) several hypothetical genes of
unknown function.

Of the genes down-regulated upon adherence, several of them (22/57) encode
proteins involved in central housekeeping functions such as glycolysis, amino acid
metabolism, nitrogen assimilation, respiration, and cell division. This suggests that there
might be a general shift in cellular metabolism in response to host-cell contact, indicating
a mechanism for the bacterium to begin to adapt to different nutrient availability in the
new host and/or the different environment inside of the host cell. Other down-regulated
genes encode proteins involved in DNA replication, recombination, and repair (RecD,
RecN, DnaB, chB), transformation competence (ComE), and four potential
transcriptional regulators. Interestingly, one of the down-regulated genes, NGl473,
encodes MdaB, a protein thought to be a drug activity modulator (43). However, MdaB
has recently been shown to be an NADPH quinone reductase that plays an important role
in managing oxidative stress in bacteria, suggestive of a role in pathogenesis (421).

In addition to the hypothetical genes up-regulated upon adherence (16/37) were
several genes encoding proteins involved in cellular metabolism and general gene
expression, again suggesting a general shift in bacterial metabolism in response to host-
cell contact. N61587, which was slightly up-regulated on adherence, is annotated as
mafB2, one of several genes of the multiple adhesin family (maf) which encode
glycolipid adhesins (276). This up-regulation of mafBZ could indicate a need for MafBZ
at subsequent steps of an infection, consistent with reports that MafB2 is involved in

Opa-independent invasion (276).

124

Of particular interest was the observation that several putative transcriptional
regulators were differentially regulated upon host cell contact, two up-regulated (Table 4-
1) and four down-regulated (Table 4-2). The identification of multiple putative
transcriptional regulators as repressed as well as induced upon adherence to epithelial
cells suggests that there are indeed complex regulatory networks involved in the
modulation of gene expression in gonococci in response to host cell contact.
Interestingly, NGOI 77, which is annotated as cpxR (43) is down-regulated upon host cell
contact. In E. coli, CpxR is a regulator that responds to cell envelope stress, in particular
to mis-folded proteins at the inner membrane, (314)]. However, NGOI 7 7 is also
annotated as ompR in N. gonorrhoeae, pth in N. meningitidis serogroup B (173), and
misR in N. meningitidis serogroup A (406). Thus, the function of the protein encoded by
NG0177 remains to be determined.

One of the highest up-regulated genes identified in our experiments was rpoH,

which encodes a heat shock-specific sigma factor (RpoH, 032). In gram-negative bacteria,
0'32 up-regulates the expression of genes encoding a number of proteins (particularly
chaperones and proteases) that are necessary for keeping proteins in the cell folded
properly under conditions of stress such as increased temperature (131, 443). groEL and
groES, which encode such molecular chaperones and are known to be transcribed from
632-dependent promoters in many gram-negative bacteria, were also identified as up-
regulated in gonococci upon adherence to host cells (Table 4-1, Figure 4-1). Our initial
hypothesis was that the up-regulation of gonococcal rpoH and the RpoH regulon would

be dependent on the extra-cytoplasmic sigma factor GE (encoded by rpoE). GE is known

to control rpoH expression in E. coli and S. typhimurium in response to misfolded extra-

125

cytoplasmic proteins (256, 328). Our rationale was that an interaction between host cells
and the outer surface of the gonococcus might be a signal to which RpoE would respond
and subsequently up-regulate rpoH expression. Construction and analysis of a
gonococcal strain with a transposon insertion in rpoE, however, showed that RpoE was
not necessary for the induction of rpoH upon host-cell contact (Figure 4-1). This may be.
due in part to the fact that there are no apparent gonococcal homologs to the anti-sigma
factor, RseA, and its accessory protein, RseB, which serve to control RpoE activity in
response to extra-cytoplasmic signals (75).

While it is clear that RpoE does not control rpoH gene expression in gonococci in
response to host cell contact, the regulatory mechanism used by gonococci to up-regulate
rpoH gene expression in response to host cell contact is still to be determined. It is
possible that transcriptional control of rpoH expression in gonococci is mediated by other
regulators in response to host cell contact, as yet to be identified. Clearly, expression of
rpoH is important in gonococci, since we (and others (205)), have been unable to
construct an rpoH null mutant, suggesting it is an essential gene.

Control of RpoH activity in gram-negative bacteria is complex, and occurs only
partially at the level of rpoH transcription (131, 443). Transcription of rpoH is usually
high under steady state conditions, but the message is not efficiently translated until a
shift in growth temperature, which results in an increase in translation of the pre-existing
rpoH mRNA by destabilization of a secondary structure in the mRNA which overlaps the
rpoH start codon (260). In addition, RpoH activity is controlled by two RpoH-dependent

chaperone systems, DnaK/J and GroEL/S which can sequester 0'32, preventing its

126

association with RNA polymerase holoenzyme (134, 215). Thus, the heat shock response
is modulated by the protein folding status of the cell.

A recent report suggests that regulation of RpoH in N. gonorrhoeae may differ
from that observed in other bacteria (205). While an increase in rpoH expression was
observed upon heat shock, the time following heat shock that the increase was observed
was less than the time observed for induction of the RpoH-dependent genes, dnaJ, dnaK,
and grpE. The level of RpoH protein, as shown by immunoblotting with antisera raised
against E. coli RpoH, correlated with mail expression, which the authors concluded
ruled out translational regulation, as has been demonstrated for E. coli rpoH (260). The
conclusion of this work was that regulation of RpoH activity is the primary level of
regulation in response to heat shock in gonococci.

Our observation that mail is induced upon host cell contact in gonococci is a
determination of the relative levels of rpoH transcript at a single point in time (3 h p.i.).
The increase in rpoH transcript levels in adherent bacteria relative to that in non-adherent
bacteria could be due to increased transcription or to an increase in rpoH mRN A stability,
which cannot be distinguished fi'om one another in these experiments. It is clear from our
results, however, that the interaction between gonococci and epithelial cells serves to
transmit a signal to the bacterium, by an as yet undefined mechanism, that results in the
induction of rpoH and at least some genes of the RpoH regulon. Regulation of RpoH
activity was not directly assessed, and could, in part, play a role in the induction of
groEL, groES, and other genes upon host cell contact. Further experiments are required to

examine this.

127

The observation that rpoH, as well as two genes of the RpoH regulon, groES and
groEL, but not dnaJ, dnaK, and grpE, which are known to be RpoH-dependent in
gonococci (205) were up-regulated in our studies could indicate the involvement of
multiple regulatory systems, as yet unidentified. RpoH may respond to more than heat
shock in the gonococcus. As a strict human pathogen with no environmental niche
outside its host, N. gonorrhoeae is likely to encounter only slight temperature
fluctuations, but numerous other environmental stresses in an infection or upon
transmission. In other bacteria, RpoH responds to stresses such as increased temperature,
as well as other factors such as increased hydrostatic pressure and hyperosmotic shock (8,
32) enabling the bacterium to detect and correct misfolded proteins in the cytoplasm
either by chaperone-assisted folding, or degradation by proteases (131).

Bacterial pathogens often up-regulate the production of virulence factors upon
infection, many of which are directly involved in host interactions, and are on the surface
of the bacterium. These proteins are often large, complex, multi-subunit proteins that
require the assistance of chaperones and other proteins in the bacterial cell to assemble
correctly and/or to be exported to their final destination. If the assembly and export
functions are not adequate, accumulation of misfolded proteins in the cytoplasm would
need to be degraded by proteases. An increase in the production of chaperones to aid in
the proper folding and assembly of virulence factors concommitant with an increase in
their expression would be critical in the adaptation process.

Our results demonstrating a role for the heat shock response in gonococcal pathogenesis
suggests that bacterial heat shock regulons might have a significant role in the regulation

of virulence. Another example is Vibrio cholerae, in which the expression of several

128

virulence genes including the cholera toxin gene (ctx) are modulated by grth
temperature as well as other environmental factors (198, 290)].

A final interesting note is that while many bacteria use several sigma factors, even
regulatory cascades of sigma factors, to control gene expression in response to a variety
of environmental conditions, N. gonorrhoeae only appears to encode four sigma factors:
05 (RpoE), 0'32 (RpoH), 0'70 (RpoD), and 054 (RpoN). (:70 (RpoD) is the primary “house-
keeping” sigma factor, highly conserved among gram-negative bacteria (222). In N.
gonorrhoeae, 054 (RpoN) has been shown to be non-functional (132). As we have shown
in this work, 0'32 (RpoH), but not 05 (RpoE) is essential for viability of gonococci, and
also plays a role in interactions between gonococci and its host. However, oE (RpoE)
does not appear to play a role in controlling expression of rpoH, at least under the
conditions tested, and preliminary experiments in this laboratory have yet to identify a
role for RpoE in gonococcal gene expression. In addition, gonococci do not appear to
have a homolog of the general stress sigma factor, 0", that in many gram-negative
bacteria regulates gene expression in resonse to stresses such as starvation,
hyperosmolarity, pH downshift, or nonoptimal temperature (153).

In summary, we have examined global gene expression in gonococci in response
to adherence to epithelial cells in culture. The results of our array experiments indicate
that the interaction between gonococci and its host results in changes in transcription of
several genes which can lead to changes in bacterial cell metabolism and other basic

functions, enabling the bacteria to quickly adapt to its new environment.

129

Chapter 5

RpoH mediates the expression of some, but not all genes induced in Neisseria

gonorrhoeae adherent to epithelial cells

This manuscript has been published in Infection and Immunity.
Du,Y and CG. Arvidson. 2006 RpoH mediates the expression of some, but not all genes

induced in Neisseria gonorrhoeae adherent to epithelial cells. Infect. Immun. 74: 2767-
2776

130

 

('0

bar
Ex;

im’a

ABSTRACT

Neisseria gonorrhoeae (gonococcus) is highly adapted to the human host, the
only known reservoir for gonococcal infection. However, since it is sexually-transmitted,
infection of a new host likely requires a regulatory response on the part of the
gonococcus to respond to this significant change in environment. We previously showed
that adherence of gonococci to epithelial cells results in changes of gene expression in the
bacteria that presumably prepares them for subsequent steps in the infection process.
Expression of the heat shock sigma factor gene, rpoH, was shown to be important for the
invasion step, as gonococci depleted for rpoH were reduced in their ability to invade
epithelial cells. Here, we show that of the genes induced in adherent gonococci, two are
part of the gonococcal RpoH regulon. When RpoH is depleted, expression of these genes
is no longer induced by host cell contact, indicating that RpoH is mediating the host cell
induction response of these genes. One RpoH-dependent gene, N003 76, is shown to be
important for invasion of epithelial cells, consistent with earlier observations that RpoH
is necessary for this step of an infection. Two genes, NG1684 and NGO340, while greatly
induced by host cell contact, were found to be RpoH-independent, indicating that more
than one regulator is involved in the response to host cell contact. Furthermore, NGO340,
but not N01 684, was shown to be important for both adherence and invasion of epithelial
cells, suggesting a complex regulatory network in the response of gonococci to contact

with host cells.

131

INTRODUCTION

Neisseria gonorrhoeae (GC, gonococcus) is a Gram-negative diplococcus which
causes the sexually-transmitted disease, gonorrhea. It is an obligate human pathogen
which typically infects the urethra in men and endocervix in women, although it
occasionally infects other tissues as well. Gonorrhea is a continuing threat to public
health worldwide, especially in developing countries and some lower socioeconomic
groups in developed countries. According to the World Health Organization (WHO;
www.who.org), about 30 million incidents of gonococcal disease occur each year, a
figure that is likely underestimated due to the large reservoir of asymptomatic carriers
(404). Another serious problem, in light of the global AIDS epidemic, is the finding that
gonorrhea can significantly enhance the transmission of HIV (202, 447). Unfortunately,
no effective vaccine for gonococcal infection is available, due in part to the high
variability of its surface antigens (46, 352, 370, 445) and treatment has heavily depended
on antibiotics. This too is a problem in that gonococci show an incredibly rapid ability to
develop resistance to antibiotics (23, 100, 361, 425).

The initial step of a gonococcal infection is colonization of mucosal tissues, a
complex process involving multiple components of both the bacteria and host cells. This
process is critical for the production of an infection. If gonococci are unable to adhere to
mucosal epithelial cells, they will be washed away with the normal flow of vaginal fluid
(in women) or urine (in men). The primary adhesins on the gonococcal cell surface are
the type IV pili, without which gonococci do not cause infection in human volunteers

(380, 381). Binding of gonococcal pili to host cells results in the induction of signal

132

transduction pathways in the eukaryotic cell that affects multiple cellular processes (20,
165, 180, 209, 276, 278, 304) and adherence of piliated gonococci results in a variety of
rearrangements in the components of the cellular cytoskeleton (reviewed in (249)). Little
is known about the response of N. gonorrhoeae to this interaction, although our previous
studies have begun to address this issue (84). In this work, we further these studies of the
examination of gonococcal gene expression in response to contact with epithelial cells to
test the hypothesis that attachment to epithelial cells signals the bacterium to modulate
the expression of genes necessary for proliferation in the host.

Our previous studies showed that adherence to epithelial cells in culture results in
changes of gene expression in gonococci, and that this response is in part mediated by
RpoH, a heat shock sigma factor (84). RpoH is a general transcriptional regulator in
several bacteria, regulating a broad range of genes responding to different stimuli inside
or outside the bacterium (8, 32, 269, 439, 443). Our previous work showed that RpoH
played a role in virulence in N. gonorrhoeae in that depletion of RpoH reduced invasion
of, but not adherence to epithelial cells in culture. Here we show that some of the genes
whose expression induced in adherent gonococci require RpoH for this induction, while
others are RpoH-independent for induction. In addition, analysis of insertion mutants
show that one of the RpoH-dependent genes is important for invasion of but not
adherence to epithelial cells, consistent with the previous conclusion that RpoH is
important for the induction of one or more genes upon adherence that is important for
subsequent steps in gonococcal infection. In addition, we show that one of the RpoH-

independent genes is important for both adherence to and invasion of epithelial cells,

133

suggesting the existence of multiple regulators in N. gonorrhoeae to mediate the response

to host cell contact.

134

MATERIALS AND METHODS

Bacterial strains and growth conditions. E. coli strain DHSa was used for all
recombinant DNA manipulations and was grown in Luria broth supplemented as
necessary with chloramphenicol (Cm) at 30 mg/liter, kanamycin (Kn) at 50 mg/liter, or
erythromycin (Em) at 300 mg/liter. Neisseria gonorrhoeae strain MSll (P+, Tr (349))
was grown in GC medium (Difco Laboratories, Sparks, MD) with Kellogg’s supplements
(187) in a humidified 5% C02 atmosphere. Em (3 mg/liter) and/or Cm (7 mg/liter) were

added when necessary.

DNA manipulations. Polymerase chain reaction (PCR) was performed in a GeneAmp
9700 thermocycler (Applied Biosystems, Foster City, CA) using Taq DNA polymerase as
described (84, 91). PCR products were purified using the QIAEX 11 Gel Extraction kit
(Qiagen, Valencia, CA) when necessary. Oligonucleotide primers were synthesized at the
Michigan State University Research Technology Support Facility (MSU RTSF). DNA
sequencing was done by the MSU RTSF. Restriction enzymes and T4 DNA ligase were
from New England Biolabs (Beverly, MA) and used as recommended. Plasmid pKH35
(142) was used for molecular complementation constructions. N. gonorrhoeae
transformation and genomic DNA isolation was performed as described previously (349,

355)

Construction of mutants. Plasmids containing genes of interest were from the N.

gonorrhoeae clone set (44) and used as targets for transposon mutagenesis. In vitro

transposition was carried out using EZ::'I'N'M transposase (Epicentre Technologies,

135

Madison, WI) and the modified transposon, TnErmUP (83). Determination of the
position of transposon insertions was determined by PCR using primers homologous to
the ends of transposon (SqFP and SqRP; Epicentre) and to the ends of the gene in

question and corroborated by restriction analysis.

Infection Assays. Human epithelial cell line A431 (ATCC CRL 1555) was grown at
37°C with 5% C02 in Dulbecco’s modified Eagle medium (DMEM; Gibco/Invitrogen,
Carlsbad, CA) supplemented with 5% fetal calf serum (FCS; Gibco). Adhesion and
invasion assays were performed as described previously (84). Assays were performed
three or more times using fi'esh bacterial isolates, with each sample assayed in triplicate
in each experiment. Adhesion frequencies were scored as the number of cell-associated
colony forming units (CFU) divided by the number of total CFU per well in that
experiment. Invasion frequencies were calculated as the number of gentamicin-resistant
(GmR) CFU per cell-associated CFU at the end of the experiment. Statistical analysis of

data was done using the Student’s t test, with p values indicated where appropriate.

RNA isolation and cDNA synthesis. RNA was isolated using TRIzolTM reagent
(Invitrogen). An additional DNase treatment was performed using Turbo DNA-free kit
(Ambion, Austin, TX) to remove any remaining contaminating DNA. Purified RNA was
eluted in RNAse-free H20 and stored at -80°C until further use. RNA was quantified
spectrophotometrically, and quality assessed by agarose gel electrophoresis. cDNA was
synthesized by reverse transcription using Superscript H RNase H' reverse transcriptase

(RT; Invitrogen) using random hexamers (Invitrogen). Briefly, RNA was mixed with

136

random hexamers and denatured at 75°C for 5 min. Mixtures were cooled to room
temperature, and reaction buffer, dNTPs and reverse transcriptase were then added to a
total volume of 20 p1. First strand synthesis was performed at 42°C for 50 min and
reaction terminated by heating to 75°C for 15 min. Controls without RT were included

for all samples.

Microarray hybridization and data analysis. Global gene expression was measured
using N. gonorrhoeae DNA microarrays composed of PCR amplicons of 2035 of the
2250 predicted open reading frames (ORFS) of the N. gonorrhoeae genome spotted onto
glass slides and have been described previously (44). Ten micrograms of total bacterial
RNA was reverse transcribed and labeled using the CyScribe post labeling kit
(Amersham Biosciences, Piscataway, NJ). Cy3 and Cy5 labeled cDNAs were purified on
QIAquick Cleanup columns separately (Qiagen), and combined and concentrated to 15 pl
using Microcon YM-30 microcentrifugation units (Millipore, Billerica, MA). Labelled
probes were mixed with 1 pl of 1 mg/ml sheared, sonicated herring sperm DNA and
denatured by boiling for 5 min, followed by chilling on ice. DNA array slides were pre-
hybridized, hybridized with labeled probes at 42°C overnight and washed as described
(84). Slides were scanned using a GenePix 40008 scanner (Axon Instruments, Union
City, CA) and images were processed and analyzed using GenePix version 4.1 software.
Four independent experiments (including a dye swap) were performed and data analysis

was performed as described previously (47, 84).

137

Quantitative RT-PCR. Oligonucleotides for genes of interest (rpoH, groES, NGO372,
N003 76, and NG1684) were designed using Primer Express software (Applied
Biosystems) and sequences will be provided on request. Oligonucleotide primers for 16S
rRNA were described previously (93, 322). Real-time PCR was performed using the
SYBR Green detection method (Applied Biosystems). Amplification reactions were in a
total volume of 15 pl and contained 1.5 pl 10X SYBR Green PCR buffer, 1.5 pl 25 mM
MgCl2, 1.2 pl 10 mM dNTP, 20 nM of each Oligonucleotide, 0.15 p1 AmpErase® UNG
(lU/pl), 0.075 pl AmpliTaq Gold® DNA polymerase (SU/pl) and appropriate diluted
template. The reactions were held at 50°C for 2 min, then heated to 95°C for 10 min and
then cycled 40 times using parameters of 95°C for 15 s and 60°C for 30 s. Reactions were
run on an ABI Prism model 7900HT Sequence Detection System (Applied Biosystems)
at the MSU RTSF.

Three independent experiments were performed using freshly extracted RNA for
each experiment (biological replicates). In each single experiment, dilutions of each
reverse transcription product were loaded in triplicate for each pair of Oligonucleotides.
Data generated from real-time PCR experiments was processed and analyzed using
Microsoft Excel. Differential gene expression was calculated using the relative
expression method as recommended (Applied Biosystems). The relative amount of target
was normalized to the internal control 16S rRNA (322), a house-keeping gene not
regulated by RpoH or host cell contact (84). Data were subjected to statistical analysis

using the Student’s t test, and p values are indicated where appropriate.

138

RESULTS

Transcriptional analysis of gonococcal genes in an rpoH conditional strain. In our
previous work we identified several genes, including rpoH and two genes (groEL and
groES) known to be regulated by RpoH in N. gonorrhoeae (391) as induced upon contact
with epithelial cells (84). Based on these observations, we hypothesized that RpoH would
mediate the induction of expression of some of these genes upon adherence to epithelial
cells. To identify genes in addition to groEL and groES that are regulated by RpoH,
global gene expression was examined in a gonococcal strain conditionally expressing
rpoH (MPD288 [rpoH::TnEmUP, lacIOP-rpoHCm] (84). In this strain, the wild-type
copy of rpoH on the chromosome was disrupted by insertion of the transposon TnEmUP
(83), and an additional copy of rpoH under control of a lac promoter was placed between
aspC and lctP on the chromosome (142). MPD288 was grown in GC broth either with or
without 20 pM IPTG for 6 h and RNA isolated from each culture. RNA was then labeled
and used to hybridize to N. gonorrhoeae DNA microarrays as described in Materials and
Methods. Four independent experiments (including one dye swap) were performed and
data was analyzed as described previously (84).

Table 5-1 lists the average ratios of expression of genes identified previously as
induced upon host-cell contact (84). As expected, the expression of rpoH was highly
induced in the presence of IPTG (5.33-fold), consistent with the fact that rpoH is under
control of a lac promoter in this strain. Also as expected, expression of groES and groEL
were significantly increased (2.58-fold and 2.06-fold respectively) in IPTG-treated
samples. In addition, expression of N603 76, predicted to encode a homolog of peptidyl-

prolyl cis-trans isomerase B (148), was induced 2.71-fold in the presence of IPTG,

139

indicating that this gene is regulated by RpoH. The expression ratios for the remainder of
the genes induced upon host cell contact (84) were not significantly larger than 2-fold in
the presence of IPTG, suggesting that they are not regulated by RpoH, at least not under

the conditions tested.

Quantitative PCR analysis of selected host cell contact-induced genes in a
conditional rpaH strain. To corroborate the microarray results and confirm that
expression of N003 76, but not N603 72 or NGI684, are induced by RpoH, real-time RT-
PCR was performed. N. gonorrhoeae strain MPD288, was grown with and without the
inducer IPTG as for the microarray experiment described above. RNA was isolated and
cDNA synthesized using random primers as described in Materials and Methods. Gene
specific primers for N003 72, NG03 76, NGI684, as well as groES and rpoH (as controls)
were designed and used in real-time PCR to determine the relative levels of transcription
of these genes in IPTG-treated and untreated samples. The relative amounts of products
were normalized to an internal 16S rRNA control, as it is a housekeeping gene whose
expression is not influenced by RpoH (84).

As expected, the expression of rpoH was highly induced bacteria grown in the
presence of IPTG (7 .5-fold), consistent with the fact that rpoH is under control of a lac
promoter in this strain. N01 684, annotated as encoding a conserved hypothetical protein
(43), is the gene whose expression was most highly induced in gonococci adherent to
A431 cells (3.27-fold, Table 5-1) (84). Microarray analysis of the conditional rpoH strain
MPD288 showed that this gene was not increased in expression in inducing conditions,

suggesting that it is not regulated by RpoH (ratio 0.80 i 0.13; Table 5-1). Consistent with

140

this observation, real-time PCR analyses show that N61 684 is not induced in MPD288
grown with IPTG, and may actually be slightlyrepressed under these conditions (ratio
0.43 i 0.12, p < 0.0001; Figure 5-1). NGO372, annotated as encoding a periplasmic
amino acid binding component of an ABC-transporter (43), was also shown to be
significantly up-regulated in adherent gonococci (1.97-fold, Table 5-1) (84). Like
N61 684, N603 72 was not significantly induced by IPTG in the DNA microarray
analysis of the conditional rpoH strain, MPD288 (ratio 1.06 i- 0.48, Table 5-1) nor was it
significantly differentially expressed in the real-time PCR analyses (ratio 0.99 i: 0.40, p =
0.958; Figure 5-1). In contrast, groES, known to be RpoH-regulated in several bacterial
species, including N. gonorrhoeae (84), (391) was induced more than 3-fold in IPTG-
treated samples as determined by real-time PCR (ratio 3.39 i 0.92, p = 0.0002; Figure 5-
1). DNA microarray analyses showed expression of N603 76 to be induced nearly 3-fold
in IPTG-induced samples, and real-time PCR results are consistent with this observation
in that an expression ratio of 3.12 i 1.09 was determined (p = 0.0004; Figure 5-1). These

results indicate that while N60376 and groES are regulated by RpoH, NGI684 and

N603 72 are not.

141

Table 5-1. Expression ratios of host cell contact-induced genes in the presence or

 

 

absence of RpoH
a n roduct expression ratio expression rage SD c
ORF '0 9°” 9° ° " (+cellsl-cells)‘ (+lPTGl-IPTG) )
NGOZBB rpoH RpoH, 032 2.61 5.33 (2.56)
NG2094 groES GroES, heat shock chaperone 2.39 2.58 (0.47)
NG2095 groEL GroEL, heat shock chaperone 2.10 2.06 (0.17)
NGO376 p piB pBeptidyl-prolyl cus-trans Isomerase 1.87 2.71 (0.60)
NG1684 conserved hypothetical protein 3.27 0.80 (0.13)
NG1363 mtrE MtrE, multidrug efflux pump 2.48 0.95 (0.06)
Neisseria-specific protein,
NG1989 uncharacterized 2.38 0.71 (0.11)
NGO340 cysK cysteine synthase 2.34 1.06 (0.36)
N60238 conserved hypothetical protein 2.34 1.01 (0.33)
NG1210 conserved hypothetical protein 2.24 N,A,d -
NG0634 hypothetical protein 2.03 1.16 (0.51)
N60372 ABC transporter 1.97 1.06 (0.48)
NG0791 conserved hypothetical protein 1.93 NA. -
Neisseria-specific protein,
N61927 uncharacterize d 1.91 1.04 (0.51)
NG0635 hypothetical protein 1.91 1.19 (0.35)
NG1083 hypothetical protein 1.89 1.04 (0.44)
N60633 nifU Fe-S scaffold protein 1.89 NA -
NG1988 conserved hypothetical protein 1.88 0.68 (0.21)
NGOZ34 hemN porphyrin oxidoreductase 1.88 1.03 (0.09)
NGO721 Neisseria-specific protein 1.85 1.49 (0.76)
NG1366 mtrR MtrR, multidrug efflux regulator 1.84 0.99 (0.16)
NG1291 conserved hypothetical protein 1.83 1.01 (0.21)
N GO 1 99 rho tirqarrlscnption termmation factor 1.79 0.70 (0.17)
NG1515 ter prephenate dehydrogenase Ter 1.76 NA. -
NGt493 rpsT 30$ ribosomal protein S20 1.73 0.96 (0.43)
NG1439 aqu ABC transporter, ATP-binding 1.72 0.87 (0.14)
NG0989 conserved hypothetical protein 1.69 NA. -
NG1831 rme 508 ribosomal protein L29 1.68 NA. -
NG1841 rspJ 30$ ribosomal protein 810 1.68 1.08 (0.15)
NG1668 pgi figtigsefi'pms‘mate 's°'"°'as° 1.67 1.01 (0.56)
NG0637 conserved hypothetical protein 1.67 0.98 (0.41)
NG1773 sdaA L-serine dehydratase; L-serine 1.65 1.20 (0.50)
deaminase
NG2180 conserved hypothetical protein 1.59 0.82 (0.26)
N60962 pncB nicotinate 1 .59 1 .10 (0.15)
phosphonbosyltransferase
NG5041 yth hypothetical protein, MS11 island 1.46 1.28 (0.53)
NG1587 maf82 adhesin MafBZ 1.41 1.01 (0.23)

 

 

142

 

N60276] comA competence protein (ComA) 1.32 0.96 (0.26)

a Gene list and ratios are from (84), and ratios are gene expression in gonococci adherent
to A431 cells relative to expression in gonococci grown in the absence of cells.

b +1PTG indicates growth in GCB + 20 pM mo, - IPTG indicates growth in GCB
alone.

° SD: standard deviation

d N.A.: not available

(Table 5-1 cont’d)

 

12

10~

Average Ratio (+l- IPTG)
O)

 

 

 

NG1684 NGO372 NGO376 groES IpoH

Figure 5-1. Real-time quantitative PCR analysis of expression of N61 684, N603 72,
N603 76, gorES and rpoH in the conditional moH strain MPD288 grown with and
without inducer (IPT G). The expression of each gene was normalized to that of the
house-keeping gene 16S rRNA. Ratios were calculated as the expression level of a gene
in MPD288 grown with 20 pM IPTG divided by that from MPD288 grown without
IPTG. Data presented is the average of three independent experiments and error bars

represent standard deviation.

143

Induction of N603 76 and groESL upon host cell contact is mediated by RpoH.
Previous results showed that N603 76 was induced in gonococci adherent to epithelial
cells (84). Results obtained by both microarray (Table 5-1) and real-time PCR (Figure 5-
1) showed that N603 76 was regulated by RpoH in standard laboratory medium (GCB).
Thus, we next asked whether the induction of N603 76 expression in adherent gonococci
was dependent on RpoH. N. gonorrhoeae strain MPD288, which conditonally expresses
rpoH, was grown in the absence of the inducer, IPTG, to deplete RpoH in the cell, and
then used to infect A431 cells. For comparison, RpoH-depleted bacteria were grown in
cell culture media without cells. At 3 h post-infection (p.i.), cells were washed to remove
non-adherent bacteria, and bacteria adherent to cells lifted by lysing the epithelial cells
with saponin. Adherent bacteria and grown in the absence of cells were harvested by
centrifiigation and RNA was isolated as described in Materials and Methods. cDNA was
made from the RNA using random primers and levels of expression from N603 76,
groES, and N61 684 in the samples were quantified by real-time PCR. N61 684 and
groES were used as negative and positive controls respectively. N61 684, while induced
in gonococci adherent to epithelial cells (84), did not appear to be regulated by RpoH in
either microarray (Table 5-1) or quantitative PCR experiments (Figure 5-1). groES
expression has been shown to be regulated by RpoH in N. gonorrhoeae (Table 5-1,
Figure 5-1) (84, 391); as well as induced upon host-cell contact (84).

As expected, the expression of groES in adherent gonococci depleted of RpoH
was similar to the levels observed in gonococci grown in cell culture medium alone,
indicating that groES was no longer induced by contact with host cells in the absence of

RpoH (ratio 1.22 i 0.17, p = 0.040; Figure 5-2). A similar expression pattern was

144

observed for N60376, which was also no longer induced in adherent gonococci in the
absence of RpoH (ratio 1.39 i 1.20, p = 0.357; Figure5-2). Interestingly, expression of
N61 684 was still induced in adherent gonococci depleted of RpoH (ratio 4.84 i 3.47, p =
0.042; Figure 5-2), suggesting that another regulatory mechanism, independent of RpoH,
is involved in modulating the expression of this gene in response to contact with

epithelial cells. Taken together, these results show that host-cell contact induction of

N603 76 and groES, but not N61 684, is mediated by RpoH.

Identification of a putative RpoH-like promoter upstream of N603 76. Several genes
have been identified in E. coli as being transcribed from 032 (RpoH)-containing RNA
polymerase, leading to the determination of a consensus RpoH-regulated promoter (195,
212, 442). Tauschek and coworkers identified a putative RpoH-binding site upstream of
groES in N. gonorrhoeae, which is near a transcriptional start site that was more active
following a 10 min pulse at 42°C (391). This promoter sequence shares high similarity
(13/20 nt match) to the consensus RpoH promoter identified in E. coli (442). Analysis of
the promoter region of N603 76 revealed a sequence with high similarity to both the
gonococcal groES promoter and the consensus E. coli RpoH promoter (13/20 nt match).
The identification of a putative RpoH-binding site upstream of N603 76 is consistent with
the observations that this gene is regulated by RpoH and suggests that the regulation is
direct, with N603 76 transcribed from a 032(RpoH)-containing RNA polymerase in N.

gonorrhoeae.

145

 

Average ratio
(+/- A431 cells)

 

 

 

 

NG1684 N60376 groES

Figure 5-2. Real-time quantitative PCR analysis of expression of N61 684, N603 76
and groES in MPD288 depleted for RpoH and adherent to A431 cells. RNA isolated
from MPD288 depleted for RpoH adherent to A431 cells (+) or grown in the absence of
A431 cells (-) was reverse transcribed using random primers. Real-time PCR was
performed with primers specific for N61 684, N603 76, groES and 16S rRNA as described
in the text. Ratios were calculated as the expression level of a gene in adherent MPD288
divided by that of MPD288 grown in cell-free culture. Data presented is the average of

three independent experiments and error bars represent standard deviation.

146

Construction of gonococcal strains with mutations in host cell contact-induced
genes. Since our previous results indicated that several genes were induced in gonococci
adherent to epithelial cells (84), we hypothesized that one or more of those genes would
be important for subsequent steps of a gonococcal infection. To test this, several host cell
contact-induced genes were selected for further investigation of their roles in gonococcal
infection. The rationale for the selection of genes to mutate was as follows: NGO340,
N60634, N61363, N61 684, N61989, and N62094 were selected based on the
observation that they showed a 2 2-fold increase in expression in adherent gonococci
(Table 5-1) (84). While N60238 and N61210 were also induced _>. 2-fold upon
adherence, neither was present in the clone set used as targets for mutagenesis (44), and
therefore we were not able to mutagenize them. NG2095 (groEL) is the second gene of
the groES—groEL operon (391), and only N62094 (groES) was selected for mutational
analysis. N603 72 was selected as it was induced nearly 2-fold upon adherence and was
also found to be differentially expressed in other experiments (Lenz, J. and C. G.
Arvidson, unpublished results), and was therefore of interest. N603 76 was selected since
it was found to be RpoH-dependent for induction in adherent gonococci (Table 5-1,
Figure 5-1 and 5-2), and N61366 (mtrR) was selected as it encodes a previously
characterized transcriptional regulator (139).

Plasmids containing genes of interest were insertionally inactivated by in vitro
transposition using the transposon TnErmUP as described previously (83, 84). Plasmids
containing mutated genes were then used to transform N. gonorrhoeae strain MSll to

EmR. Allelic exchange between the wild-type copy on the chromosome and transforming

147

DNA containing the mutant copy containing the EmR determinant should result in null
mutations of each of these genes. Not surprisingly, despite several transformation
attempts, we were unable to construct a gonococcal groES null mutant in that no EmR
transformants were obtained. Since groES is essential in other bacterial species (94), it is
likely that it is essential in gonococci as well, although additional experiments will be
required to prove this. We were also unable to contruct null mutations in three additional
genes, N603 72, N60634, and N61363. The reasons for this were not clear, but for this
work these genes were not studied further. Once mutants were successfully constructed in
N. gonorrhoeae strain MS11, each mutant was backcrossed with the parent strain by
transformation with genomic DNA. This was to insure that the mutation was caused by
the insertion of the transposon in the gene of interest. A list of the mutant strains

constructed and the gene mutated in each is in Table 5-2.

Analysis of mutants for interactions with epithelial cells in culture. Once gonococcal
strains were constructed with insertion mutations in individual genes identified as
induced upon contact with epithelial cells, we next asked whether any of the mutations
affected the ability of gonococci to adhere to and/or invade A431 cells in culture. Wild-
type MS11 and mutant strains were grown overnight on GC agar and then used to infect
A431 cells at an MOI of 10 as described in Materials and Methods.

Figure 5-3A summarizes the results of three independent experiments in which
adherence was measured at 3 h p.i, and adhesion fiequencies expressed as the percent
cell-associated CFU as a function of the total CFU in the well at the end of the

experiment. These results show that insertion mutations in N603 76, N61366, N61 684,

148

and N61989 had no appreciable affect on gonococcal adherence to A431 cells compared
to the wild-type parent, MS11. However, mutant M0340 was significantly reduced in
adherence, with an adhesion frequency more than 3-fold lower than that determined for
MS11 (32.9 i 9.3%, p < 0.0001). A possible explanation for this phenotype could be that
the mutant expressed a variant piIE gene, which encodes pilin, the major subunit of the
gonococcal pilus (374). Type IV pili are the primary adhesins for gonococci (380) and
undergo antigenic variation at a high fi'equency (353), thus a variant pilin could alter the
ability of the strain to adhere to cells in culture. To test this possibility, the pilE genes
from M0340 and from the wild-type parent strain, MS11, were PCR amplified and DNA
sequences were determined. The sequencing results showed that the two pilE sequences
were identical at nucleotide level, indicating that the reduced adhesion phenotype of
M0340 is not due to the production of a variant pilin in the M0340 mutant strain.

Figure 5-3B summarizes the invasion assay results obtained from three
independent assays performed in triplicate. Assays were performed as described (84) and
invasion was scored as the number of GrnR CFU per cell-associated CFU at 7 h p.i. The
results of these experiments show that two mutants, M0340 and M0376, were
significantly reduced in their ability to invade A431 cells at 7 h p.i.. Mutant M0340,
which was reduced in adherence to A431 cells (Figure 5-3A), also invaded cells ~ 70%
(70.1 i 10.4%, p < 0.0001) as well as the wild-type parent strain, indicating that this
mutant is generally deficient in its ability to interact with epithelial cells. A second
mutant, M0376, was also reduced in its ability to invade A431 cells, invading ~40% (40.9

i 8.5%, p < 0.0001) as well as the wild-type MSll, although it appeared to adhere to

149

cells as well as MS11 (Figure 5-3A). The other three mutants assayed, M1366, M1684,

and M1989, invaded with essentially the same frequencies as the wild-type strain.

Molecular complementation of the N60340 and N60376 mutations. In order to
demonstrate that the reduced adhesion and invasion phenotypes observed for strains
M0340 and M0376 were indeed due to insertion mutations in NGO340 and N60376,
molecular complementation experiments were performed in which each of the genes was
cloned and expressed in trans in the corresponding insertion mutant strains. NGO340 and
N603 76, along with 400-500 bp of 5’ and 3’ flanking sequences were PCR amplified and
cloned into a derivative of the plasmid pKH35 in which the Plac-laCIQ fragment had been
removed, to generate pC340 and pC376. Adjacent to the cloned genes on these plasmids
is a marker for CmR, and flanking the cloned gene-marker construct are two genes, aspC
and lctP of the N. gonorrhoeae chromosome between which is has been reported that
large sequences can be inserted without being deleterious to the cell (84), (142).
Transformation of N. gonorrhoeae with linearized pC340 and pC376 and selection for
CmR resulted in recombination between the incoming DNA and the aspC-lctP region of
the chromosome. The resulting heterodiploids are such that the chromosomal copy of
NGO340 or N603 76 had the TnErmUP insertion, and an additional wild-type copy of the
corresponding gene is at the aspC-lctP locus. Transforrnants were confirmed as having
both a wild-type and Tn-mutated copy of NGO340 or N603 76 by PCR using

combinations of gene-specific and transposon-specific primers.

150

P

 

Relative adhesion frequency

 

MS11 C0340

M1684 M1366
M0340 M0376 M1989

 

 

Relative invasion frequency

0 ,
MS11 60340 C0376 M1989
M0340 M0376 M1684

M1366

Figure 5-3. Interaction of N. gonorrhoeae mutants with A431 cells. (A) Adherence to
A431 cells. Adhesion data are presented as relative adhesion frequency, normalized to the
wild-type control (MS11), which was arbitrarily set at 100%. (B) Invasion of A431 cells.
Invasion data are presented as relative invasion frequency, normalized to MS11, which
was arbitrarily set at 100%. These data are averages of three independent experiments

performed in triplicate, and error bars indicate standard deviation.

151

Table 5-2. Mutants constructed in host cell contact induced genes.

 

 

gene ORF ID Mutant Eggdenta
cysK NGO340 M0340 no
NGO372 N.o.° no
ppiB N60376 M0376 yes
NG0634 N.D. no
mtrE NG1363 N .D. no
mtrR NG1366 M1 366 no
NG1 684 M1684 no
NG1 989 M1989 no
groES NG2094 N.D. yes

 

3 Regulation is mediated by RpoH as determined by DNA microarray (Table 5-1) and 0-
PCR (Figure 5-1).

b N.D.: no mutant obtained

152

The resulting strains, C340 and C376, were then used to infect A431 cells to
measure adherence and invasion as described in Materials and Methods, and the data is
shown graphically in Figure 5-3. The complemented mutant, C340, adhered to and
invaded A431 cells as efficiently as the original wild-type parent strain, MS11 (p = 0.436
and 0.296, respectively). The complemented mutant, C376, also invaded A431 cells as
efficiently as the original wild-type parent strain (p = 0.798). These results indicate that
providing the wild-type NGO340 and N603 76 in trans indeed complemented the

mutations, restoring the adherence and invasion capabilities of the two mutant strains.

153

DISCUSSION

Adherence to mucosal epithelial cells is a criticial first step in a gonococcal
infection. As gonorrhea is a sexually-transmitted disease, transmission to a new host
often results in the bacterium entering a very different environment fi'om which it came,
and the initial interaction with epithelial cells has the potential to serve as a signal to the
bacterium that it is in a new host. Previous studies from our laboratory have demonstrated
that attachment of gonococci to epithelial cells results in the modulation of expression of
several genes (84). Genes differentially expressed in adherent gonococci included those
involved in a variety of ftmctions such as glycolysis, amino acid metabolism, protein
synthesis, protein transport, as well as regulation of gene expression. In particular, we
found that rpoH, which encodes the heat shock sigma factor, 032 (RpoH), was induced in
adherent gonococci and that this induction was necessary for optimal invasion of
epithelial cells.

RpoH is a critical regulator of the bacterial heat shock response, and is highly
conserved in bacteria (271, 452). Studies in other bacteria have shown that RpoH is a
general transcription factor not only involved in the heat shock response, but also
involved in the response to osmotic pressure, misfolded proteins, nutrient limitation and
other stimuli (8, 18, 32, 444). Our observation that RpoH is involved in modulating gene
expression in gonococci upon contact with host cells suggests that this interaction is yet
another stress response mediated by RpoH in a bacterium.

The goals of this work were two-fold. First, we sought to determine which genes

induced upon adherence to epithelial cells require RpoH for this induction. Second, we

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examined several host cell contact-induced genes to determine which, if any, play a role
in adherence to and invasion of epithelial cells.

N603 76, encoding a putative rotarnase, was found to be dependent on RpoH for
induction upon contact with epithelial cells. Three pieces of evidence support this
conclusion. First, in a standard laboratory medium (GCB), expression of this gene was
significantly induced in the conditional rpoH strain grown in the presence of inducer as
determined by DNA microarray analysis (Table 5-1) and quantitative RT-PCR (Figure 5-
1) indicating regulation by RpoH. Second, when the conditional rpoH strain, MPD288,
was grown in RpoH-depletion conditions (no IPTG), induction of N603 76 expression in
adherent gonococci was abated (Figure 5-2), indicating that RpoH was required for the
cell contact-mediated induction. Third, a putative RpoH-binding site with significant
sequence similarity to RpoH-dependent promoters was identified upstream of N603 76
(data not shown). Similar observations were made for groES, a well-characterized RpoH-
dependent gene in several bacteria including N. gonorrhoeae (84, 105, 199, 391, 440).
Taken together, these results demonstrate that like groEL and groES, N603 76 is part of
the gonococcal RpoH regulon and requires RpoH for induction upon contact with
epithelial cells.

In contrast to our observations that groEL and groES and N603 76 are induced in
adherent gonococci in an RpoH-dependent manner, N61 684, whose expression was the
most highly induced in gonococci adherent to A431 cells (84) appeared to be RpoH-
independent. Expression of N61 684 in the conditional rpoH strain in the presence of
inducer was essentially the same as in its absence (Table 5-1). However, when the

conditional rpoH strain, MPD288, was grown in RpoH-depletion conditions and used to

155

infect A431 cells, expression of N61 684 in adherent gonococci was still induced (Figure
5-2). These results clearly indicate that the induction of N61 684 in adherent gonococci is
independent of RpoH and is likely mediated by another, as yet unidentified regulator.

We previously showed that several genes were induced in gonococci adherent to
epithelial cells (84). This led to the hypothesis that one or more of those genes would be
important for adherence to and/or invasion of epithelial cells. To test this, mutations in
several host cell contact-induced genes were constructed and analyzed for their effects on
adherence to and invasion of A431 cells in culture.

We were successful in isolating null mutants of NGO340, N603 76, N61366,
N61 684, and N61989 (listed in Table 5-2) but were unable to isolate transformants with
insertion mutations in groES, N60634, N603 72, and N61363, in spite of several
attempts. It is possible that these genes are essential in N. gonorrhoeae, and is very likely
for groES, which is known to be essential in other bacterial species (218). However, there
have been reports of mtrE (N61363) mutants isolated in other N. gonorrhoeae strains
(76), thus there may be other explanations for our inability to construct a mtrE mutant in
MS11. N603 72 encodes a putative ABC-transporter component (43), and is induced in
adherent gonococci in an RpoH-independent manner. Whether or not the putative ABC-
transport system is essential will require further experimentation. Another explanation for
the inability to obtain some of these mutants is that there may be polar effects on genes
downstream of those we attempted to mutate. Additional experiments, such as using
plasmid constructs with more flanking sequences on either side of the transposon

insertion to facilitate homologous crossover and/or the development and use of a non-

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polar transposon might result in viable mutants in these genes. To prove that either of
these is essential will require the construction of conditional mutants.

Assays for adherence to A431 epithelial cells using the six transposon insertion
mutants isolated (listed in Table 5-2) showed that one mutant, M0340, was significantly
reduced in its ability to adhere to cells (Figure 5-3A). This mutant was also reduced in its
ability to invade A431 cells (Figure 5-3B). Since invasion is scored as a function of
adherence (GmR per cell associated CFU), the observed reduction in invasion frequency
indicates that each step (adherence and invasion) is defective in this mutant, and that the
protein encoded by this gene, thought to be involved in cysteine biosynthesis, is
important for gonococcal-host cell interactions. While expression of NGO340 is induced
in adherent gonococci, this induction is independent of RpoH (Table 5-1, Figure 5-1),
therefore, our previous observation that depletion of RpoH in gonococci reduces its
ability to invade A431 cells (84) is not due to a lack of induction of NGO340 expression.
However, this does provide evidence for the existence of additional regulators that are
involved in the host cell contact response.

In the gonococcal genome database (43), NGO340 is annotated as encoding an 0-
acetylserine (thiol)-lyase-A, which catalyzes the last step in the synthesis of L-cysteine
(EC 2.5.1.47). In E. coli and the closely related Salmonella typhimurium, there are two
genes encoding O-acetylserine (thiol)-lyase-A, cysK and cysM (53), however only one
(annotated as cysK) has been identified in the gonococcal genome. Cysteine has been
shown to be very important for growth of gonococci in that cysteine is required for
colony formation on chemically defined agar medium (56) and cystine (formed from

cysteine in oxidizing conditions) is a limiting factor in continuous culture of gonococci

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(185). Cysteine is an essential amino acid, not synthesized by the human host, thus it is
likely that cysteine will be limiting in the natural environment of the gonococcus.
Cysteine, as one of two sulfur-containing amino acids serves as a source of organic sulfur
in the bacterial cell, and is also critical for the folding of many proteins via the formation
of disulfide bonds. It is this feature that may account for our observations that cysK is
necessary for gonococcal host cell interactions. The interaction between gonococci and
host cells involves several proteinaceous structures on the surface of the bacterium,
including pili (380), opacity proteins (232, 419, 427), and porins (176, 194). Cysteine
residues are highly conserved in the otherwise highly antigenicially variable pilin, the
major protein component of the gonococcal pilus (357), and are critical in the formation
of disulfide bonds during assembly of the pilus fiber (288). As pili are critical for
gonococcal attachment to epithelial cells this could explain why cysK mutants do not
adhere well to A431 cells (Figure 5-3A), although since pili do not promote invasion, and
may even impede the process (232, 360), this does not explain our observation that
M0340 invades A431 cells less efficiently than the wild-type. The reason for this
observation is less clear and will require additional experimentation.

A second mutant, M0376, adhered to A431 cells essentially the same as the wild-
type (Figure 5-3A). However, this mutant did not invade A431 cells well at all, ~40% as
efficiently as the wild-type (Figure 5-3B). Furthermore, the induction of expression of
N603 76 in adherent gonococci is dependent on RpoH (Table 5-1, Figure 5-1 and 5-2),
consistent with our prior observation that RpoH is necessary for optimal invasion of
A431 cells (84). In the gonococcal genome database (43), N60376 is annotated as

encoding a peptidyl-prolyl cis-trans isomerase B (also referred to as rotamase B, PPIase,

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PpiB). Rotamases facilitate the folding of proteins by catalyzing the cis-trans
isomerization of proline imidic peptide bonds of proline-containing peptides and were
first identified as cyclophilins in higher organisms, targets of the immunosuppressive
drug, cyclosporin (96, 388). Analysis of the many sequenced genomes shows that
rotarnases are present in nearly all organisms prokaryotic and eukaryotic, many having
several rotamases with slightly differing functions and characteristics (107). Rotamases
can be divided into three families: 1) the cyclophilin family, which are homologous to
cyclosporin binding proteins; 2) the FKBP ($506 binding protein) family, which bind
another immunosuppressive drug, FK506, and are not similar in sequence to cyclophilins
(143, 362); and 3) a recently identified family called parvulins, so named for their small
size (10.1 kDa) (313). Bacterial rotarnases are found in the cytoplasm, periplasm, and can
also be surface exposed (87, 148, 155).

Rotamases play a role in the proper folding of many bacterial proteins, including
virulence factors in bacterial pathogens. A Legionella pneumophila rotarnase mutant was
shown to invade the amoeba, Acanthamoeba castellanii lO-fold less well than wild-type
(343). Hermans and co-workers recently identified a surface exposed rotarnase, Ser, in
Streptococcus pneumoniae that was important for colonization, but not invasive disease
in a mouse model of infection (155). In addition, sIrA mutant pneumococci were
impaired in their ability to adhere to multiple cell lines in culture, clearly demonstrating a
role for Ser in pneumococcal-host cell interactions. 0f the nine rotarnases identified in
E. coli, five are cytoplasmic and four are periplasmic (87). A quadruple mutant in which
each periplasmic rotarnase gene was inactivated was viable, but reduced in its ability to

assemble pili (both type I and P) (178). As pili are adhesins in uropathogenic E. coli

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(UPEC) that mediate their attachment to cells in the urogenital tract, a defect in pilus
assembly would likely affect the ability of UPEC to interact with host cells in an
infection. Our observation that a null mutation in a gene encoding a putative rotarnase in
N. gonorrhoeae results in a defect in invasion is consistent with these observations that
rotarnases are important in pathogen-host interactions.

There are five putative rotarnases encoded in the gonococcal genome (43): one of
the parvulin family (N60766, ppiD), two of the cyclophilin family (N60376, ppiB;
N60544, ppiA), and two of the FKBP family (N60981, stD; N61225, mip). Leuzzi and
co-workers recently reported that N61225, which encodes a FKBP-type rotarnase, is
involved in persistance of gonococci in macrophages (213). Indeed, N61225 is annotated
as encoding a macrophage infectivity potentiator (MIP), a family of proteins found on the
surface of several intracellular pathogens that plays a role in the interactions between the
pathogen and phagocytic cells. Null mutations in N61225 are viable, and the mutants
appear not to be defective in production of pili or Opa proteins, and are bound by and
taken up by macrophages as well as the wild-type parent strain (213). However, N61225
mutants appeared to be more sensitive to killing by the macrophages, as fewer
intracellular bacteria were recovered from macrophages infected with the mutant
compared to the wild-type. This phenotype was specific for macrophages, as the mutant
behaved similarly to the wild-type in adhesion, invasion, and intracellular survival assays
using ME-180 (human cervical epithelial) cells. Little is known of the roles played by the
other gonococcal rotarnases.

N603 76 is the only rotarnase gene identified as regulated upon contact with

epithelial cells (84). DNA microarray experiments showed that expression of N60544,

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N60766, N60981, and N61225 were essentially the same in gonococci grown either in
the absence of or adherent to epithelial cells. Regulation of N603 76 upon contact with
epithelial cells is dependent on RpoH (Table 5-1, Figure 5-1 and 5-2), similar to
regulation of expression of rotarnase genes in E. coli, which varies for the many
rotarnases identified in this bacterium. Not surprisingly, most are controlled by stress
regulators such as CpxR and the heat-shock sigma factors, 0'32 (RpoH) and GE (RpoE)
(69,71,300)

In this work, we showed that a N603 76/ppiB mutation affects invasion of, but not
adherence to epithelial cells (Figure 5-3). Our previous studies showed that ppiB
expression is increased when gonococci are adherent to cells (84). These observations
suggest that gonococci may be responding to an increase in the synthesis of proline-
containing proteins that are important for the invasion process and need to increase the
production of PpiB to facilitate proper folding of these proteins. That PpiB is cytoplasmic
suggests that its target proteins are at least folded in the cytoplasm, if not remaining there.
The identity of these targets remains a mystery.

In summary, we have shown that adherence of gonococci to epithelial cells, a
critical early step in an infection, results in the modulation of expression of several genes
that are important for subsequent steps. In particular, two of these genes, NGO340 and
N603 76, encoding cysteine synthetase and rotarnase, respectively, are likely important in
the proper synthesis and assembly of proteins that are needed following the attachment
step. That the heat shock sigma factor, RpoH, in part mediates this regulation is
consistent with observations in other bacteria that this regulatory system responds to

environmental stresses, and indicating that signalling is occurring across the bacterial

161

membrane. Additionally, the identification of genes that are induced by host cell contact
independent of RpoH indicates that there are multiple regulators involved in this
response. Further characterization of this regulatory network will be key to a more in

depth understanding of the communication between gonococci and the human host.

162

Chapter 6

Summary and Future work

163

Neisseria gonorrhoeae infects more than 60 million people each year worldwide
(www.who.org), posing a significant public health threat. Treatment has heavily
depended on antibiotics; however, N. gonorrhoeae’s susceptibility to these antibiotics has
been declining. Thus, developing new strategies to prevent and treat gonococcal
infections is necessary in this modern society. Although much effort has been made to
understand the pathogenesis of this pathogen, our knowledge about this pathogen,
particularly the interaction between gonococcus and host cells is still limited.

In order to fully understand the pathogenesis of N. gonorrhoeae, this dissertation
investigated the interaction between this pathogen and epithelial cells using a tissue
culture model for infection. While N. gonorrhoeae typically establishes infection in the
subepithelial space of the urogenital mucosa, adherence to epithelial cells is the critical
first step in a gonococcal infection. The underlying hypothesis of this dissertation is that
this initial attachment to host cells transmits signals into the bacterium, resulting in the
modulation of gene expression to facilitate its subsequent survival and amplification in
the host.

The important tool used for the experiments in this dissertation is the N.
gonorrhoeae DNA microarray, which was designed and constructed in our lab in
collaboration with several research groups around the country (44, Appendix A). This
technique now allows us to measure gene expression at the genome-wide level.

Using the gonococcal DNA microarray and a cell culture model of infection, this
dissertation reports the changes of gene expression within N. gonorrhoeae in response to
host cell interaction. The genes differentially regulated upon host cell contact encode

products with a broad range of functions, including transcriptional regulation, bacterial

164

metabolism, protein transport, and cell division, etc. (Table 3-1 & 3-2). The shift in
expression of many metabolic genes suggests a general shift in the cellular metabolism in
response to host cell contact, likely to allow the pathogen to adapt to the different
environment inside the host cell. These observations are consistent with our hypothesis
and show that host cell contact does indeed serve as an important signal to induce
changes of gene expression inside the bacterium. Characterizing each single gene
differentially expressed would be intriguing, as it will provide more information on the
pathogenesis of N. gonorrhoeae.

In this work, RpoH was identified as an important regulator in gonococcal
infection, as depletion of RpoH from the bacteria reduces gonococcal ability to invade
epithelial cells. This phenotype is partially due to the failure of induction of an RpoH-
regulated gene, N603 76, as the N603 76 mutant was not able to invade epithelial cells as
efficiently as the wild-type. In addition to the identification of rpoH and N60376, two
genes, N61 684 and NGO340, which were greatly induced upon host cell contact, were
not dependent on RpoH for this regulation. Futhennore, NGO340 was identified as
important in both adhesion and invasion steps. These results indicate that RpoH is not the
only regulator involved in gonococci-host cell interactions. It is likely that a complex
regulatory network is engaged in coordinating gonococcal responses to host cell contact
(Chapter 5).

This dissertation has added some new information to our understanding of the
complex regulatory network of N. gonorrhoeae in gonococcal infections. However, the

more we learn about this pathogen, the more questions have emerged.

165

First, NGO340 and RpoH-regulated N603 76 were identified as important in
infection, as the N603 76 mutant was reduced in its ability to invade epithelial cells and
NGO340 mutant was compromised in both adhesion and invasion steps (Chapter 5).
According to the annotated gonococcal genome (43), N603 76 encodes a putative
peptidyl-prolyl cis-trans isomerase B (rotarnase B or PpiB) and NGO340 encodes a
putative 0-acetylserine(thiol)-lyase A (Cysteine synthase). However, these predictions
are solely based on sequence homologies to characterized genes in E. coli and other
bacteria. Their functions in N. gonohorroeae are still unclear. If N603 76 does encode a
peptidyl-prolyl cis-trans isomerase B (PpiB) in N. gonorrhoeae, what are the targets? The
same question also applies to NGO340. Is the gene product of NGO340 a cysteine
synthase? It is likely that some proteins important in a gonococcal infection require
cysteine to maintain their conformation. If so, what are these targets? As NG0376 and
NGO340 are likely involved in protein modification, techniques such as 2D SDS-PAGE
and mass spectroscopy may be used to identify their target proteins and to analyze the
locations of these proteins. Overall, characterizing the roles of these two genes in N.
gonohorroeae would be necessary to shed light on why they are required in gonococcal
pathogenesis.

Second, of the genes that were differentially regulated upon host cell contact, this
dissertation only characterized a few of genes that were induced. In fact, our results have
identified 37 genes as induced (Table 4-1) and 57 genes as respressed (Table 4-2) in
adherent gonococci in response to host cell contact (Chapter 5). The products of these
genes are predicted to be involved in a variety of cellular functions, transcriptional

regulation, bacterial metabolism, molecular transport and so on. Particularly, of the genes

166

down-regulated upon adherence, more than half of them encode proteins involved in
central housekeeping functions, such as glycolysis, amino acid metabolism, nitrogen
assimilation, respiration and cell division (Chapter 5), which again indicates a general
shift in cellular metabolism within gonococci in response to host cell contact. In addition,
some of them encode putative drug activity modulator and proteins important in DNA
replication, recombination or transformation competence. However, our knowledge about
roles of these gene products in gonococcal infection is still incomplete. What are their
roles in gonococcal infection? Are they required in the subsequent steps and why are they
required? It will be necessary and intriguing to further individually characterize the genes
up- and down regulated upon adherence, in particular, their roles in the subsequent
infection steps. Transposon mutagenesis, DNA arrays and cell culture models may be
used to characterize these genes.

Another aspect worthy of mention is that nearly half of genes differentially
regulated upon host contact encode hypothetical proteins, and some are Neisseria
specific. Those genes are intriguing to study, as they may contribute to the unique
characteristics of this pathogen and are potential drug targets or vaccine candidates.

In addition to characterizing each single gene, another critical question is how
these genes are co-regulated upon adherence to the host cell, that is, what are the
regulators involved in modulating gene expression? In this work, RpoH was identified as
a critical transcription factor regulating gene expression in response to adherence
(Chapter 5). However, many genes induced in adherent gonococci were not dependent on
RpoH for this regulation, such as N61 684, the gene induced most upon adherence.

Furthermore, an RpoH-independent gene (NGO340) was found to be important in both

167

L _11

 

 

adhesion and invasion steps. These results clearly indicate that regulators other than
RpoH contribute to the changes of gene expression upon host cell contact. Thus,
identifying and characterizing these regulators will be of great significance, as it will help
us to dissect the signaling pathways and piece together the complicated network involved
in a gonococcal infection. In the long run, it will help us to design and develop new
strategies to treat this disease.

However, the current techniques may not be sensitive enough to identify these
regulators, as typically in response to stimuli the regulators themselves may not
necessarily undergo dramatic changes to regulate subsequent cascades and thus may pose
challenges for detection and identification. In the gonococcal genome sequence database,
there are about 50 genes encoding putative transcription factors in N. gonorrhoeae
(Chapter 1, Table 1-1), some of which are likely involved in the regulation of gene
expression upon contact with host cells. Thus, transposon mutagenesis, cell culture
models of infection and microarray analysis can be used to define the regulon of each
individual regulator. Then, its regulon can be compared to the list of genes differentially
regulated upon host cell contact. Thus, genes present in both lists will suggest that these
genes are regulated by this specific regulator. In Chapter 5 we have used this method to
identify RpoH as the transcription factor required for the induction of N603 76 upon
adherence.

In this work, the analysis of gonococcal gene expression is limited to gonococci
adhered to A431 cells (110), which only represents a single tissue that the gonococcus
can colonize. Clinical studies show that gonococci can infect the mucous membrane of

the cervix, throat, rectum and conjunctiva. Occasionally, this bacterium can also infect

168

the joints and cause arthritis. It is likely that N. gonorrhoeae can evolve slightly distinct
responses upon attaching to different tissues. Thus, it is necessary to use other cell lines
to investigate the adhesion step in the future studies.

Finally, in this dissertation we only examine gene expression profiles of N.
gonorrhoeae at a 3-h post infection (p.i.). Gonococcal infection is known as a dynamic
process (249). Upon initial adhesion, gonococci undergo consecutive changes, forming
and dispersing microcolonies and the subsequent loss of pili from the surface.
Additionally, gonococci can trigger host cell responses rapidly after attachment (240).
For example the transportation of the transcription factor nuclear factor kappa B (NF -KB)
into the nucleus happens very quickly and NF-KB can be detected in the nucleus within
minutes of attachment of gonococci (240). Therefore, different genes are likely required
at different times of infection and gene expression is likely coordinated within the
bacterium. Thus, it will be important to examine gene expression in this bacterium during
the course of the adhesion process. Hence a time course study is necessary to perform
towards this aim. The finding will be crucial in disclosing why and how gonococcus

responses to host cell contact over time.

169

Appendix A

Verification of gonococcal DNA microarray

170

ABSTRACT

A gonococcal DNA microarray was developed in our lab in collaboration with
several research groups around the country. This DNA array represents 90% (2035/2250)
of the predicted open reading frames of N. gonorrhoeae, including 1977 ORFs from the
sequenced N. gonorrhoeae FA1090 genome and 58 of the gonococcal genetic island from
the N. gonorrhoeae strain MS11. In this work, validation experiments were performed to
compare gene expression in otherwise isogenic piliated (P+) and non-piliated (P-) strains
of N. gonorrhoeae strain M81 1. Consistent with previous research results, our results
showed that pilE, encoding the major pilus subunit was the most highly differentially
expressed gene and demonstrated that this array is suitable for genome wide

transcriptional analysis in gonococci.

171

INTRODUCTION

Neisseria gonorrhoeae (GC, gonococcus) is an obligate human pathogen, with no
known animal host, thus creating challenges for in vivo gonococcal studies. Research
characterizing gonococcal infections shows that the interaction between gonococcus and
its host involves many signaling pathways in both the pathogen and host cells (146, 249).
To thoroughly understand this interaction, it will be necessary to study gene expression
profiles of N. gonorrhoeae during infection. DNA microarray technology allows one to
examine changes of gene expression at a genome wide level in a single experiment. This
technique has been successfully used for several pathogens to demonstrate that the
modulation of gene expression is important and required for a pathogen to establish an
infection in its host (9, 88).

Taking advantage of the complete sequence of the N. gonorrhoeae FA1090
genome (43), we developed a PCR amplicon-based gonococcal DNA microarray spotted
onto glass slides (44). This DNA microarray represents more than 90% (1977/2035) of
the predicted ORFS from the sequenced genome of N. gonorrhoeae strain FA1090
(www.stdgen.lanl. gov) and 58 of the 61 ORFs of a gonococcal genetic island (GGI) of N.
gonorrhoeae strain MSll (78). To verify that this DNA microarray was suitable for
studying transcriptional profiles in N. gonorrhoeae, experiments were performed to
compare gene expression in otherwise isogenic piliated (P+) and non-piliated (P-) strains

of N. gonorrhoeae.

172

MATERIALS AND METHODS

RNA isolation and labeling. N. gonorrhoeae strains MS11 and MSll-307 (247) were
grown in GC broth with supplements (187) and 0.042% sodium bicarbonate in a
humidified 5% C02 environment . RNA was isolated from midlog phase cultures using
Trizol reagent (Invitrogen, Carlsbad, CA). RNA was analyzed quantitatively by
spectrophotometry, and for quality by agarose gel electrophoresis. RNA was labeled with
either Cy3 or Cy5 dyes using the CyScribe first-strand labeling kit (Amersham
Biosciences, Piscataway, NJ) and the cDNA probes were purified using the Qiagen PCR

cleanup kit (Qiagen, Valencia, CA). Probes were combined and concentrated to 15 p1

using Microcon® YM-30 centrifugal filtration units (Millipore, Billerica, Massachusetts).

Hybridizations and data analysis. DNA array slides were UV-crosslinked at 60 m]
energy, and then washed twice in 0.1% SDS for 3 min at room temperature to remove the
unbound DNA. The slide was then washed in H20 for 2 min at room temperature and
boiled in 1120 for 3 min to denature the double-stranded DNA on the slide. The slide was
plunged briefly into room temperature H20 to cool and dried by centrifugation for l min
at 800 xg in a 50 ml conical tube. Prehybridization was performed by incubating the slide
with 60 pl pre-warmed prehybridization buffer (5X SSC, 0.1% SDS, 1% BSA) in a
Corning hybridization chamber (Coming Life Sciences, Acton, MA) at 42°C for 45
minutes. The labelled probes were denatured by boiling for 5 min, along with 1 pl 1
mg/ml sheared, sonicated herring sperm DNA and chilled on ice. DNA was then mixed
with 15 p1 4X hybridization buffer (Amersham) and 30 p1 formamide, pipetted onto the

prehybridized array slide, assembled in the hybridization chamber, and hybridized at

173

42°C overnight. Following hybridization, the coverslip was removed and the slide was
washed as follows: once in 1X SSC; 0.2% SDS at 42°C, twice in 0.1X SSC; 0.2% SDS at
room temperature, once in 0.1X SSC. After the last wash, the slide was dried by
centrifugation and scanned using a GenePix 4000B scanner (Axon Instruments, Union
City, CA). Images were processed and analyzed using GenePix 4.0 software. Data

normalization and analysis were done as described (Chapter 3 and 4).

174

RESULTS AND DISCUSSION
Gene expression in P+ /- MSll strains

Gonococcal pili play very important roles in gonococcal pathogenesis and are the
major surface structure mediating the adhesion between gonococci and host cells (169,
380, 383). Without pili, gonococci are non-adherent and avirulent (311, 380). Gonococcal
pili are of the type IV class (287, 377) and the major subunit, pilin, is encoded by the pilE
expression loci on the genome.

In this work, we used a gonococcal DNA microarray to compare gene expression
between wild-type gonococcal strain M811 and a mutant, MS11-307, which cannot
produce type IV pili on its surface, due to deletions in the pilin expression loci, pilE
(247). Strains were grown in GC broth, total RNA isolated and labeled with Cy3 or Cy5
by reverse transcription. The labeled cDNAs were then hybridized to the gonococcal
DNA microarray slide, as described in Materials and Methods. A total of three
independent experiments (including one dye swap) were done and data normalization and
analysis was performed as described (47). Outliers were identified as those with
expression ratios (log2 transformed) greater than 2.5 standard deviations from the mean
and are listed in Tables A-1 and -2.

As expected, pilE was the most highly expressed gene in the wild-type and up-
regulated about 70-fold. Several pilS loci also had high expression ratios (5-15 fold) due
to homologous sequences they share with pilE (Table A-l). Additionally, bfrA and bfiB
were also significantly increased in expression in the wildtype strain, with ratios of 3.8
and 4.0 respectively. These two gene products comprise bacterioferritin, an important

iron storage protein for bacteria. However, whether pili have some role in iron-

175

uptake/storage in N. gonorrhoeae remains to be determined. The up-regulation of several
other genes in wild-type gonococci is also worth mentioning here. One such gene is ppiB,
which encodes a putative rotarnase, an enzyme involved in protein folding (96, 388). This
gene was induced 2.82-fold in wild-type relative to non-piliated gonococci. As piliated
gonococci express abundant pilin proteins, the induction of this gene in the piliated
gonococcal strain may suggest the requirement of this gene product in the folding of
pilin. Additionally, himA and recN are slightly induced in wild-type gonococci. HimA
encodes a subunit of II-IF (Integration Host Factor), a site-specific DNA-binding protein
that with roles in DNA recombination, transcription and DNA replication (453). It has
been shown that IHF is required for pilE transcription (453), which is consistent with our
observation of the induction of himA in wild-type gonococci. RecN encodes a DNA repair
protein RecN, required for both DNA repair and DNA transformation in N. gonorrhoeae
(454). Research has shown that DNA transformation is important in pilus antigenic
variation in N. gonorrhoeae by providing pilin sequences for recombination (3 54). Thus,
the induction of recN in this study may further indicate that RecN plays an active role in
the DNA transformation-related pilus antigenic variation.

Compared to the number of genes up-regulated in P+ gonococci, fewer genes were
identified as significantly up-regulated in P' bacteria (MS11-307, Table A-2). Moreover,
several genes were consistently induced ~1.5 fold in P' strains and most of them were
involved in transcription and translation. This may be due to the observation that unlike
P+ gonococci, in which pilus biosynthesis and assembly takes lots of energy, P’
gonococci don’t require such energy and usually grow faster. The increased grth is

consistent with the up-regulation of transcription and translation systems.

176

In this work we used the newly developed gonococcal DNA arrays to compare
gene expression in a wild-type strain and a pilE mutant. The deletion of the complete pilE
locus in this mutant completely abolishes pilE expression. As expected, our results are
highly consistent with this prediction, showing that pilE had the highest expression ratio
and was essentially expressed only in the wild—type. These results demonstrate that this
DNA array is suitable for analyzing gonococcal gene expression at a genome-wide level.

In summary, this first version of a gonococcal DNA array provides a feasible
means to analyze global gene expression in N. gonorrhoeae. With the advent of this DNA
array, it is now possible to examine gene expression profiles during the course of
gonococcal infection, which will substantially increase our knowledge about this
pathogen and the disease it causes and hence provide more opportunities to design new

strategies to prevent and treat it.

177

Table A-1. Genes expressedhigher in 1’+ (MSllA) than P’ (M811-307)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

gene comments ORF ID P+IP- ave Stdev
pilE pilin expression locus N62061 67.13 53.66
piIS @entpilin locus NG1512.1 15.09 8.67
pilS silent pilin locus N62062 12.04 6.91
island NG3002 6.58 3.55
bfrB bacterioferritin NGO795 4.04 0.77
bfrA bacterioferritin NGO794 3.79 0.82
island NG5001 3.44 0.30
prB endopeptidase Clp ATP-binding chain B NG1046 2.86 0.71
peptidyI-prolyl cis-trans isomerase B (rotarnase
ppiB BL NG0376 2.82 0.39
grpE HSP-7O cofactor; nucleotide exchange factor NG1422 2.77 0.50
island NG5041 2.36 0.79
pgi glucose-S-phosphate isomerase NG1668 2.32 0.80
conserved hypothetical protein NG0904 2.26 0.20
recN DNA repair protein RecN NG0318 2.18 0.36
int gene product NG0733 2.12 0.35
robable iron-sulphur protein N60906 2.11 0.25
conserved hypothetical protein NG0905 2.11 0.38
hypothetical protein NG0634 2.04 0.64
ggIC ilin glycosylation protein NGOO84 2.02 0.14
thA cytochrome c peroxidase NG1769 2.02 0.50
heat shock protein (probable membrane-bound
hth zinc metallopeptidase) NGO399 1.97 0.71
m hosphgglummutase NG0375 1 .95 0.42
hypothetical protein N60635 1.92 0.55
hypothetical protein NG1633 1 .90 0.33
himA IHF alpha subunit NG0305 1.89 0.57
groEL heat shock protein, 60 Kd subunit NG2095 1.85 0.42
dnaJ HSP-40/chaperone DnaJ NG1901 1.80 0.21
IS1016 transposase fragment NGO186.1 1 .78 0.12

 

 

 

 

178

Table A-2. Genes expressed higher in P' (M811-307) than P+ (MSllA)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

gene comments [ORF ID P-I-IP- ave Stdev
type III restriction/modification system [
mod modification methylase NG0641 0.36 0.09
lip Ieucine-responsive rgqulatory protein ING1294 0.42 0.11
mdaB drgq activity modulator B NG1473 0.51 0.16
rp/U 50$ ribosomal protein L21 (Rpltfl NG1676 0.56 0.06
rpID 50$ ribosomal protein L4 NG1837 0.57 0.05
DNA-directed RNA polymerase alpha chain
LpoA (RpoA) iNG1818 0.57 0.22
secY preprotein translocase SecY NG1822 0.59 0.19
galW 508 ribosomal protein L23 NG1836 0.60 0.04
ipIT 50$ ribosomal protein L20 NG0298 0.60 0.21
rplA 50$ ribosomal protein L1 lNo1854 0.60 0.23
IplF 50$ ribosomal protein L6 ING1625 0.61 0.19
rpie 50$ ribosomal protein L2 lNG1835 0.61 0.18
rpsM 30S ribosomal protein S13 NG1821 0.62 0.26
rpIQ 508 ribosomal protein L17 NG1817 0.62 0.19
rpIO 50$ ribosomal protein L15 lNG1823 0.63 0.23
QsH 30$ ribosomal protein 88 NG1826 0.64 0.20
boIA BoIA/YrbA family protein NG1657 0.64 0.21
R814 308 ribosomal protein S14 NG1826.1 0.64 0.27
fusA translation elongation factor EF-G ING1843 0.65 0.12
rhlE TP-dependent RNA helicase ING0650 0.65 0.14
rpm 508 ribosomal protein L11 [NG1855 0.65 0.15
rplL 50$ ribosomal protein L7/L12 IN61852 0.66 0.16
rpmA 50$ ribosomal protein L27 N61677 0.68 0.19
MN 508 ribosomal protein L14 ]NG1829 0.68 0.21
rpiE 5os ribosomal protein L5 lNG1827 0.69 0.23
periplasmic iron-binding protein (major iron-
fpr regulated protein), ABC solute binding protein iNG0217 0.77 0.52

 

179

REFERENCES:

1. 2004. Centers for Disease Control and Prevention. Sexually transmitted disease
surveillance 2003 supplement: Gonococcal Isolate Surveillance Project (GISP) annual
report -2003. Atlanta: US Department of Health and Human Services.

2. 2002. Increases in fluoroquinolone-resistant Neisseria gonorrhoeae --- Hawaii
and California, 2001. Morbidity and Mortality Weekly Report 51 : 1041-1044.

3. STADEN DNA sequence analysis package [<http://sta_den.sourcefgge.nefl.

4. 2002. Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO
Western Pacific Region, 2001. World Health Organization. Commun Dis Intell
26:541-545.

5. 2003. Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO
Western Pacific Region, 2002. Commun Dis Intell 27:488-491.

6. World Health Organization [<httfl/www.who.org>l.

7. Addinall, S. G., Cao, C., and Lutkenhaus, J. 1997. FtsN, a late recruit to the
septum in Escherichia coli. Mol Microbiol 25:303-309.

8. Aertsen, A., Vanoirbeek, K., De Spiegeleer, P., Sermon, J., Hauben, K.,
Farewell, A., Nystr, T., and Michiels, C. W. 2004. Heat shock protein-mediated

resistance to high hydrostatic pressure in Escherichia coli. Appl. Environ. Microbiol.
70:2660-2666.

9. Allan, E., Clayton, C. L., McLaren, A., Wallace, D. M., and Wren, B. W.
2001. Characterization of the low-pH responses of Helicobacter pylori using genomic
DNA arrays. Microbiology 147:2285-2292.

10. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller,
W., and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs. Nucleic Acids Res 25:3389-3402.

1 1. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller,
W., and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs. Nucl Acids Res 25:3389 - 3402.

12. Andersson, H., Bakker, E., and von Heijne, G. 1992. Different positively
charged amino acids have similar effects on the topology of a polytopic
transmembrane protein in Escherichia coli. J Biol Chem 267: 1491-1495.

13. Aug, 8., Lee, C. Z., Peck, K., Sindici, M., Matrubutham, U., Gleeson, M. A.,
and Wang, J. T. 2001. Acid-induced gene expression in Helicobacter pylori: study in
genomic scale by microarray. Infect Immun 69:1679-1686.

180

l4. Apicella, M. A., Ketterer, M., Lee, F. K., Zhou, D., Rice, P. A., and Blake, M.
S. 1996. The pathogenesis of gonococcal urethritis in men: confocal and
immunoelectron microscopic analysis of urethral exudates from men infected with

Neisseria gonorrhoeae. J Infect Dis 173:636-646.

15. Apicella, M. A., and Mandrell, R. E. 1989. Molecular mimicry as a factor in the
pathogenesis of human neisserial infections: in vitro and in vivo modification of the
lipooligosaccharide of Neisseria gonorrhoeae by N-acetylneuraminic acid. Pediatr

Infect Dis J 8:901-902.

16. Apicella, M. A., Shero, M., Jarvis, G. A., Griffiss, J. M., Mandrell, R. E., and
Schneider, H. 1987. Phenotypic variation in epitope expression of the Neisseria
gonorrhoeae lipooligosaccharide. Infect Immun 55: 1 755-1 7 61.

17. Archibald, F. S., and Duong, M. N. 1986. Superoxide dismutase and oxygen
toxicity defenses in the genus Neisseria. Infect Immun 51:631-641.

18. Arsene, F., Tomoyasu, T., and Bukau, B. 2000. The heat shock response of
Escherichia coli. Int J Food Microbiol 55:3-9.

19. Arvidson, C. G., Kirkpatrick, R., Witkamp, M. T., Larson, J. A., Schipper,
C. A., Waldbeser, L. S., O'Gaora, P., Cooper, M., and So, M. 1999. Neisseria
gonorrhoeae mutants altered in toxicity to human fallopian tubes and molecular
characterization of the genetic locus involved. Infect Immun 67:643-652.

20. Ayala, B. P., Vasquez, B., Clary, S., Tainer, J. A., Rodland, K., and So, M.
2001. The pilus-induced Ca2+ flux triggers lysosome exocytosis and increases the
amount of Lampl accessible to Neisseria IgAl protease. Cell Microbiol 3:265-27 5.

21. Ayala, B. P., Vasquez, B., Wetzler, L. M., and So, M. 2002. Neisseria
gonorrhoeae porin P1.B induces endosome exocytosis and a redistribution of Larnpl
to the plasma membrane. Infect. Immun. 70:5965-5971.

22. Ayala, P., Wilbur, J. S., Wetzler, L. M., Tainer, J. A., Snyder, A., and So, M.
2005. The pilus and porin of Neisseria gonorrhoeae cooperatively induce Ca”
transients in infected epithelial cells. Cell Microbiol 7: 1736-1748.

23. Bal, C. 1999. Increasing antimicrobial resistance in STDs and the need for
surveillance: Neisseria gonorrhoeae as a model. FEMS Immun. and Med. Microbiol.
24:447-453.

24. Banerjee, A., Wang, R., Uljon, S. N., Rice, P. A., Gotschlich, E. C., and Stein,
D. C. 1998. Identification of the gene (lgt6) encoding the lipooligosaccharide beta

chain synthesizing glucosyl transferase from Neisseria gonorrhoeae. Proc Natl Acad
Sci USA 95:10872-10877.

181

25. Batchelor, E., and Goulian, M. 2003. Robustness and the cycle of
phosphorylation and dephosphorylation in a two-component regulatory system. Proc
Natl Acad Sci USA 100:691-696.

26. Beck, S. C., and Meyer, T. F. 2000. IgAl protease from Neisseria gonorrhoeae
inhibits TNFalpha-mediated apoptosis of human monocytic cells. FEBS Lett 472:287-
292.

27. Benevento, W. J., Murray, P., Reed, C. A., and Pepose, J. S. 1990. The
sensitivity of Neisseria gonorrhoeae, Chlamydia trachomatis, and herpes simplex type
II to disinfection with povidone-iodine. Am J Ophthalrnol 109:329-333.

28. Bengoechea, J. A., Zhang, L., Toivanen, P., and Skurnik, M. 2002. Regulatory
network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica
includes cell envelope-dependent signals. Mol Microbiol 44:1045-1062.

29. Berish, S. A., Subbarao, S., Chen, C. Y., Trees, D. L., and Morse, S. A. 1993.
Identification and cloning of a fur homolog from Neisseria gonorrhoeae. Infect
Immun 61 :4599-4606.

30. Bernatchez, S., Francis, F. M., Salimnia, H., Beveridge, T. J., Li, H., and
Dillon, J. A. 2000. Genomic, transcriptional and phenotypic analysis of ftsE and ftsX
of Neisseria gonorrhoeae. DNA Res 7:75-81.

31. Bhat, K. S., Gibbs, C. P., Barrera, 0., Morrison, S. G., Jahnig, F., Stern, A.,
Kupsch, E. M., Meyer, T. F., and Swanson, J. 1991. The opacity proteins of

Neisseria gonorrhoeae strain MSll are encoded by a family of 11 complete genes.
Mol Microbiol 5:1889-1901.

32. Bianchi, A. A., and Baneyx, F. 1999. Hyperosmotic shock induces the 0'32 and
6‘5 stress regulons of Escherichia coli. Mol Microbiol 34:1029-1038.

33. Binnicker, M. J., Williams, R. D., and Apicella, M. A. 2004. Gonococcal porin
IB activates NF-kappaB in human urethral epithelium and increases the expression of
host antiapoptotic factors. Infect Immun 72:6408 - 6417.

34. Binnicker, M. J., Williams, R. D., and Apicella, M. A. 2003. Infection of
human urethral epithelium with Neisseria gonorrhoeae elicits an upregulation of host

anti-apOptotic factors and protects cells from staurosporine-induced apoptosis. Cell
Microbiol 5:549-560.

35. Biswas, G. D., and Sparling, P. F. 1995. Characterization of lpr, the structural
gene for a lactoferrin receptor in Neisseria gonorrhoeae. Infect Immun 63:2958-2967.

36. Blake, M. S., Gotschlich, E. C., and Swanson, J. 1981. Effects of proteolytic
enzymes on the outer membrane proteins of Neisseria gonorrhoeae. Infect Immun
33:212-222.

182

37. Blake, M. S., Wetzler, L. M., Gotschlich, E. C., and Rice, P. A. 1989.
Developing a gonococcal protein I vaccine. Adv Exp Med Biol 251 :315-327.

38. Blake, M. S., Wetzler, L. M., Gotschlich, E. C., and Rice, P. A. 1989. Protein
111: structure, function, and genetics. Clin Microbiol Rev 2 Suppl:S60-63.

39. Blattner, F. R., Plunkett, G., 3rd, Bloch, C. A., Perna, N. T., Burland, V.,
Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor,
J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B., and
Shae, Y. 1997. The complete genome sequence of Escherichia coli K-12. Science
277:1453-1474.

40. Bonnah, R. A., Lee, S. W., Vasquez, B. L., Enns, C. A., and So, M. 2000.
Alteration of epithelial cell transferrin—iron homeostasis by Neisseria meningitidis and
Neisseria gonorrhoeae. Cell Microbiol 2:207-21 8.

41. Boslego, J. W., Tramont, E. C., Chung, R. C., McChesney, D. G., Ciak, J.,
Sadoff, J. C., Piziak, M. V., Brown, J. D., Brinton, C. C., Jr., Wood, S. W., and et

al. 1991. Efficacy trial of a parenteral gonococcal pilus vaccine in men. Vaccine
9:154—162.

42. Boyle-Vavra, S., and Seifert, H. S. 1996. Uptake-sequence-independent DNA
transformation exists in Neisseria gonorrhoeae. Microbiology 142:2839-2845.

43. Brettin, T. 2002. Neisseria gonorrhoeae annotated genome sequence database.

44. Brettin, T., Altherr, M. R., Du, Y., Mason, R. M., Friedrich, A., Potter, L.,
Langford, C., Keller, T. J., Jens, J., Howie, H., Weyand, N. J., Clary, S.,
Prichard, K., Wachocki, S., Sodergren, E., Dillard, J. P., Weinstock, G., So, M.,
and Arvidson, C. G. 2005. Expression capable library for studies of Neisseria
gonorrhoeae, version 1.0. BMC Microbiol 5:50.

45. Brevet, J. 1974. Direct assay for sigma factor activity and demonstration of the
loss of this activity during sporulation in Bacillus subtilis. Mol Gen Genet 128:223-
221.

46. Brinton, C., Bryan, J., Dillon, J., Guerina, N., Jacobson, L., Labik, A., Lee,
S., Levine, A., S., L., McMichael, J., Polen, S., Rogers, K., To, A., and To, S. 1978.
Uses of pili in gonorrhea control: role of bacterial pili in disease. Purification and
properties of gonococcal pili, and progress in the development of gonococcal pilus
vaccine for gonorrhea, p. 155-177. In G. Brooks, E. Gotschlich, K. Hohnes, W.
Sawyer, and F. Young (ed.), Irnmunobiology of Neisseria gonorrhoeae. American
Society for Microbiology, Washington, DC.

47. Britton, R. A., Eichenberger, P., Gonzalez-Pastor, J. E., Fawcett, P., Monson,
R., Losick, R., and Grossman, A. D. 2002. Genome-wide analysis of the stationary-

phase sigma factor (sigma-H) regulon of Bacillus subtilis. J. Bacteriol. 184:4881-
4890.

183

48. Brodeur, B. R., Johnson, W. M., Johnson, K. G., and Diena, B. B. 1977. In
vitro interaction of Neisseria gonorrhoeae type 1 and type 4 with tissue culture cells.
Infect Immun 15:560-567.

49. Brown, S., and Fournier, M. J. 1984. The 4.5 S RNA gene of Escherichia coli is
essential for cell growth. J Mol Biol 178:533-550.

50. Buddelmeijer, N., Judson, N., Boyd, D., Mekalanos, J. J., and Beckwith, J.
2002. ngQ, a cell division protein in Escherichia coli and Vibrio cholerae, localizes
in codependent fashion with FtsL to the division site. Proc Natl Acad Sci USA
99:6316-6321.

51. Buttner, M. J. 1989. RNA polymerase heterogeneity in Streptomyces coelicolor
A3(2). Mol Microbiol 3:1653-1659.

52. Bygdeman, S. M., Mardh, P. A., and Sandstrom, E. G. 1984. Susceptibility of
Neisseria gonorrhoeae to rifampicin and thiamphenicol: correlation with protein I
antigenic determinants. Sex Transm Dis 11:366-370.

53. Byrne, C. R., Monroe, R. S., Ward, K. A., and Kredich, N. M. 1988. DNA
sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and
linkage of the cysK regions to ptsH. J Bacteriol 170:3150-3157.

54. Cannon, J. G., and Sparling, P. F. 1984. The genetics of the gonococcus. Annu
Rev Microbiol 38:111-133.

55. Carbonetti, N. H., Simnad, V. 1., Seifert, H. S., So, M., and Sparling, P. F.
1988. Genetics of protein I of Neisseria gonorrhoeae: construction of hybrid porins.
Proc Natl Acad Sci USA 85:6841-6845.

56. Carifo, K., and Catlin, W. 1973. Neisseria gonorrhoeae auxotyping:
Differentiation of clinical isolates based on growth responses on chemically defined
media. Appl Microbiol 26:223-230.

57. Chang, C., and Stewart, R. C. 1998. The two-component system. Regulation of
diverse signaling pathways in prokaryotes and eukaryotes. Plant Physiol 117:723-731.

58. Chen, A., Boulton, I. C., Pongoski, J., Cochrane, A., and Gray-Owen, S. D.
2003. Induction of HIV -1 long terminal repeat-mediated transcription by Neisseria
gonorrhoeae. AIDS 17:625-628.

59. Chen, C. J., Sparling, P. F., Lewis, L. A., Dyer, D. W., and Elkins, C. 1996.
Identification and purification of a hemoglobin-binding outer membrane protein from
Neisseria gonorrhoeae. Infect Immun 64:5008-5014.

60. Chen, J. C., Bavoil, P., and Clark, V. L. 1991. Enhancement of the invasive
ability of Neisseria gonorrhoeae by contact with HecIB, an adenocarcinoma
endometrial cell line. Mol Microbiol 5:1531-1538.

184

61. Chen, T., Belland, R. J., Wilson, J., and Swanson, J. 1995. Adherence of pilus-
Opa+ gonococci to epithelial cells in vitro involves heparan sulfate. J Exp Med
182:511-517.

62. Chen, T., Grunert, F., Medina-Marine, A., and Gotschlich, E. C. 1997.
Several carcinoembryonic antigens (CD66) serve as receptors for gonococcal opacity
proteins. J Exp Med 185:1557 - 1564.

63. Collins, R. F., Davidsen, L., Derrick, J. P., Ford, R. C., and Tonjum, T. 2001.
Analysis of the PilQ secretin from Neisseria meningitidis by transmission electron
microscopy reveals a dodecameric quaternary structure. J Bacteriol 183:3825-3 832.

64. Cornelissen, C. N., and Sparling, P. F. 1996. Binding and surface exposure
characteristics of the gonococcal transferrin receptor are dependent on both
transferrin-binding proteins. J Bacteriol 178:1437-1444.

65. Cotter, P. A., and Miller, J. F. 1998. In vivo and ex vivo regulation of bacterial
virulence gene expression. Curr Opin Microbiol 1:17 - 26.

66. Cozzone, A. J. 1998. Regulation of acetate metabolism by protein
phosphorylation in enteric bacteria. Annu Rev Microbiol 52: 127-164.

67. Dagberg, B., and Uhlin, B. E. 1992. Regulation of virulence-associated plasmid
genes in enteroinvasive Escherichia coli. J Bacteriol 174:7606-7612.

68. Danaher, R. J., Levin, J. C., Arking, D., Burch, C. L., Sandlin, R., and Stein,
D. C. 1995. Genetic basis of Neisseria gonorrhoeae lipooligosaccharide antigenic
variation. J Bacteriol 177:7275-7279.

69. Danese, P. N., and Silhavy, T. J. 1997. The GE and the Cpx signal transduction
systems control the synthesis of periplasmic protein-folding enzymes in Escheriechia
coli. Genes Dev 11:1183-1193.

70. Dartigalongue, C., Missiakas, D., and Raina, S. 2001. Characterization of the
Escherichia coli sigma E regulon. J Biol Chem 276:20866-20875.

71. Dartigalongue, C., and Raina, S. 1998. A new heat-shock gene, ppiD, encodes a
peptidyl-prolyl isomerase required for folding of outer membrane proteins in
Escherichia coli. EMBO J 17:3968-3980.

72. David, G. 1993. Integral membrane heparan sulfate proteoglycans. Faseb J
7:1023-1030.

73. de Gier, J. W., Mansournia, P., Valent, Q. A., Phillips, G. J., Luirink, J., and
von Heijne, G. 1996. Assembly of a cytoplasmic membrane protein in Escherichia
coli is dependent on the signal recognition particle. F EBS Lett 399:307-309.

185

74. de la Paz, H., Cooke, S. J., and Heckels, J. E. 1995. Effect of sialylation of
lipopolysaccharide of Neisseria gonorrhoeae on recognition and complement-
mediated killing by monoclonal antibodies directed against different outer-membrane
antigens. Microbiology 141 (Pt 4):913-920.

75. De Las Penas, A., Connolly, L., and Gross, C. 1997. Sigma E is an essential
sigma factor in Escherichia coli. J. Bacteriol. 179:6862-6864.

76. Delahay, R. M., Robertson, B. D., Balthazar, J. T., Shafer, W. M., and Ison,
C. A. 1997. Involvement of the gonococcal MtrE protein in the resistance of Neisseria
gonorrhoeae to toxic hydrophobic agents. Microbiology 143:2127-2133.

77. Dempsey, J. A., Litaker, W., Madhure, A., Snodgrass, T. L., and Cannon, J.
G. 1991. Physical map of the chromosome of Neisseria gonorrhoeae FA1090 with
locations of genetic markers, including opa and pi] genes. J Bacteriol 173:5476-5486.

78. Dillard, J. P., and Seifert, H. S. 2001. A variable genetic island specific for
Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is
found more often in disseminated infection isolates. Mol Microbiol 41 :263-277.

79. Dorman, C. J., and Porter, M. E. 1998. The Shigella flexneri virulence gene
regulatory cascade: A paradigm of bacterial control mechanisms. Mol Microbiol
29:67 7 - 684.

80. Drake, S. L., and Koomey, M. 1995. The product of the pilQ gene is essential
for the biogenesis of type IV pili in Neisseria gonorrhoeae. Mol Microbiol 18:975-
986.

81. Drake, S. L., Sandstedt, S. A., and Koomey, M. 1997. PilP, a pilus biogenesis
lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high-molecular-
mass multimer. Mol Microbi0123:657-668.

82. Drazek, E. S., Stein, D. C., and Deal, C. D. 1995. A mutation in the Neisseria
gonorrhoeae tfaD homolog results in altered lipooligosaccharide expression. J
Bacteriol 177:2321-2327.

83. Du, Y., and Arvidson, C. G. 2003. Identification of ZipA, a signal recognition
particle-dependent protein from Neisseria gonorrhoeae. J. Bacteriol. 185:2122-2130.

84. Du, Y., Lenz, J., and Arvidson, C. G. 2005. Global gene expression and the role
of sigma factors in Neisseria gonorrhoeae in interactions with epithelial cells. Infect.
Immun. 73:4834-4845.

85. Ducey, T. F., Carson, M. B., Orvis, J., Stintzi, A. P., and Dyer, D. W. 2005.
Identification of the iron-responsive genes of Neisseria gonorrhoeae by microarray
analysis in defined medium. J Bacteriol 187 :4865-4874.

186

 

86. Duensing, T. D., and van Putten, J. P. 1997. Vitronectin mediates
internalization of Neisseria gonorrhoeae by Chinese hamster ovary cells. Infect
Immun 65:964-970.

87. Duguay, A. R., and Silhavy, T. J. 2004. Quality control in the bacterial
periplasm. Biochim Biophys Acta 1694:121-134.

88. Eckmann, L., Smith, J. R., Housley, M. P., Dwinell, M. B., and Kagnoff, M.
F. 2000. Analysis by high density cDNA arrays of altered gene expression in human

intestinal epithelial cells in response to infection with the invasive enteric bacteria
Salmonella. J Biol Chem 275:14084-14094.

89. Edwards, J. L., and Apicella, M. A. 2005. I-domain-containing integrins serve
as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells. Cell
Microbiol 7:1197-1211.

90. Edwards, J. L., and Apicella, M. A. 2002. The role of lipooligosaccharide in
Neisseria gonorrhoeae pathogenesis of cervical epithelia: lipid A serves as a C3
acceptor molecule. Cell Microbiol 4:585-598.

91. Engelke, D. R., Krikos, A., Bruck, M. E., and Ginsburg, D. 1990. Purification
of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Anal Biochem
191:396-400.

92. Falke, J. J., Bass, R. 8., Butler, S. L., Chervitz, S. A., and Danielson, M. A.
1997. The two-component signaling pathway of bacterial chemotaxis: a molecular

view of signal transduction by receptors, kinases, and adaptation enzymes. Annu Rev
Cell Dev Biol 13:457-512.

93. Farrell, D. J. 1999. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR
using cch nested PCR and 16S rRNA PCR. J Clin Microbiol 37 :386-390.

94. Fayet, 0., Ziegelhoffer, T., and Georgopoulos, C. 1989. The groES and groEL
heat shock gene products of Escherichia coli are essential for bacterial grth at all
temperatures. J Bacteriol 171 : 1379-1385.

95. Feng, J., Atkinson, M. R., McCleary, W., Stock, J. B., Wanner, B. L., and
Ninfa, A. J. 1992. Role of phosphorylated metabolic intermediates in the regulation of
glutamine synthetase synthesis in Escherichia coli. J Bacteriol 174:6061-6070.

96. Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., and Schmid, F.
X. 1989. Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical
proteins. Nature 337 :476-478.

97. Fischer, S. H., and Rest, R. F. 1988. Gonococci possessing only certain P.II

outer membrane proteins interact with human neutrophils. Infect Immun 56:1574 -
1579.

187

98. Fletcher, J. D., Stratton, J., Chandler, C. S., and Sparling, P. F. 1983.
Epidemiologic notes and reports penicillin-resistant gonorrhea -- North Carolina.
MMWR 32:273 - 275.

99. Forest, K. T., Bernstein, S. L., Getzoff, E. D., So, M., Tribbick, G., Geysen, H.
M., Deal, C. D., and Tainer, J. A. 1996. Assembly and antigenicity of the Neisseria
gonorrhoeae pilus mapped with antibodies. Infect Immun 64:644-652.

100. Fox, K. K., Knapp, J. S., Holmes, K. K., Hook, E. W. r., Judson, F. N.,
Thompson, S. E., Washington, J. A., and Whittington, W. L. 1997. Antimicrobial
resistance in Neisseria gonorrhoeae in the United States, 1988-1994: the emergence of
decreased susceptibility to the fluoroquinolones. J Infect Dis 175:1396-1403.

101. Francis, F., Ramirez-Arcos, S., Salimnia, H., Victor, C., and Dillon, J. R.
2000. Organization and transcription of the division cell wall (dcw) cluster in
Neisseria gonorrhoeae. Gene 251 : 141-15 1.

102. Frasz, C., and Arvidson, C. G. 2003. Role for both DNA and RNA in GTP
hydrolysis by the Neisseria gonorrhoeae signal recognition particle receptor. J
Bacteriol 185:801-808.

103. Freissler, E., Meyer auf der Heyde, A., David, G., Meyer, T. F., and Dehio, C.
2000. Syndecan-l and syndecan-4 can mediate the invasion of OpaHSPG-expressing
Neisseria gonorrhoeae into epithelial cells. Cell Microbiol 2:69-82.

104. Freitag, N. E., Seifert, H. S., and Koomey, M. 1995. Characterization of the
pilF-pilD pilus-assembly locus of Neisseria gonorrhoeae. Mol Microbiol 16:575-586.

105. Fujita, M., Amemura, A., and Aramaki, H. 1988. Transcription of the groESL
operon in Pseudomonas aeruginosa PAOl. FEMS Microbiol Lett 163:237-242.

106. Fussenegger, M., Rudel, T., Barten, R., Ryll, R., and Meyer, T. F. 1997.
Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae--a
review. Gene 192:125-134.

107. Galat, A. 1999. Variations of sequences and amino acid compositions of proteins
that sustain their biological functions: An analysis of the cyclophilin family of
proteins. Arch Biochem Biophys. 371 : 149-162.

108. Gallegos, M. T., Schleif, R., Bairoch, A., Hofmann, K., and Ramos, J. L.
1997. Arac/XylS family of transcriptional regulators. Microbiol Mol Biol Rev 61:393-
410.

109. Ghigo, J. M., Weiss, D. S., Chen, J. C., Yarrow, J. C., and Beckwith, J. 1999.
Localization of FtsL to the Escherichia coli septal ring. Mol Microbi0131:725-737.

110. Giard, D. J., Aaronson, S. A., Todaro, G. J., Arnstein, P., Kersey, J. H.,
Dosik, H., and Parks, W. P. 1973. In vitro cultivation of human tumors:

188

establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst
51:1417-1423.

111. Giardina, P. C., Williams, R., Lubaroff, D., and Apicella, M. A. 1998.
Neisseria gonorrhoeae induces focal polymerization of actin in primary human
urethral epithelium. Infect Immun 66:3416-3419.

112. Gibson, B. W., Webb, J. W., Yamasaki, R., Fisher, S. J., Burlingame, A. L.,
Mandrell, R. E., Schneider, H., and Griffiss, J. M. 1989. Structure and
heterogeneity of the oligosaccharides from the lipopolysaccharides of a pyocin-
resistant Neisseria gonorrhoeae. Proc Natl Acad Sci USA 86:17 -21.

113. Gill, D. R., and Salmond, G. P. 1990. The identification of the Escherichia coli
ftsY gene product: an unusual protein. Mol Microbiol 4:575-583.

114. Gomez-Duarte, 0. G., Dehio, M., Guzman, C. A., Chhatwal, G. S., Dehio, C.,
and Meyer, T. F. 1997. Binding of Vitronectin to opa-expressing Neisseria
gonorrhoeae mediates invasion of HeLa cells. Infect Immun 65:3857-3866.

115. Goodman, S. D., and Scocca, J. J. 1988. Identification and arrangement of the

DNA sequence recognized in specific transformation of Neisseria gonorrhoeae. Proc
Natl Acad Sci USA 85:6982-6986.

116. Gorby, G., Simon, D., and Rest, R. F. 1994. Escherichia coli that express
Neisseria gonorrhoeae opacity-associated proteins attach to and invade human
fallopian tube epithelium. Ann NY Acad Sci 730:286-289.

117. Goryshin, I. Y., and Reznikoff, W. S. 1998. Tn5 in vitro transposition. J Biol
Chem 273:7367-7374.

118. Gotschlich, E. C. 1994. Genetic locus for the biosynthesis of the variable portion
of Neisseria gonorrhoeae lipooligosaccharide. J Exp Med 180:2181-2190.

119. Gotschlich, E. C., Blake, M. S., Lytton, E. J., and Seiff, M. 1987. Gonococcal
protein 111: Purification and chemical characterization of the protein, and the DNA
sequence of the structural gene. Antonie Van Leeuwenhoek 53:455-459.

120. Gotschlich, E. C., Seiff, M., and Blake, M. S. 1987. The DNA sequence of the
structural gene of gonococcal protein 111 and the flanking region containing a

repetitive sequence. Homology of protein 111 with enterobacterial OmpA proteins. J
Exp Med 165:471-482.

121. Gotschlich, E. C., Seiff, M. E., Blake, M. S., and Koomey, M. 1987. Porin
protein of Neisseria gonorrhoeae: cloning and gene structure. Proc Natl Acad Sci
USA 84:8135-8139.

189

122. Grassme, H., Gulbins, E., Brenner, B., Ferlinz, K., Sandhoff, K., Harzer, K.,
Lang, F., and Meyer, T. F. 1997. Acidic sphingomyelinase mediates entry of N.
gonorrhoeae into nonphagocytic cells. Cell 91 :605-615.

123. Grassme, H. U., Ireland, R. M., and van Putten, J. P. 1996. Gonococcal
opacity protein promotes bacterial entry-associated rearrangements of the epithelial
cell actin cytoskeleton. Infect Immun 64: 1621-1630.

124. Gray-Owen, S. D., Dehio, C., Haude, A., Grunert, F., and Meyer, T. F. 1997.
CD66 carcinoembryonic antigens mediate interactions between Opa-expressing

Neisseria gonorrhoeae and human polymorphonuclear phagocytes. EMBO J 16:3435-
3445.

125. Gray-Owen, S. D., Lorenzen, D. R., Haude, A., Meyer, T. F., and Dehio, C.
1997. Differential Opa specificities for CD66 receptors influence tissue interactions
and cellular response to Neisseria gonorrhoeae. Mol Microbiol 26:971-980.

126. Gregg, C. R., Melly, M. A., Hellerqvist, C. G., Coniglio, J. G., and McGee, Z.
A. 1981. Toxic activity of purified lipopolysaccharide of Neisseria gonorrhoeae for
human fallopian tube mucosa. J Infect Dis 143:432-439.

127. Griffiss, J. M., Schneider, H., Mandrell, R. E., Jarvis, G. A., Kim, J. J.,
Gibson, B., and Apicella, M. A. 1987. The immunochemistry of neisserial LOS.
Antonie Van Leeuwenhoek 53:501-507.

128. Griffiss, J. M., Schneider, H., Mandrell, R. E., Yamasaki, R., Jarvis, G. A.,
Kim, J. J., Gibson, B. W., Hamadeh, R., and Apicella, M. A. 1988.

Lipooligosaccharides: the principal glycolipids of the neisserial outer membrane. Rev
Infect Dis 10 Suppl 2:S287-295.

129. Groisman, E. A. 2001. The pleiotropic two-component regulatory system PhoP-
Pth. J Bacteriol 183:1835-1842.

130. Groisman, E. A., Chiao, E., Lipps, C. J., and Heffron, F. 1989. Salmonella
typhimurium phoP virulence gene is a transcriptional regulator. Proc Natl Acad Sci
USA 86:7077-7081.

131. Gross, C. A. 1996. Function and regulation of the heat shock proteins, p. 1382-
1399. In F. C. Neidhart, R. Curtiss III, I. L. Ingraham, E. C. C. Lin, K. B. Low, B.
Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.),
Escherichia coli and Salmonella. Cellular and Molecular Biology, vol. 1. ASM Press,
Washington, DC.

132. Gross, C. A., Lonetto, M., and Losick, R. 1992. Bacterial sigma factors. In S. L.

McKnight and K. R. Yamamoto (ed.), Transcriptional Regulation. Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY.

190

133. Gueneau de Novoa, P., and Williams, K. P. 2004. The tmRNA website:
reductive evolution of tmRNA in plastids and other symbionts. Nucleic Acids Res
32:D104 - 108.

134. Guisbert, E., Herman, C., Lu, C. Z., and Gross, C. A. 2004. A chaperone
network controls the heat shock response in E. coli. Genes & Dev 18:2812-2821.

135. Guo, M., Harvey, 1., Yang, W., Coghill, L., Campopiano, D. J., Parkinson, J.
A., MacGillivray, R. T., Harris, W. R., and Sadler, P. J. 2003. Synergistic anion
and metal binding to the ferric ion-binding protein from Neisseria gonorrhoeae. J Biol
Chem 278:2490-2502.

136. Haas, R., and Meyer, T. F. 1986. The repertoire of silent pilus genes in
Neisseria gonorrhoeae: evidence for gene conversion. Cell 44:107-115.

137. Hagblom, P., Segal, E., Billyard, E., and So, M. 1985. Intragenic recombination
leads to pilus antigenic variation in Neisseria gonorrhoeae. Nature 315: 156-158.

138. Hagman, K. E., Pan, W., Spratt, B. G., Balthazar, J. T., Judd, R. C., and
Shafer, W. M. 1995. Resistance of Neisseria gonorrhoeae to antimicrobial

hydrophobic agents is modulated by the mtrRCDE efflux system. Microbiology 141
(Pt 3):611-622.

139. Hagman, K. E., and Shafer, W. M. 1995. Transcriptional control of the mtr
efflux system of Neisseria gonorrhoeae. J Bacteriol 177:4162-4165.

140. Hale, C. A., and de Boer, P. A. 1997. Direct binding of FtsZ to ZipA, an
essential component of the septa] ring structure that mediates cell division in E. coli.
Cell 88:175-185.

141. Halter, R., Pohlner, J., and Meyer, T. F. 1984. IgA protease of Neisseria
gonorrhoeae: isolation and characterization of the gene and its extracellular product.
EMBO J 3:1595 -1601.

142. Hamilton, H. L., Dominguez, N. M., Schwartz, K. J., Hackett, K. T., and
Dillard, J. P. 2005. Neisseria gonorrhoeae secretes chromosomal DNA via a novel
type IV secretion system. Mol Microbiol 55:1704 - 1721.

143. Harding, M. W., Galat, A., Uehling, D. E., and Schreiber, S. L. 1989. A
receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase.
Nature 341 :758-760.

144. Hauck, C. R., Lorenzen, D., Saas, J., and Meyer, T. F. 1997. An in vitro-
differentiated human cell line as a model system to study the interaction of Neisseria
gonorrhoeae with phagocytic cells. Infect Immun 65: 1 863-1869.

191

145. Hauck, C. R., and Meyer, T. F. 1997. The lysosomal/phagosomal membrane
protein h-lamp-l is a target of the IgAl protease of Neisseria gonorrhoeae. FEBS Lett
405:86-90.

146. Hauck, C. R., and Meyer, T. F. 2003. 'Small' talk: Opa proteins as mediators of
Neisseria-host-cell communication. Current Opinion in Microbiology 6:43-49.

147. Hauck, C. R., Meyer, T. F., Lang, F., and Gulbins, E. 1998. CD66-mediated
phagocytosis of Opa52 Neisseria gonorrhoeae requires a Src-like tyrosine kinase- and
Racl-dependent signalling pathway. EMBO J 17:443-454.

148. Hayano, T., Takahashi, N., Kato, S., Maki, N., and Suzuki, M. 1991. Two
distinct forms of peptidylprolyl-cis-trans-isomerase are expressed separately in

periplasmic and cytoplasmic compartments of Escherichia coli cells. Biochemistry
30:3041-3048.

149. Hebeler, B. H., and Young, F. E. 1975. Autolysis of Neisseria gonorrhoeae. J
Bacteriol 122:385-392.

150. Hedges, S. R., Mayo, M. S., Mestecky, J., Hook, E. W., 111, and Russell, M.
W. 1999. Limited local and systemic antibody responses to Neisseria gonorrhoeae
during uncomplicated genital infections. Infect. Immun. 67:3937-3946.

151. Heithoff, D. M., Conner, C. P., and Mahan, M. J. 1997 . Dissecting the biology
of a pathogen during infection. Trends Microbiol 5:509-513.

152. Henderson, B., and Wilson, M. 1996. Cytokine induction by bacteria: beyond
lipopolysaccharide. Cytokine 8:269-282.

153. Hengge—Aronis, R. 2002. Signal transduction and regulatory mechanisms

involved in control of the 0'5 subunit of RNA polymerase in Escherichia coli.
Microbiol. Mol. Biol. Rev. 66:373-395.

154. Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D., Dalton, E., and Holden, D.
W. 1995. Simultaneous identification of bacterial virulence genes by negative
selection. Science 269:400-403.

155. Hermans, P. W. M., Adrian, P. V., Albert, C., Estevao, S., Hoogenboezem, T.,
Luijendijk, I. H. T., Kamphausen, T., and Hammerschmidt, S. 2006. The
streptococcal lipoprotein rotarnase A (Ser) is a functional peptidyl-prolyl isomerase
involved in pneumococcal colonisation. J Biol Chem 281: 968-976.

156. Heusipp, G., Schmidt, M., and Miller, V. 2003. Identification of rpoE and nadB
as host responsive elements of Yersinia enterocolitica. FEMS Microbiol Lett.
226:291-298.

157. Hitchcock, P. J. 1989. Unified nomenclature for pathogenic Neisseria species.
Clin Microbiol Rev 2 Suppl:S64-65.

192

158. Hobbs, M., and Mattick, J. S. 1993. Common components in the assembly of
type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion
apparatus: a general system for the formation of surface-associated protein complexes.

Mol Microbiol 10:233-243.

159. Hoffmann, K. M., Williams, D., Shafer, W. M., and Brennan, R. G. 2005.
Characterization of the multiple transferable resistance repressor, MtrR, from
Neisseria gonorrhoeae. J Bacteriol 187:5008-5012.

160. Holtje, J. V., Kopp, U., Ursinus, A., and Wiedemann, B. 1994. The negative
regulator of beta-lactamase induction AmpD is a N-acetyl-anhydromuramyl-L—alanine
amidase. FEMS Microbiol Lett 122:159—164.

161. Honda, K., Nakamura, K., Nishiguchi, M., and Yamane, K. 1993. Cloning and
characterization of a Bacillus subtilis gene encoding a homolog of the 54-kilodalton
subunit of mammalian signal recognition particle and Escherichia coli F fh. J Bacteriol
175:4885-4894.

162. Hook, E. W., 3rd, Judson, F. N., Handsfield, H. H., Ehret, J. M., Holmes, K.
K., and Knapp, J. S. 1987. Auxotype/serovar diversity and antimicrobial resistance

of Neisseria gonorrhoeae in two mid-sized American cities. Sex Transm Dis 14:141-
146.

163. Hopper, S., Vasquez, B., Merz, A., Clary, S., Wilbur, J. S., and So, M. 2000.
Effects of the immunoglobulin A1 protease on Neisseria gonorrhoeae trafficking
across polarized T84 epithelial monolayers. Infect Immun 68:906-911.

164. Hopper, S., Wilbur, J. S., Vasquez, B. L., Larson, J., Clary, S., Mehr, I. J.,
Seifert, H. S., and So, M. 2000. Isolation of Neisseria gonorrhoeae mutants that show

enhanced trafficking across polarized T84 epithelial monolayers. Infect Immun
68:896-905.

165. Howie, H. L., Glogauer, M., and So, M. 2005. The N. gonorrhoeae type IV
pilus stimulates mechanosensitive pathways and cytoprotection through a pilT-
dependent mechanism. PLoS Biol 3:e100.

166. Humphreys, S., Stevenson, A., Bacon, A., Weinhardt, A. B., and Roberts, M.
1999. The alternative sigma factor, sigmaE, is critically important for the virulence of
Salmonella typhimurium. Infect Immun 67:1560-1568.

167. liver, D., Kallstrom, H., Normark, S., and Jonsson, A. B. 1998. Transcellular
passage of Neisseria gonorrhoeae involves pilus phase variation. Infect Immun
66:469-473.

168. James, J. F., and Swanson, J. 1978. Studies on gonococcus infection. XIII.

Occurrence of color/opacity colonial variants in clinical cultures. Infect Immun
19:332-340.

193

169. Jephcott, A. E., Reyn, A., and Birch-Andersen, A. 1971. Neisseria
gonorrhoeae 3. Demonstration of presumed appendages to cells fi‘om different colony
types. Acta Pathol Microbiol Scand [B] Microbiol Irnmunol 79:437-439.

170. Jerse, A. E. 1999. Experimental gonococcal genital tract infection and opacity
protein expression in estradiol-treated mice. Infect Immun 67 :5699 - 5708.

171. Jerse, A. E., Cohen, M. S., Drown, P. M., Whicker, L. G., Isbey, S. F., Seifert,
H. S., and Cannon, J. G. 1994. Multiple gonococcal opacity proteins are expressed
during experimental urethral infection in the male. J Exp Med 179:911-920.

172. Jerse, A. E., Sharma, N. D., Simms, A. N., Crow, E. T., Snyder, L. A., and
Shafer, W. M. 2003. A gonococcal efflux pump system enhances bacterial survival in
a female mouse model of genital tract infection. Infect Immun 71:5576-5582.

173. Johnson, C. R., Newcombe, J., Thorne, S., Borde, H. A., Eales-Reynolds, L.-
J., Gorringe, A. R., Funnel], S. G. P., and McFadden, J. J. 2001. Generation and

characterization of a PhoP homologue mutant of Neisseria meningitidis. Mol
Microbiol 39:1345-1355.

174. Johnson, S. R., Steiner, B. M., and Perkins, G. H. 1996. Cloning and
characterization of the catalase gene of Neisseria gonorrhoeae: use of the gonococcus
as a host organism for recombinant DNA. Infect Immun 64:2627-2634.

175. Joiner, K. A., Scales, R., Warren, K. A., Frank, M. M., and Rice, P. A. 1985.
Mechanism of action of blocking immunoglobulin G for Neisseria gonorrhoeae. J
Clin Invest 76:1765-1772.

176. Judd, R. C. 1989. Protein 1: structure, function, and genetics. Clin Microbiol Rev
2 Suppl:S41-48.

177. Judd, R. C. 1982. Surface peptide mapping of protein I and protein 111 of four
strains of Neisseria gonorrhoeae. Infect Immun 37 :632-641.

178. Justice, S. S., Hunstad, D. A., Harper, J. R., Duguay, A. R., Pinkner, J. S.,
Bann, J., Frieden, C., Silhavy, T. J., and Hultgren, S. J. 2005. Periplasmic peptidyl
prolyl cis-trans isomerases are not essential for viability, but SurA Is required for pilus
biogenesis in Escherichia coli. J Bacteriol 187:7680-7686.

179. Kallstrom, H., Hansson-Palo, P., and Jonsson, A. B. 2000. Cholera toxin and
extracellular Ca2+ induce adherence of non-piliated Neisseria: evidence for an

important role of G-proteins and Rho in the bacteria-cell interaction. Cell Microbiol
2:341-351.

180. Kallstrom, H., Islam, M. S., Berggren, P. 0., and Jonsson, A. B. 1998. Cell
signaling by the type IV pili of pathogenic Neisseria. J Biol Chem 273:21777-21782.

194

181. Kallstrom, H., and Jonsson, A. B. 1998. Characterization of the region
downstream of the pilus biogenesis gene pilCI in Neisseria gonorrhoeae. Biochim
Biophys Acta 1397: 137-140.

182. Kallstrom, H., Liszewski, M. K., Atkinson, J. P., and Jonsson, A. B. 1997.
Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic
Neisseria. Mol Microbiol 25:639-647.

183. Kampmeier, R. H. 1978. Identification of the gonococcus by Albert Neisser.
1879. Sex Transm Dis 5:71-72.

184. Kane, M. D., Jatkoe, T. A., Stumpf, C. R., Lu, J., Thomas, J. D., and Madore,
S. J. 2000. Assessment of the sensitivity and specificity of Oligonucleotide (50mer)
microarrays. Nucleic Acids Res 28:4552-4557.

185. Keevil, C., Major, N., Davies, D., and Robinson, A. 1986. Physiology and
virulence determinants of Neisseria gonorrhoeae grown in glucose-, oxygen-, or
cystine—limited continuous culture. J. Gen. Microbiol. 132:3289-3302.

186. Kellogg, D. S., Jr., Cohen, 1. R., Norins, L. C., Schroeter, A. L., and Reising,
G. 1968. Neisseria gonorrhoeae. II. Colonial variation and pathogenicity during 35
months in vitro. J Bacteriol 96:596-605.

187. Kellogg, D. S., Jr., Peacock, W. L., J r., Deacon, W. E., Brown, L., and Pirkle,
D. I. 1963. Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation.
J Bacteriol 85:1274-1279.

188. Kidd, S. P., Potter, A. J., Apicella, M. A., Jennings, M. P., and McEwan, A.
G. 2005. leR of Neisseria gonorrhoeae: a novel redox responsive transcription
factor from the MerR family. Mol Microbiol 57:1676-1689.

189. Kilian, M., Mestecky, J., and Schrohenloher, R. E. 1979. Pathogenic species of
the genus Haemophilus and Streptococcus pneumoniae produce immunoglobulin A1
protease. Infect Immun 26: 143-149.

190. Kirchner, M., and Meyer, T. F. 2005. The PilC adhesin of the Neisseria type IV
pilus-binding specificities and new insights into the nature of the host cell receptor.
Mol Microbiol 56:945-957.

191. Klimpel, K. W., Lesley, S. A., and Clark, V. L. 1989. Identification of subunits
of gonococcal RNA polymerase by immunoblot analysis: evidence for multiple sigma
factors. J Bacteriol 171:3713-3718.

192. Knapp, J., and Hook, E. I. 1988. Prevalence and presistence of Neisseria
cinerea and other Neisseria spp. in adults. J Clin Microbiol 26:896-900.

193. Knapp, J. S. 1988. Historical perspectives and identification of Neisseria and
related species. Clin Microbiol Rev 1:415-431.

195

194. Knapp, J. S., Tam, M. R., Nowinski, R. C., Holmes, K. K., and Sandstrom, E.
G. 1984. Serological classification of Neisseria gonorrhoeae with use of monoclonal
antibodies to gonococcal outer membrane protein 1. J Infect Dis 150:44-48.

195. Kornitzer, D., Teff, D., Altuvia, S., and Oppenheim, A. B. 1991. Isolation,
characterization, and sequence of an Escherichia coli heat shock gene, hth. J
Bacteriol 173:2944-2953.

196. Koumans, E., Johnson, R., Knapp, J., and St Louis, M. 1998. Laboratory
testing for Neisseria gonorrhoeae by recently introduced nonculture tests: a
performance review with clinical and public health considerations. Clin Infect Dis.
27:1171-1180.

197. Kovacikova, G., and Skorupski, K. 2002. The alternative sigma factor B plays
an important role in intestinal survival and virulence in Vibrio cholerae. Infect.
Immun. 70:5355-5362.

198. Krukonis, E. S., and DiRita, V. J. 2003. From motility to virulence: sensing and
responding to enviromnental signals in Vibrio cholerae. Curr Opin Microbiol 6:186-
190.

199. Kuchanny-Ardigo, D., and Lipinska, B. 2003. Cloning and characterization of
the groE heat-shock operon of the marine bacterium Vibrio harveyi. Microbiology
149:1483-1492.

200. Kupsch, E. M., Knepper, B., Kuroki, T., Heuer, 1., and Meyer, T. F. 1993.
Variable opacity (Opa) outer membrane proteins account for the cell tropisms
displayed by Neisseria gonorrhoeae for human leukocytes and epithelial cells. Embo J
12:641-650.

201. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227 :680 - 685.

202. Laga, M., Manoka, A., Kivuvu, M., Malele, B., Tuliza, M., Nzila, N.,
Goeman, J., Behets, F., Batter, V., Alary, M., Heyward, W. L., Ryder, R. W., and
Fiat, P. 1993. Non-ulcerative sexually transmitted diseases as risk factors for HIV-1
transmission in women: results from a cohort study. AIDS 7:95 - 102.

203. Laga, M., Plummer, F. A., Nzanze, H., Namaara, W., Brunham, R. C.,
Ndinya-Achola, J. 0., Maitha, G., Ronald, A. R., D'Costa, L. J., Bhullar, V. B.,
and et al. 1986. Epidemiology of ophthalmia neonatorum in Kenya. Lancet 2:1145-
1149.

204. Laskos, L., Dillard, J. P., Seifert, H. S., Fyfe, J. A., and Davies, J. K. 1998.
The pathogenic Neisseriae contain an inactive rpoN gene and do not utilize the pilE
sigrna54 promoter. Gene 208:95-102.

196

 

205. Laskos, L., Ryan, C. S., Fyfe, J. A. M., and Davies, J. K. 2004. The RpoH-
mediated stress response in Neisseria gonorrhoeae is regulated at the level of activity.
J. Bacteriol. 186:8443-8452.

206. Lauer, P., Albertson, N. H., and Koomey, M. 1993. Conservation of genes
encoding components of a type IV pilus assembly/two-step protein export pathway in
Neisseria gonorrhoeae. Mol Microbiol 8:357-368.

207. Lee, E. H., Rouquette-Loughlin, C., Folster, J. P., and Shafer, W. M. 2003.
FarR regulates the farAB-encoded efflux pump of Neisseria gonorrhoeae via an MtrR
regulatory mechanism. J Bacteriol 185:7145-7152.

208. Lee, E. H., and Shafer, W. M. 1999. The farAB-encoded efflux pump mediates
resistance of gonococci to long-chained antibacterial fatty acids. Mol Microbiol
33:839-845.

209. Lee, S. W., Bonnah, R. A., Higashi, D. L., Atkinson, J. P., Milgram, S. L., and
So, M. 2002. CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells
by Neisseria gonorrhoeae. J Cell Biol 156:951-957.

210. Lee, S. W., Higashi, D. L., Snyder, A., Merz, A. J., Potter, L., and So, M.
2005. PilT is required for PI(3,4,5)P3-mediated cross-talk between N. gonorrhoeae
and epithelial cells. Cell Microbiol in press.

211. Lehman, I. R. 1974. DNA ligase: structure, mechanism, and function. Science
186:790-797.

212. Lesley, S. A., Jovanovich, S. B., Tse—Dinh, Y. C., and Burgess, R. R. 1990.
Identification of a heat shock promoter in the topA gene of Escherichia coli. J
Bacteriol 172:6871-6874.

213. Leuzzi, R., Serino, L., Scarselli, M., Savino, S., Fontana, M. R., Monaci, E.,
Taddei, A., Fischer, G., Rappuoli, R., and Pizza, M. 2005. Ng-MIP, a surface-
exposed lipoprotein of Neisseria gonorrhoeae, has a peptidyl-prolyl cis/trans

isomerase (PPIase) activity and is involved in persistence in macrophages. Mol
Microbiol 58:669-681.

214. Lewis, L. A., Gillaspy, A. F., McLaughlin, R. E., Gipson, M., Ducey, T.,
Ownbey, T., Hartman, K., Nydick, C., Carson, M., Vaughn, J., Thomson, C.,
Song, L., Lin, S., Yuan, X., Najar, F., Zhan, M., Ren, Q., Zhu, H., Qi, S., Kenton,
S. M., Lai, H., White, J. D., Clifton, S., Roe, B. A., and Dyer, D. W. The
gonococcal genome sequencing project. unpublished.

215. Liberek, K., and Georgopoulos, C. 1993. Autoregulation of the Escherichia coli
heat shock response by the DnaK and DnaJ heat shock proteins. Proc Natl Acad Sci
USA 90:11019-11023.

197

216. Lin, L., Ayala, P., Larson, J., Mulks, M., Fukuda, M., Carlsson, S. R., Enns,
C., and So, M. 1997. The Neisseria type 2 IgAl protease cleaves LAMPl and
promotes survival of bacteria within epithelial cells. Mol Microbiol 24:1083-1094.

217. Lindberg, A. A., Karnell, A., and Weintraub, A. 1991. The lipopolysaccharide
of Shigella bacteria as a virulence factor. Rev Infect Dis 13 Suppl 4:S279-284.

218. Lipinska, B., Fayet, 0., Baird, L., and Georgopoulos, C. 1989. Identification,
characterization, and mapping of the Escherichia coli htrA gene, whose product is

essential for bacterial growth only at elevated temperatures. J Bacteriol 171:1574-
1584.

219. Lissenden, S., Mohan, S., Overton, T., Regan, T., Crooke, H., Cardinale, J.
A., Householder, T. C., Adams, P., O'Conner, C. D., Clark, V. L., Smith, H., and
Cole, J. A. 2000. Identification of transcription activators that regulate gonococcal

adaptation from aerobic to anaerobic or oxygen-limited growth. Mol Microbiol
37:839-855.

220. Litwin, C. M., and Calderwood, S. B. 1993. Cloning and genetic analysis of the
Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker
exchange. J Bacteriol 175:706-715.

221. Litwin, C. M., and Calderwood, S. B. 1993. Role of iron in regulation of
virulence genes. Clin Microbiol Rev 6:137-149.

222. Lonetto, M., Gribskov, M., and Gross, C. A. 1992. The sigma 70 family:
sequence conservation and evolutionary relationships. J Bacteriol 174:3843-3 849.

223. Long, C. D., Tobiason, D. M., Lazio, M. P., Kline, K. A., and Seifert, H. S.
2003. Low-level pilin expression allows for substantial DNA transformation
competence in Neisseria gonorrhoeae. Infect Immun 71 :6279-6291.

224. Lucas, C. E., Balthazar, J. T., Hagman, K. E., and Shafer, W. M. 1997. The
MtrR repressor binds the DNA sequence between the mtrR and mtrC genes of

Neisseria gonorrhoeae. J Bacteriol 179:4123-4128.

225. Lucas, R. L., and Lee, C. A. 2000. Unravelling the mysteries of virulence gene
regulation in Salmonella typhimurium. Mol Microbiol 36:1024 - 1033.

226. Luirink, J., and Sinning, I. 2004. SRP-mediated protein targeting: structure and
function revisited. Biochim Biophys Acta 1694:17-35.

227. Luirink, J., ten Hagen-Jongman, C. M., van der Weijden, C. C., Oudega, B.,
High, S., Dobberstein, B., and Kusters, R. 1994. An alternative protein targeting
pathway in Escherichia coli: studies on the role of FtsY. EMBO J 13:2289-2296.

228. Lukashkov, V. M., and Maianskii, A. N. 1980. Immunologic characteristics of
protein antigens of the Neiseriaceae family. 111. Study of delayed hypersensitivity

198

 

cross reactions between different species of Neisseria. Zh Mikrobiol Epidemiol
Immunobiol:75-78.

229. Lytton, E. J., and Blake, M. S. 1986. Isolation and partial characterization of the
reduction-modifiable protein of Neisseria gonorrhoeae. J Exp Med 164:1749-1759.

230. Macfarlane, J., and Muller, M. 1995. The functional integration of a polytopic
membrane protein of Escherichia coli is dependent on the bacterial signal-recognition
particle. Eur J Biochem 233:766-771.

231. Makepeace, B. L., Watt, P. J., Heckels, J. E., and Christodoulides, M. 2001.
Interactions of Neisseria gonorrhoeae with mature human macrophage opacity

proteins influence production of proinflammatory cytokines. Infect Immun 69:1909—
1913.

232. Makino, S., van Putten, J. P., and Meyer, T. F. 1991. Phase variation of the
opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into
human epithelial cells. Embo J 10:1307-1315.

233. Mandrell, R., Schneider, H., Apicella, M., Zollinger, W., Rice, P. A., and
Griffiss, J. M. 1986. Antigenic and physical diversity of Neisseria gonorrhoeae
lipooligosaccharides. Infect Immun 54:63-69.

234. Mandrell, R. E., and Apicella, M. A. 1993. Lipo-oligosaccharides (LOS) of
mucosal pathogens: molecular mimicry and host-modification of LOS.
Irnmunobiology 187 :382-402.

235. Maness, M. J., and Sparling, P. F. 1973. Multiple antibiotic resistance due to a
single mutation in Neisseria gonorrhoeae. J Infect Dis 128:321-330.

236. Matsuyama, S., Mizuno, T., and Mizushima, S. 1986. Interaction between two
regulatory proteins in osmoregulatory expression of ompF and ompC genes in

Escherichia coli: a novel ompR mutation suppresses pleiotropic defects caused by an
envZ mutation. J Bacteriol 168:1309-1314.

237. Mayer, L. W. 1982. Rates in vitro changes of gonococcal colony opacity
phenotypes. Infect Immun 37:481-485.

238. McDade, R. L., Jr., and Johnston, K. H. 1980. Characterization of serologically
dominant outer membrane proteins of Neisseria gonorrhoeae. J Bacteriol 141:1183-
1191.

239. McGee, Z. A., Johnson, A. P., and Taylor-Robinson, D. 1976. Human fallopian
tubes in organ culture: preparation, maintenance, and quantitation of damage by
pathogenic microorganisms. Infect Immun 13:608 - 618.

240. McGee, Z. A., Johnson, A. P., and Taylor-Robinson, D. 1981. Pathogenic
mechanisms of Neisseria gonorrhoeae: observations on damage to human fallopian

199

 

tubes in organ culture by gonococci of colony type 1 or type 4. J Infect Dis 143:413-
422.

241. McGee, Z. A., Stephens, D. S., Hoffman, L. H., Schlech, W. F., 3rd, and
Horn, R. G. 1983. Mechanisms of mucosal invasion by pathogenic Neisseria. Rev
Infect Dis 5 Suppl 4:S708-714.

242. McKevitt, M., Patel, K., Smajs, D., Marsh, M., McLaughlin, M., Norris, S. J.,
Weinstock, G. M., and Palzkill, T. 2003. Systematic cloning of T reponema pallidum

open reading frames for protein expression and antigen discovery. Genome Res
13:1665 - 1674.

243. Mellies, J., Jose, J., and Meyer, T. F. 1997. The Neisseria gonorrhoeae gene
aniA encodes an inducible nitrite reductase. Mol Gen Genet 256:525-532.

244. Melly, M. A., Gregg, C. R., and McGee, Z. A. 1981. Studies of toxicity of
Neisseria gonorrhoeae for human fallopian tube mucosa. J Infect Dis 143:423-431.

245. Melly, M. A., McGee, Z. A., and Rosenthal, R. S. 1984. Ability of monomeric
peptidoglycon fragments from Neisseria gonorrhoeae to damage human fallopian-tube
mucosa. J Infect Dis 149:378 - 386.

246. Merz, A. J., Enns, C. A., and So, M. 1999. Type IV pili of pathogenic
Neisseriae elicit cortical plaque formation in epithelial cells. Mol Microbiol 32:1316-
1332.

247. Merz, A. J., Rifenbery, D. B., Arvidson, C. G., and So, M. 1996. Traversal of a
polarized epithelium by pathogenic Neisseriae: facilitation by type IV pili and
maintenance of epithelial barrier function. Mol Med 2:745-754.

248. Merz, A. J., and So, M. 1997. Attachment of piliated, Opa- and Opc- gonococci
and meningococci to epithelial cells elicits cortical actin rearrangements and clustering
of tyrosine-phosphorylated proteins. Infect Immun 65:4341-4349.

249. Merz, A. J., and So, M. 2000. Interactions of pathogenic Neisseriae with
epithelial cell membranes. Annu Rev Cell Dev Biol 16:423-457.

250. Merz, A. J., So, M., and Sheetz, M. P. 2000. Pilus retraction powers bacterial
twitching motility. Nature 407:98-102.

251. Meyer, T. F., Billyard, E., Haas, R., Storzbach, S., and So, M. 1984. Pilus
genes of Neisseria gonorrheae: chromosomal organization and DNA sequence. Proc
Natl Acad Sci USA 81:6110-6114.

252. Meyer, T. F., Mlawer, N., and So, M. 1982. Pilus expression in Neisseria
gonorrhoeae involves chromosomal rearrangement. Cell 30:45-52.

200

253. Miettinen, A., Saikku, P., Jansson, E., and Paavonen, J. 1986. Epidemiologic
and clinical characteristics of pelvic inflammatory disease associated with

Mycoplasma hominis, Chlamydia trachomatis, and Neisseria gonorrhoeae. Sex
Transm Dis 13:24-28.

254. Miller, S. 1., Kukral, A. M., and Mekalanos, J. J. 1989. A two-component
regulatory system (phoP pth) controls Salmonella typhimurium virulence. Proc Natl
Acad Sci USA 86:5054—5058.

255. Miller, V. L., and Mekalanos, J. J. 1988. A novel suicide vector and its use in
construction of insertion mutations: osmoregulation of outer membrane proteins and
virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170:2575-2583.

256. Missiakas, D., and Raina, S. 1998. The extracytoplasmic function sigma factors:
role and regulation. Mol Microbiol 28:1059-1066.

257. Mizuno, T. 1997. Compilation of all genes encoding two-component
phosphotransfer signal transducers in the genome of Escherichia coli. DNA Res
4:161-168.

258. Mizuno, T., Chou, M. Y., and Inouye, M. 1983. A comparative study on the
genes for three porins of the Escherichia coli outer membrane. DNA sequence of the
osmoregulated ompC gene. J Biol Chem 258:6932-6940.

259. Morand, P. C., Bille, E., Morelle, S., Eugene, E., Beretti, J. L., Wolfgang, M.,
Meyer, T. F., Koomey, M., and Nassif, X. 2004. Type IV pilus retraction in
pathogenic Neisseria is regulated by the PilC proteins. EMBO J 23:2009-2017.

260. Morita, M. T., Tanaka, Y., Kodama, T. S., Kyogoku, Y., Yanagi, H., and

Yura, T. 1999. Translational induction of heat shock transcription factor 0'”. Genes &
Dev 13:655-665.

261. Mosleh, I. M., Boxberger, H. J., Sessler, M. J., and Meyer, T. F. 1997.
Experimental infection of native human ureteral tissue with Neisseria gonorrhoeae:

adhesion, invasion, intracellular fate, exocytosis, and passage through a stratified
epithelium. Infect Immun 65:3391-3398.

262. Mulks, M. H., and Knapp, J. S. 1987. Irnmunoglobulin A1 protease types of
Neisseria gonorrhoeae and their relationship to auxotype and serovar. Infect Immun
55:931-936.

263. Mulks, M. H., and Plant, A. G. 1978. IgA protease production as a characteristic
distinguishing pathogenic from harmless Neisseriaceae. N Engl J Med 299:973-976.

264. Mulks, M. H., Plaut, A. G., Feldman, H. A., and Frangione, B. 1980. IgA
proteases of two distinct specificities are released by Neisseria meningitidis. J Exp
Med 152:1442-1447.

201

265. Muller, A., Gunther, D., Brinkmann, V., Hurwitz, R., Meyer, T. F., and
Rudel, T. 2000. Targeting of the pro-apoptotic VDAC-like porin (PorB) of Neisseria
gonorrhoeae to mitochondria of infected cells. EMBO J 19:5332-5343.

266. Muller, A., Gunther, D., Dux, F., Naumann, M., Meyer, T. F., and Rudel, T.
1999. Neisseria] porin (PorB) causes rapid calcium influx in target cells and induces
apoptosis by the activation of cysteine proteases. EMBO J 18:339-352.

267. Muller, A., Rassow, J., Grimm, J., Machuy, N., Meyer, T. F., and Rudel, T.
2002. VDAC and the bacterial porin PorB of Neisseria gonorrhoeae share
mitochondrial import pathways. EMBO J 21:1916-1929.

268. Murphy, G. L., Connell, T. D., Barritt, D. S., Koomey, M., and Cannon, J. G.
1989. Phase variation of gonococcal protein 11: regulation of gene expression by
slipped-strand mispairing of a repetitive DNA sequence. Cell 56:539-547.

269. Nagai, H., Yano, R., Erickson, J. W., and Yura, T. 1990. Transcriptional
regulation of the heat shock regulatory gene rpoH in Escherichia coli: involvement of
a novel catabolite-sensitive promoter. J Bacteriol 172:2710-2715.

270. Naids, F. L., Belisle, B., Lee, N., and Rest, R. F. 1991. Interactions of Neisseria
gonorrhoeae with human neutrophils: studies with purified PII (Opa) outer membrane
proteins and synthetic Opa peptides. Infect Immun 59:4628-4635.

271. Nakahigashi, K., Yanagi, H., and Yura, T. 1998. Regulatory conservation and
divergence of 0'32 homologs from Gram-negtive bacteria: Serratia marcescens,

Proteus mirabilis, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. J
Bacteriol 180:2402-2408.

272. Nakai, K., and Kanehisa, M. 1991. Expert system for predicting protein
localization sites in gram-negative bacteria. Proteins 11:95-110.

273. Nakamura, K., Imai, Y., Nakamura, A., and Yamane, K. 1992. Small
cytoplasmic RNA of Bacillus subtilis: ftmctional relationship with human signal
recognition particle 7S RNA and Escherichia coli 4.58 RNA. J Bacteriol 174:2185-
2192.

274. Nassif, X., Beretti, J. L., Lowy, J., Stenberg, P., O'Gaora, P., Pfeifer, J.,
Normark, S., and So, M. 1994. Roles of pilin and PilC in adhesion of Neisseria

meningitidis to human epithelial and endothelial cells. Proc Natl Acad Sci USA
91:3769-3773.

275. Nassif, X., and So, M. 1995. Interaction of pathogenic Neisseriae with
nonphagocytic cells. Clin. Microbiol. Rev. 8:376-388.

276. Naumann, M., Rudel, T., and Meyer, T. F. 1999. Host cell interactions and
signalling with Neisseria gonorrhoeae. Curr Opin Microbiol 2:62-70.

202

277. Naumann, M., Rudel, T., Wieland, B., Bartsch, C., and Meyer, T. F. 1998.
Coordinate activation of activator protein 1 and inflammatory cytokines in response to

Neisseria gonorrhoeae epithelial cell contact involves stress response kinases. J Exp
Med 188:1277-1286.

278. Naumann, M., Wessler, S., Bartsch, C., Wieland, B., and Meyer, T. F. 1997.
Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the
transcription factors nuclear factor kappaB and activator protein 1 and the induction of
inflammatory cytokines. J Exp Med 186:247-258.

279. Neumann-Haefelin, C., Schafer, U., Muller, M., and Koch, H. G. 2000. SRP-
dependent co-translational targeting and SecA-dependent translocation analyzed as
individual steps in the export of a bacterial protein. EMBO J 19:6419-6426.

280. Nicholson, T. L., Olinger, L., Chong, K., Schoolnik, G., and Stephens, R. S.
2003. Global stage-specific gene regulation during the developmental cycle of
Chlamydia trachomatis. J Bacteriol 185:3179 - 3189.

281. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. 1997.
Identification of prokaryotic and eukaryotic signal peptides and prediction of their
cleavage sites. Protein Engineering 10:1 - 6.

282. Nikaido, H. 1968. Biosynthesis of cell wall lipopolysaccharide in gram-negative
enteric bacteria. Adv Enzymol Relat Areas Mol Biol 31 :77-124.

283. N orel, F., Robbe-Saule, V., Popoff, M. Y., and Coynault, C. 1992. The putative
sigma factor KatF (RpoS) is required for the transcription of the Salmonella

typhimurium virulence gene spo in Escherichia coli. FEMS Microbiol Lett 78:271-
276.

284. Nunn, D. 1999. Bacterial type 11 protein export and pilus biogenesis: more than
just homologies? Trends Cell Biol 9:402-408.

285. Olesky, M., Hobbs, M., and Nicholas, R. A. 2002. Identification and analysis of
amino acid mutations in porin IB that mediate intermediate-level resistance to

penicillin and tetracycline in Neisseria gonorrhoeae. Antimicrob Agents Chemother
46:2811 - 2820.

286. Oshima, T., Aiba, H., Masuda, Y., Kanaya, S., Sugiura, M., Wanner, B. L.,
Mori, H., and Mizuno, T. 2002. Transcriptome analysis of all two-component
regulatory system mutants of Escherichia coli K-12. Mol Microbiol 46:281-291.

287. Ottow, J. C. 1975. Ecology, physiology, and genetics of fimbriae and pili. Annu
Rev Microbiol 29:79-108.

288. Parge, H. E., Forest, K. T., Hickey, M. J., Christensen, D. A., Getzoff, E. D.,
and Tainer, J. A. 1995. Structure of the fibre-fonning protein pilin at 2.6 A
resolution. Nature 378:32-38.

203

 

289. Parsons, N. J., Andrade, J. R., Patel, P. V., Cole, J. A., and Smith, H. 1989.
Sialylation of lipopolysaccharide and loss of absorption of bactericidal antibody
during conversion of gonococci to serum resistance by cytidine 5'-monophospho-N-
acetyl neurarninic acid. Microb Pathog 7:63-72.

290. Parsot, C., and Mekalanos, J. J. 1990. Expression of ToxR, the transcriptional
activator of the virulence factors in Vibrio cholerae, is modulated by the heat shock
response. Proc Natl Acad Sci USA 87:9898-9902.

291. Pearce, W. A., and Buchanan, T. M. 1978. Attachment role of gonococcal pili.
Optimum conditions and quantitation of adherence of isolated pili to human cells in
vitro. J Clin Invest 61:931-943.

292. Pelnso, P., Herschlag, D., Neck, 8., Freymann, D. M., Johnson, A. E., and
Walter, P. 2000. Role of 4.5S RNA in assembly of the bacterial signal recognition
particle with its receptor. Science 288:1640-1643.

293. Pettersson, J., Nordfelth, R., Dubinina, E., Bergman, T., Gustafsson, M.,
Magnusson, K. E., and Wolf-Watz, H. 1996. Modulation of virulence factor
expression by pathogen target cell contact. Science 273:1231 - 1233.

294. Phillips, G. J., and Silhavy, T. J. 1992. The E. coli flh gene is necessary for
viability and efficient protein export. Nature 359:744-746.

295. Plant, L., Asp, V., Lovkvist, L., Sundqvist, J., and Jonsson, A. B. 2004.
Epithelial cell responses induced upon adherence of pathogenic Neisseria. Cell
Microbiol 6:663-670.

296. Plant, L., and Jonsson, A.-B. 2003. Contacting the host: insights and
implications of pathogenic Neisseria cell interactions. Scand J Infect Dis 35:608-613.

297. Plant, A. G., Genco, R. J., and Tomasi, T. B., Jr. 1974. Isolation of an enzyme
from Streptococcus sanguis which specifically cleaves IgA. J Irnmunol 113:589-591.

298. Plant, A. G., Wistar, R., Jr., and Capra, J. D. 1974. Differential susceptibility
of human IgA immunoglobulins to streptococcal IgA protease. J Clin Invest 54:1295-
1300.

299. Plummer, F. A., Chnbb, H., Simonsen, J. N., Bosire, M., Slaney, L., Maclean,
1., Ndinya-Achola, J. 0., Waiyaki, P., and Brunham, R. C. 1993. Antibody to Rmp

(outer membrane protein 3) increases susceptibility to gonococcal infection. J Clin
Invest 91 :339-343.

300. Pogliano, J., Lynch, A. S., Belin, D., Lin, E. C., and Beckwith, J. 1997.
Regulation of Escherichia coli cell envelope proteins involved in protein folding and
degradation by the Cpx two-component system. Genes Dev 11:1169-1182.

204

301. Pohlner, J., Halter, R., Beyrenther, K., and Meyer, T. F. 1987. Gene structure
and extracellular secretion of Neisseria gonorrhoeae IgA protease. Nature 325:458-
462.

302. Pohlner, J., Halter, R., and Meyer, T. F. 1987. Neisseria gonorrhoeae IgA
protease. Secretion and implications for pathogenesis. Antonie Van Leeuwenhoek
53:479-484.

303. Pool, M. R. 2005. Signal recognition particles in chloroplasts, bacteria, yeast and
mammals (review). Mol Membr Biol 22:3-15.

304. Popp, A., Billker, 0., and Rudel, T. 2001. Signal transduction pathways induced
by virulence factors of Neisseria gonorrhoeae. Int J Med Microbiol 291 :307-314.

305. Posada, D., Crandall, K. A., Nguyen, M., Demma, J. C., and Viscidi, R. P.
2000. Population genetics of the porB gene of Neisseria gonorrhoeae: different
dynamics in different homology groups. Mol Biol Evol 17:423-436.

306. Powers, T., and Walter, P. 1995. Reciprocal stimulation of GTP hydrolysis by
two directly interacting GTPases. Science 269: 1422-1424.

307. Prentice, M. J., Hutchinson, G. R., and Taylor-Robinsin, D. 1977. A
microbiological study of neonatal conjunctivae and conjunctivitis. Br J Ophthalmol
61:601-607.

308. Price, G. A., Hobbs, M. M., and Cornelissen, C. N. 2004. Irnmunogenicity of
gonococcal transferrin binding proteins during natural infections. Infect. Immun.
72:277-283.

309. Pngsley, A. P. 1993. The complete general secretory pathway in gram-negative
bacteria. Microbiol Rev 57:50-108.

310. Pnjol, C., Eugene, E., Morand, P., and Nassif, X. 2000. Do pathogenic
Neisseriae need several ways to modify the host cell cytoskeleton? Microbes Infect
2:821 - 827.

311. Pnnsalang, A. P., and Sawyer, W. D. 1973. Role of pili in the virulence of
Neisseria gonorrhoeae. Infect Immun 8:255 - 263.

312. Rabin, R. S., and Stewart, V. 1993. Dual response regulators (NarL and NarP)
interact with dual sensors (NarX and NarQ) to control nitrate- and nitrite-regulated
gene expression in Escherichia coli K-12. J Bacteriol 175:3259-3268.

313. Rahfeld, J.-U., Schierhorn, A., Mann, K., and Fischer, G. 1994. A novel
peptidyl-prolyl cis/trans isomerase from Escherichia coli. FEBS Lett 343:65-69.

314. Raivio, T. L., and Silhavy, T. J. 1999. The GE and Cpx regulatory pathways:
overlapping but distinc envelop stress responses. Curr Opin Microbiol 2: 159-165.

205

 

315. Ramirez-Arcos, S., Szeto, J., Beveridge, T., Victor, C., Francis, F., and
Dillon, J. 2001. Deletion of the cell-division inhibitor MinC results in lysis of
Neisseria gonorrhoeae. Microbiology 147:225-237.

316. Ray K, B. M., Knmari S, Narain JP. 2005. Antimicrobial resistance of
Neisseria gonorrhoeae in selected World Health Organization Southeast Asia region
countries: an overview. Sex Transm Dis. 32:178-184.

317. Reizer, J., Romano, A. H., and Dentscher, J. 1993. The role of phosphorylation
of HPr, a phosphocarrier protein of the phosphotransferase system, in the regulation of
carbon metabolism in gram-positive bacteria. J Cell Biochem 51:19-24.

318. Rice, P. A., Vayo, H. E., Tam, M. R., and Blake, M. S. 1986. Immunoglobulin
G antibodies directed against protein IH block killing of serum-resistant Neisseria
gonorrhoeae by immune serum. J Exp Med 164:1735-1748.

319. Rice, R. J., Biddle, J. W., JeanLouis, Y. A., DeWitt, W. E., Blonnt, J. H., and
Morse, S. A. 1986. Chromosomally mediated resistance in Neisseria gonorrhoeae in
the United States: results of surveillance and reporting, 1983-1984. J Infect Dis
153:340-345.

320. Richardson, W. P., and Sadoff, J. C. 1988. Induced engulfinent of Neisseria
gonorrhoeae by tissue culture cells. Infect Immun 56:2512-2514.

321. Roe, B. A., Lin, S. P., Song, L., Yuan, X., Clifton, S., and Dyer, D. 2002.
Gonococcal genome sequencing project. URL http://www.genome.ou.edu/gono/html.

322. Rossan, R., Heyndricks, L., and van Henverswyn, H. 1988. Nucleotide
sequence of a 16S ribosomal RNA gene fi'om Neisseria gonorrhoeae. Nucleic Acids
Res 16:6227.

323. Rostand, K. S., and Esko, J. D. 1997. Microbial adherence to and invasion
through proteoglycans. Infect Immun 65:1-8.

324. Rothbard, J. B., Fernandez, R., Wang, L., Teng, N. N., and Schoolnik, G. K.
1985. Antibodies to peptides corresponding to a conserved sequence of gonococcal
pilins block bacterial adhesion. Proc Natl Acad Sci USA 82:915-919.

325. Rothfield, L., Justice, S., and Garcia-Lara, J. 1999. Bacterial cell division.
Annu Rev Genet 33:423-448.

326. Ronqnette—Longhlin, C. E., Balthazar, J. T., and Shafer, W. M. 2005.
Characterization of the MacA-MacB efflux system in Neisseria gonorrhoeae. J
Antimicrob Chemother.

327. Rouquette, C., Harmon, J. B., and Shafer, W. M. 1999. Induction of the
mtrCDE-encoded efflux pump system of Neisseria gonorrhoeae requires MtrA, an
AraC-like protein. Mol Microbiol 33:651-658.

206

328. Ronviere, P. E., De Las Penas, A., Mecsas, J., Lu, C. Z., Rudd, K. E., and
Gross, C. A. 1995. rpoE, the gene encoding the second heat-shock sigma factor,
sigma E, in Escherichia coli. EMBO J 14:1032-1042.

329. Rozen, S., and Skaletsky, H. 2000. Primer3 on the WWW for general users and
for biologist programmers. Bioinforrnatics Methods and Protocols:365 - 386.

330. Rudel, T., Facius, D., Barten, R., Schenerpflng, 1., Nonnenmacher, E., and
Meyer, T. F. 1995. Role of pili and the phase-variable PilC protein in natural
competence for transformation of Neisseria gonorrhoeae. Proc Natl Acad Sci USA
92:7986-7990.

331. Rudel, T., Schenrerpflng, 1., and Meyer, T. F. 1995. Neisseria PilC protein
identified as type-4 pilus tip-located adhesin. Nature 373:357-359.

332. Salimnia, H., Radia, A., Bernatchez, S., Beveridge, T. J., and Dillon, J. R.
2000. Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae:
expression in Escherichia coli and N. gonorrhoeae. Arch Microbiol 173:10-20.

333. Sambrook, J., Fritsch, E. F., and Maniatis., T. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY.

334. Sambrook, J., and Russell, D. 2001. Molecular Cloning, a laboratory manual,
third ed, vol. 1. Cold Spring Harbor Press, Cold Spring Harbor, NY.

335. Samuelsson, T., Olsson, M., Wikstrom, P. M., and Johansson, B. R. 1995. The
GTPase activity of the Escherichia coli F flr protein is important for normal growth.
Biochim Biophys Acta 1267 :83-91.

336. Sarker, S., and Oliver, D. 2002. Critical regions of secM that control its

translation and secretion and promote secretion-specific secA regulation. J Bacteriol
184:2360-2369.

337. Sarnbbi, F. A., Jr., Blackman, E., and Sparling, P. F. 1974. Genetic mapping

of linked antibiotic resistance loci in Neisseria gonorrhoeae. J Bacteriol 120:1284—
1292.

338. Schell, M. A. 1993. Molecular biology of the LysR family of transcriptional
regulators. Annu Rev Microbiol 47:597-626.

339. Scherer, C. A., and Miller, S. I. 2001. Molecular pathogenesis of Salmonellae,
p. 265-333. In E. A. Groisman (ed.), Principles of Bacterial Pathogenesis. Academic
Press, San Diego.

340. Schmidt, M. G., Dolan, K. M., and Oliver, D. B. 1991. Regulation of
Escherichia coli secA mRNA translation by a secretion-responsive element. J
Bacteriol 173:6605-661 1.

207

341. Schneider, H., Hale, T. L., Zollinger, W. D., Seid, R. C., Jr., Hammack, C. A.,
and Griffiss, J. M. 1984. Heterogeneity of molecular size and antigenic expression
within lipooligosaccharides of individual strains of Neisseria gonorrhoeae and
Neisseria meningitidis. Infect Immun 45:544-549.

342. Schneider, H., Hale, T. L., Zollinger, W. D., Seid, R. C. J., Hammack, C. A.,
and Griffiss, J. M. 1996. Does an experimental gonococcal infection protect human
volunteers from subsequent reinfection? Abst Tenth Intnl Path Neisseria Conf 10:13.

343. Schneider, H., Schmidt, K. A., Skillman, D. R., Van De Verg, L., Warren, R.
L., Wylie, H. J., Sadoff, J. C., Deal, C. D., and Cross, A. S. 1996. Sialylation
lessens the infectivity of Neisseria gonorrhoeae MS11ka. J Infect Dis 173:1422-
1427.

344. Schryvers, A. B., and Stojiljkovic, I. 1999. Iron acquisition systems in the
pathogenic Neisseria. Mol Microbiol 32:11 17 - 1123.

345. Schweizer, M., and Henning, U. 1977. Action of a major outer cell envelope
membrane protein in conjugation of Escherichia coli K-12. J Bacteriol 129:1651-
1652.

346. Schweizer, M., Hindennach, 1., Garten, W., and Henning, U. 197 8. Major
proteins of the Escherichia coli outer cell envelope membrane. Interaction of protein 11
with lipopolysaccharide. Eur J Biochem 82:21 1-217.

347. Scopelitis, E., and Martinez-Osnna, P. 1993. Gonococcal arthritis. Rheum Dis
Clin North Am 19:363-377.

348. Sebastian, S., Agarwal, S., Murphy, J. R., and Genco, C. A. 2002. The
gonococcal fur regulon: identification of additional genes involved in major catabolic,
recombination, and secretory pathways. J Bacteriol 184:3965-3974.

349. Segal, E., Billyard, E., So, M., Storzbach, S., and Meyer, T. F. 1985. Role of
chromosomal rearrangement in N. gonorrhoeae pilus phase variation. Cell 40:293-
300.

350. Segal, E., Hagblom, P., Seifert, H. S., and So, M. 1986. Antigenic variation of
gonococcal pilus involves assembly of separated silent gene segments. Proc Natl Acad
Sci USA 83:2177-2181.

351. Seifert, H. S. 1997. Insertionally inactivated and inducible recA alleles for use in
Neisseria. Gene 188:215-220.

352. Seifert, H. S. 1992. Molecular mechanisms of antigenic variation in Neisseria
gonorrhoeae. Mol Cell Biol Hum Dis Ser 1:1-22.

353. Seifert, H. S. 1996. Questions about gonococcal pilus phase- and antigenic
variation. Mol Microbiol 21:433-440.

208

354. Seifert, H. S., Ajioka, R. S., Marchal, C., Sparling, P. F., and So, M. 1988.
DNA transformation leads to pilin antigenic variation in Neisseria gonorrhoeae.
Nature 336:392-395.

355. Seifert, H. S., Ajioka, R. S., Parnchnri, D., Heffron, F., and So, M. 1990.
Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA
transformation competence. J Bacteriol 172:40-46.

356. Seifert, H. S., Chen, E. Y., So, M., and Heffron, F. 1986. Shuttle mutagenesis: a
method of transposon mutagenesis for Saccharomyces cerevisiae. Proc Natl Acad Sci
USA 83:735-739.

357. Seifert, H. S., and 80, M. 1988. Genetic mechanisms of bacterial antigenic
variation. Microbiol Rev 52:327-336.

358. Selnanov, A., and Bibi, E. 1997. F tsY, the prokaryotic signal recognition particle
receptor homologue, is essential for biogenesis of membrane proteins. J Biol Chem
272:2053-2055.

359. Shafer, W. M., Balthazar, J. T., Hagman, K. E., and Morse, S. A. 1995.
Missense mutations that alter the DNA-binding domain of the MtrR protein occur

frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to faecal lipids.
Microbiology 141 (Pt 4):907-911.

360. Shaw, J. H., and Falkow, S. 1988. Model for invasion of human tissue culture
cells by Neisseria gonorrhoeae. Infect Immun 56:1625-1632.

361. Shlaes, D., Levy, S., and Archer, G. 1991. Antimicrobial resistance: new
directions. ASM News 57:455-458.

362. Siekierka, J. J., Hung, S. H., Poe, M., Lin, C. S., and Sigal, N. H. 1989. A
cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl
isomerase activity but is distinct fi'om cyclophilin. Nature 341:755-757.

363. Simon, D., and Rest, R. F. 1992. Escherichia coli expressing a Neisseria
gonorrhoeae opacity-associated outer membrane protein invade human cervical and
endometrial epithelial cell lines. Proc Natl Acad Sci USA 89:5512-5516.

364. Simpson, D. A., Hansinger, R. P., and Mulks, M. 1988. Purification,
characterization, and comparison of the immunoglobulin A1 proteases of Neisseria
gonorrhoeae. J Bacteriol 170:1866 - 1873.

365. Skerman, V. 1967. p.218 - 220. A guide to the identification of the genera of
bacteria. The Williams & Wilkins Co., Baltimore, MD.

366. Skorupski, K., and Taylor, R. K. 1997. Control of the ToxR virulence regulon
in Vibrio cholerae by environmental stimuli. Mol Microbiol 25: 1003 - 1009.

209

367. Skorupski, K., and Taylor, R. K. 1996. Positive selection vectors for allelic
exchange. Gene 169:47-52.

368. Song, W., Ma, L., Chen, R., and Stein, D. C. 2000. Role of lipooligosaccharide
in Opa-independent invasion of Neisseria gonorrhoeae into human epithelial cells. J
Exp Med 191:949-960.

369. Sparling, P. F. 1966. Genetic transformation of Neisseria gonorrhoeae to
streptomycin resistance. J Bacteriol 92: 1364-1371 .

370. Sparling, P. F., Cannon, J. G., and So, M. 1986. Phase and antigenic variation
of pili and outer membrane protein 11 of Neisseria gonorrhoeae. J Infect Dis 153:196-
201.

371. Spence, J. M., Chen, J. C., and Clark, V. L. 1997. A proposed role for the
lutropin receptor in contact-inducible gonococcal invasion of HeclB cells. Infect
Immun 65:3736-3742.

372. Stabler, R. A., Marsden, G. L., Witney, A. A., Li, Y., Bentley, S. D., Tang, C.
M., and Hinds, J. 2005. Identification of pathogen-specific genes through microarray
analysis of pathogenic and commensal Neisseria species. Microbiology 151:2907-
2922.

373. Stern, A., Brown, M., Nickel, P., and Meyer, T. F. 1986. Opacity genes in
Neisseria gonorrhoeae: control of phase and antigenic variation. Cell 47:61-71.

374. Stern, A., Nickel, P., Meyer, T. F., and So, M. 1984. Opacity determinants of
Neisseria gonorrhoeae: gene expression and chromosomal linkage to the gonococcal
pilus gene. Cell 37:447-456.

375. Stock, A. M. 1997. Energy sensors for aerotaxis in Escherichia coli: something
old, something new. Proc Natl Acad Sci USA 94:10487-10489.

376. Stock, A. M., Robinson, V. L., and Goudrean, P. N. 2000. Two-component
signal transduction. Annu Rev Biochem 69: 183-215.

377. Strom, M. S., and Lory, S. 1993. Structure-function and biogenesis of the type
IV pili. Annu Rev Microbiol 47:565-596.

378. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dnbendorff, J. W. 1990.
Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol
185:60-89.

379. Swanson, J. 1982. Colony opacity and protein 11 compositions of gonococci.
Infect Immun 37:359-368.

380. Swanson, J. 1973. Studies on gonococcus infection. IV. Pili: their role in
attachment of gonococci to tissue culture cells. J Exp Med 137:571-589.

210

 

381. Swanson, J., Bergstrom, S., Boslego, J., and Koomey, M. 1987. Gene
conversion accounts for pilin structural changes and for reversible piliation "phase"
changes in gonococci. Antonie Van Leeuwenhoek 53:441-446.

382. Swanson, J., King, G., and Zeligs, B. 1975. Studies on gonococcus infection.
VIII. 125Iodine labeling of gonococci and studies on their in vitro interactions with
eukaryotic cells. Infect Immun 11:453-459.

383. Swanson, J., Krans, S. J., and Gotschlich, E. C. 1971. Studies on gonococcus
infection. I. Pili and zones of adhesion: their relation to gonococcal grth patterns. J
Exp Med 134:886-906.

384. Swanson, J., Robbins, K., Barrera, 0., Corwin, D., Boslego, J., Ciak, J.,
Blake, M., and Koomey, J. M. 1987. Gonococcal pilin variants in experimental
gonorrhea. J Exp Med 165:1344-1357.

385. Szeto, J., Ramirez-Arcos, S., Raymond, C., Hicks, L. D., Kay, C. M., and
Dillon, J. A. 2001. Gonococcal MinD affects cell division in Neisseria gonorrhoeae

and Escherichia coli and exhibits a novel self-interaction. J Bacteriol 183:6253-6264.

386. Taffa, N., Bjnne, G., Snndby, J., Ganstad, P., and Alestrom, A. 2002.
Prevalence of gonococcal and chlamydia] infections and sexual risk behavior among
youth in Addis Ababa, Ethiopia. Sex Transm Dis 29:828-833.

387. Taha, M. K., So, M., Seifert, H. S., Billyard, E., and Marchal, C. 1988. Pilin
expression in Neisseria gonorrhoeae is under both positive and negative
transcriptional control. EMBO J 7:4367-4378.

388. Takahashi, N., Hayano, T., and Suzuki, M. 1989. Peptidyl-prolyl cis-trans
isomerase is the cyclosporin A-binding protein cyclophilin. Nature 337 :473-475.

389. Tapsall, J. W. 2005. Antibiotic Resistance in Neisseria gonorrhoeae. Clin Infect
Dis 41 :S263-S268.

390. Tatnsova, T. A., and Madden, T. L. 1999. Blast 2 sequences - a new tool for
comparing protein and nucleotide sequences. FEMS Microbiol Lett 174:247 - 250.

391. Tauschek, M., Hamilton, C. W., Hall, L. A., Chomvarin, C., Fyfe, J. A. M.,
and Davies, J. K. 1997. Transcriptional analysis of the groESL operon of Neisseria
gonorrhoeae. Gene 189:107-1 12.

392. Testerman, T. L., Vazquez-Torres, A., Xn, Y., Jones-Carson, J., Libby, S. J.,
and Fang, F. C. 2002. The alternative sigma factor controls antioxidant defences

required for Salmonella virulence and stationary-phase survival. Mol Microbiol
43:7 71-782.

393. Thomas, C. E., and Sparling, P. F. 1996. Isolation and analysis of a fur mutant
of Neisseria gonorrhoeae. J Bacteriol 178:4224-4232.

211

394. Tian, H., Boyd, D., and Beckwith, J. 2000. A mutant hunt for defects in
membrane protein assembly yields mutations affecting the bacterial signal recognition
particle and Sec machinery. Proc Natl Acad Sci USA 97 :4730—4735.

395. Tobiason, D. M., and Seifert, H. S. 2001. Inverse relationship between pilus-
mediated gonococcal adherence and surface expression of the pilus receptor, CD46.
Microbiology 147:2333-2340.

396. Tomb, J. F., White, 0., Kerlavage, A. R., Clayton, R. A., Sutton, G. G.,
Fleischmann, R. D., Ketchnm, K. A., Klenk, H. P., Gil], S., Dongherty, B. A.,
Nelson, K., Quackenbnsh, J., Zhou, L., Kirkness, E. F., Peterson, S., Loftus, B.,
Richardson, D., Dodson, R., Khalak, H. G., Glodek, A., McKenney, K.,
Fitzegerald, L. M., Lee, N., Adams, M. D., Hickey, E. K., Berg, D. E., Gocayne, J.
D., Utterback, T. R., Peterson, J. D., Kelley, J. M., Cotton, M. D., Weidman, J.
M., Fujii, C., Bowman, C., Watthey, L., Wallin, E., Hayes, W. S., Borodovsky,
M., Karp, P. D., Smith, H. 0., Fraser, C. M., and Venter, J. C. 1997 . The complete
genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547.

397. Tonjnm, T., Freitag, N. E., Namork, E., and Koomey, M. 1995. Identification
and characterization of pilG, a highly conserved pilus- assembly gene in pathogenic
Neisseria. Mol Microbiol 16:451-464.

398. Torrnellas, J., Jackson, M. W., Pennock, J. W., and Piano, G. V. 2005. The
Yersinia pestis type III secretion needle plays a role in the regulation of Yop secretion.
Mol Microbiol 57:1719-1733.

399. Totten, P. A., Lara, J. C., and Lory, S. 1990. The rpoN gene product of
Pseudomonas aeruginosa is required for expression of diverse genes, including the
flagellin gene. J Bacteriol 172:389—396.

400. Tramont, E. C. 1976. Specificity of inhibition of epithelial cell adhesion of
Neisseria gonorrhoeae. Infect Immun 14:593-595.

40]. Trien-Cnot, P., Poyart-Salmeron, C., Carlier, C., and Courvalin, P. 1990.
Nucleotide sequence of the erythromycin resistance gene of the conjugative
transposon Tn1545. Nucleic Acids Res 18:3660.

402. Tseng, H. J., McEwan, A. G., Apicella, M. A., and Jennings, M. P. 2003.
OxyR acts as a repressor of catalase expression in Neisseria gonorrhoeae. Infect
Immun 71:550-556.

403. Tnomanen, E., Lindqnist, S., Sande, S., Galleni, M., Light, K., Gage, D., and
Normark, S. 1991. Coordinate regulation of beta-lactamase induction and
peptidoglycan composition by the amp operon. Science 251:201-204.

404. Turner, C. F., Rogers, S. M., Miller, H. G., Miller, W. C., Gribble, J. N.,
Chromy, J. R., Leone, P. A., Cooley, P. C., Quinn, T. C., and Zenilman, J. M.

212

2002. Untreated gonococcal and chlarnydial infection in a probability sample of
adults. JAMA 287 :726 - 733.

405. Tyson, M. 2005. Guidance for first-line treatment of anogenital gonorrhoea
infection. Br J Nurs. 14:646-648, 650-642.

406. Tzeng, Y. L., Datta, A., Ambrose, K., Le, M., Davies, J. K., Carlson, R. W.,
Stephens, D. S., and Kahler, C. M. 2004. The MisR/MisS two-component regulatory

system influences inner core structure and immunotype of lipooligosaccharide in
Neisseria meningitidis. J Biol Chem 279:35053-35062.

407. Ulbrandt, N. D., Newitt, J. A., and Bernstein, H. D. 1997. The E. coli signal
recognition particle is required for the insertion of a subset of inner membrane
proteins. Cell 88:187-196.

408. Valent, Q. A., de Gier, J. W., von Heijne, G., Kendall, D. A., ten Hagen-
Jongman, C. M., Oudega, B., and Luirink, J. 1997. Nascent membrane and
presecretory proteins synthesized in Escherichia coli associate with signal recognition
particle and trigger factor. Mol Microbiol 25:53-64.

409. Valent, Q. A., Scotti, P. A., High, S., de Gier, J. W., von Heijne, G., Lentzen,
G., Wintermeyer, W., Oudega, B., and Luirink, J. 1998. The Escherichia coli SRP
and SecB targeting pathways converge at the translocon. EMBO J 17:2504-2512.

410. van der Ley, P., Heckels, J. E., Virji, M., Hoogerhont, P., and Poolman, J. T.
1991. Topology of outer membrane porins in pathogenic Neisseria spp. Infect Immun
59:2963-2971.

411. van Pntten, J. P., Dnensing, T. D., and Cole, R. L. 1998. Entry of OpaA+
gonococci into HEp-2 cells requires concerted action of glycosaminoglycans,
fibronectin and integrin receptors. Mol Microbiol 29:369-379.

412. van Pntten, J. P., and Paul, S. M. 1995. Binding of syndecan-like cell surface
proteoglycan receptors is required for Neisseria gonorrhoeae entry into human
mucosal cells. EMBO J 14:2144-2154.

413. Veal, W. L., Nicholas, R. A., and Shafer, W. M. 2002. Overexpression of the
MtrC-MtrD-MtrE efflux pump due to an mtrR mutation is required for Chromosomally
mediated penicillin resistance in Neisseria gonorrhoeae. J Bacteriol 184:5619-5624.

414. Virji, M., and Everson, J. S. 1981. Comparative virulence of opacity variants of
Neisseria gonorrhoeae strain P9. Infect Immun 31:965-970.

415. Virji, M., Makepeace, K., Ferguson, D. J., and Watt, S. M. 1996.
Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors
for Opa proteins of pathogenic neisseriae. Mol Microbiol 22:941-950.

213

416. Virji, M., Watt, S. M., Barker, S., Makepeace, K., and Doyonnas, R. 1996.
The N-domain of the human CD66a adhesion molecule is a target for Opa proteins of
Neisseria meningitidis and Neisseria gonorrhoeae. Mol Microbiol 22:929-939.

417. Vogel, U., and Frosch, M. 1999. Mechanisms of neisserial serum resistance. Mol
Microbiol 32:1133-1139.

418. Wainwright, L. A., Pritchard, K. H., and Seifert, H. S. 1994. A conserved
DNA sequence is required for efficient gonococcal pilin antigenic variation. Mol
Microbiol 13:75-87.

419. Waldbeser, L. S., Ajioka, R. S., Merz, A. J., Pnaoi, D., Lin, L., Thomas, M.,
and So, M. 1994. The opaH locus of Neisseria gonorrhoeae MS11A is involved in
epithelial cell invasion. Mol Microbiol 13:919-928.

420. Walhont, A. J., Temple, G. F., Brasch, M. A., Hartley, J. L., Lorson, M. A.,
van den Henvel, S., and Vidal, M. 2000. GATEWAY recombinational cloning:

application to the cloning of large numbers of open reading frames or ORFeomes.
Methods Enzymol 328:575-592.

42]. Wang, G., and Maier, R. J. 2004. An NADPH quinone reductase of
Helicobacter pylori plays an important role in oxidative stress resistance and host
colonization. Infect Immun 72: 1391-1396.

422. Wang, J., Gray-Owen, S. D., Knorre, A., Meyer, T. F., and Dehio, C. 1998.
Opa binding to cellular CD66 receptors mediates the transcellular traversal of
Neisseria gonorrhoeae across polarized T84 epithelial cell monolayers. Mol Microbiol
30:657-671.

423. Wang, Q. P., and Kagnni, J. M. 1989. A novel sigma factor is involved in
expression of the rpoH gene of Escherichia coli. J Bacteriol 171:4248-4253.

424. Wang, R. F., and Knshner, S. R. 1991. Construction of versatile low-copy-
number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene
100:195-199.

425. Wang, S. A., Lee, M. V., O'Connor, N., Iverson, C. J., Ohye, R. G., Whiticar,
P. M., Hale, J. A., Trees, D. L., Knapp, J. S., Effler, P. V., and Weinstock, H. S.
2003. Multidrug-resistant Neisseria gonorrhoeae with decreased susceptibility to
cefixime-Hawaii, 2001. Clin Infect Dis 37:849-852.

426. Wee], J. F., Hopman, C. T., and van Pntten, J. P. 1991. Bacterial entry and
intracellular processing of Neisseria gonorrhoeae in epithelial cells:

immunomorphological evidence for alterations in the major outer membrane protein
P.1B. J Exp Med 174:705-715.

214

427. Weel, J. F., Hopman, C. T., and van Pntten, J. P. 1991. In situ expression and
localization of Neisseria gonorrhoeae opacity proteins in infected epithelial cells:
apparent role of Opa proteins in cellular invasion. J Exp Med 173:1395-1405.

428. Weel, J. F., and van Pntten, J. P. 1991. Fate of the major outer membrane
protein P.IA in early and late events of gonococcal infection of epithelial cells. Res
Microbiol 142:985-993.

429. Weiss, D. S., Chen, J. C., Ghigo, J. M., Boyd, D., and Beckwith, J. 1999.
Localization of FtsI (PBP3) to the septa] ring requires its membrane anchor, the Z ring,
FtsA, FtsQ, and FtsL. J Bacteriol 181:508-520.

430. Wessler, S., Mnenzner, P., Meyer, T. F., and Naumann, M. 2005. The anti-
inflammatory compound curcumin inhibits Neisseria gonorrhoeae-induced NF-
kappaB signaling, release of pro-inflammatory cytokines/chemokines and attenuates
adhesion in late infection. Biol Chem 386:481-490.

431. West, S., and Sparling, F. 1985. Response of Neisseria gonorrhoeae to iron
limitation: alterations in expression of membrane proteins without apparent
siderophore production. Infect Immun 47 :388 - 394.

432. Whitehead, R. N., and Cole, J. A. 2006. Different responses to nitrate and nitrite
by the model organism Escherichia coli and the human pathogen Neisseria
gonorrhoeae. Biochem Soc Trans 34:111-114.

433. Wild, K., Halic, M., Sinning, 1., and Beckmann, R. 2004. SRP meets the
ribosome. Nat Struct Mol Biol 11:1049-1053.

434. Winzeler, E., and Shapiro, L. 1995. Use of flow cytometry to identify a
Caulobacter 4.5 S RNA temperature-sensitive mutant defective in the cell cycle. J Mol
Biol 251:346-365.

435. Wolff, K., and Stern, A. 1995. Identification and characterization of specific
sequences encoding pathogenicity associated proteins in the genome of commensal
Neisseria species. FEMS Microbiol Lett 125:255-263.

436. Wolfgang, M., Lauer, P., Park, H. S., Brossay, L., Hebert, J., and Koomey,
M. 1998. PilT mutations lead to simultaneous defects in competence for natural

transformation and twitching motility in piliated Neisseria gonorrhoeae. Mol
Microbiol 29:321-330.

437. Wolfgang, M., Park, H. S., Hayes, S. F., van Pntten, J. P., and Koomey, M.
1998. Suppression of an absolute defect in type IV pilus biogenesis by loss-of-function

mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae. Proc Natl Acad
Sci USA 95:14973-1497 8.

215

438. Wolfgang, M., van Pntten, J. P., Hayes, S. F., Dorward, D., and Koomey, M.
2000. Components and dynamics of fiber formation define a ubiquitous biogenesis
pathway for bacterial pili. EMBO J 19:6408-6418.

439. Wu, J., and Newton, A. 1997. The Caulobacter heat shock sigma factor gene

rpoH is positively autoregulated from a 632-dependent promoter. J. Bacteriol.
179:514-521.

440. Yamauchi, S., Oknyama, H., Morita, E. H., and Hayashi, H. 2003. Gene
structure and transcriptional regulation specific to the groESL operon from the
psychrophilic bacterium Colwellia maris. Arch Microbiol 180:272-278.

441. Yang, Q. L., and Gotschlich, E. C. 1996. Variation of gonococcal
lipooligosaccharide structure is due to alterations in poly-G tracts in lgt genes
encoding glycosyl transferases. J Exp Med 183:323-327.

442. Yura, T., N agai, H., and Mori, H. 1993. Regulation of the heat-shock response
in bacteria. Annu Rev Microbiol 47 :321-350.

443. Yura, T., and Nakahigashi, K. 1999. Regulation of the heat-shock response.
Curr Opin Microbiol 2:153-158.

444. Yura, T., Nakahigashi, K., and Kanemori, M. 1996. Transcriptional regulation
of stress-inducible genes in procaryotes. Exs 77:165-181.

445. Zak, K., Diaz, J. L., Jackson, D., and Heckels, J. E. 1984. Antigenic variation
during infection with Neisseria gonorrhoeae: detection of antibodies to surface
proteins in sera of patients with gonorrhea. J Infect Dis 149:166-174.

446. Zarantonelli, L., Borthagaray, G., Lee, E. H., Veal, W., and Shafer, W. M.
2001. Decreased susceptibility to azithromycin and erythromycin mediated by a novel

mtrR promoter mutation in Neisseria gonorrhoeae. J Antimicrob Chemother 47 :651-
654.

447. Zhang, J., Li, G., Bafica, A., Pantelic, M., Zhang, P., Broxmeyer, H., Lin, Y.,
Wetzler, L., He, J. J., and Chen, T. 2005. Neisseria gonorrhoeae enhances infection
of dendritic cells by HIV type 1. J Irnmunol 174:7995-8002.

448. Zhang, J. P., and Normark, S. 1996. Induction of gene expression in
Escherichia coli after pilus-mediated adherence. Science 273:1234-1236.

449. Zheng, H. Y., Hassett, D. J., Bean, K., and Cohen, M. S. 1992. Regulation of
catalase in Neisseria gonorrhoeae. Effects of oxidant stress and exposure to human
neutrophils. J Clin Invest 90:1000-1006.

450. Zheng, N., and Gierasch, L. M. 1997. Domain interactions in E. coli SRP:
stabilization of M domain by RNA is required for effective signal sequence
modulation of NG domain. Mol Cell 1:79-87.

216

 

451. Zhou, D., Lee, N. G., and Apicella, M. A. 1994. Lipooligosaccharide
biosynthesis in Neisseria gonorrhoeae: cloning, identification and characterization of
the alpha 1,5 heptosyltransferase I gene tfaC. Mol Microbiol 14:609-618.

452. Zhou, Y. N., Knsnkawa, N., Erickson, J. W., Gross, C. A., and Yura, T. 1988.
Isolation and characterization of Escherichia coli mutants that lack the heat shock
sigma factor 0'32. J Bacteriol 170:3640-3649.

453. Hill, S.A., Samuels, D.S., Carlson, J.H., Wilson, J., Hogan, D., Lubke, L., and
Belland, R.J. 1997. Integration host factor is a transcriptional cofactor of pilE in
Neisseria gonorrhoeae. Mol Microbiol. 23:649-656.

454. Skaar, E. P., M. P. Lazio, and Seifert, H. S. 2002. Roles of the real and recN

genes in homologous recombination and DNA repair pathways of Neisseria
gonorrhoeae. J. Bacteriol. 184:919-927.

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