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This is to certify that the

dissertation entitled
Characterization of the genes of Actinobacillus

i pleuropneumoniae involved in oxidative stress
and pathogenesis

 

presented by
Robin Jeanne Kastenmayer

has been accepted towards fulfillment
of the requirements for

211.12. degree in Microbiology and
Molecular Genetics

Major professor

Date Mgr figQOOJ/
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MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

PLACE IN RETURN Box to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

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6/01 cJClRC/Dateouepss-sz

CHARACTERIZATION OF THE GENES OF AC T INOBA CILL US
PLEUROPNEUMONIAE INVOLVED IN OXIDATIVE STRESS AND

PATHOGENESIS

by

Robin Jeanne Kastenmayer

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

2002

I XL
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ABSTRACT

CHARACTERIZATION OF THE GENES OF AC T INOBACILL US
PLEUROPNEUMONIAE INVOLVED IN OXIDATIVE STRESS AND

PATHOGENESIS

By

Robin Jeanne Kastenmayer

Actinobacillus pleuropneumoniae is the causative agent of porcine hemorrhagic
pleuropneumonia, a severe and contagious respiratory disease. While many virulence
factors in A. pleuropneumoniae have been identified, there are still many unanswered
questions and unknown proteins which represent potential antibiotic targets or effective
vaccines. An in vivo technology system (IVET) developed in A. pleuropneumoniae was
refined so that novel virulence factors could be identified and characterized.

Using this system, twenty five unique in vivo induced (ivi) clones were identified.
These clones contained promoters that were induced during infection of the pig (in viva)
but had minimal expression on rich laboratory media (in vitro). These genes that were
upregulated during infection could be divided into four categories: proteins previously
identified as virulence factors, those required for metabolism during rapid grth and
colonization, proteins of known function that had not been previously characterized as
required for virulence, and unknown proteins.

One of these ivi genes encodes Ohr, an organic hydroperoxide reductase. Ohr

was shown to be regulated in response to organic peroxides. Ohr functions during the

oxidative stress that occurs subsequent host immune cell influx and degranulation to
release oxygen radicals. Ohr and its regulator are serotype-specific, found only in three
of the twelve serotypes. The characterization of 0hr shows the new information that can
be gained from the use of IVET screens to identify virulence genes.

In order to further characterize the identified ivi genes, as well as to construct
potential vaccines, genetically defined attenuated mutants are required. Mutant
construction was attempted for both 0hr and for ile, a gene involved in isoleucine-
leucine-valine biosynthesis.

The characterization of 0hr and initial analysis of 1'le have shown not only the
potential information that can be gained through the use of IVET, but in addition, the
many questions that arise. IVET has contributed to the understanding of A.
pleuropneumoniae pathogenesis through the identification of virulence factors and by

providing an initial platform from which more questions can be asked.

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ACKNOWLEDGMENTS

I would like to thank my mentor, Martha Mulks, for her encouragement, support
and understanding. She has been the primary factor in my growth as an independent
scientific researcher and has had a tremendous impact on my career. I wish to thank the
members of my committee, Dr. Michael Bagdasarian, Dr. Wendy Champness, Dr. Linda
Mansfield, and Dr. Larry Snyder, for their advice and guidance and unwavering support.

I would like to thank the current and former members of the Mulks’ laboratory for
making it a fun and exciting place to work. Without their help and support, I would not
have been able to accomplish this research. I wish to thank Scott Doree, Trevor Wagner,
and Traci Eagen for their enjoyable and stimulating discussions of science. I would
especially like to thank Dr. Troy Fuller and Dr. Byron Hagewood for their wise advice
and assistance in the early stages of my research. In addition, I would like to thank Dr.
Fuller and Dr. Rick Martin for two exciting and rewarding collaborations.

I also wish to thank the many people, both within and outside of the College of
Veterinary Medicine, who have made the dual degree program possible. Their moral
support and belief in myself and the program have been an unwavering strength. I would
like to thank Dr. Ron Patterson, Angie Zell, Coreena Spitzley, and Dr. Jerry Dodgson for
their aid in establishing this program. I especially wish to thank the current and former
Associate Deans for Graduate Studies and Research, in particular, Dr. John Baker.
Professional school has been a unique and challenging experience, and one that has
shaped my life both personally and professionally. I wish to thank my instructors,

classmates and the staff in the college for this wonderful and exciting portion of my life.

Co]

for

I wish to thank the Harold Wetterberg Foundation, the Jaqua Foundation, the
College of Veterinary Medicine, and the Graduate School at Michigan State University
for their financial support. Without this funding, my program would have been
impossible.

Perhaps the most important people to thank are those that are there day after day
with unconditional love and support. It is for this that I wish to thank my husband and
my family and fi'iends. Without your understanding, I would not have been able to

accomplish all that I have done. Without your love, it would not have mattered.

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TABLE OF CONTENTS

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

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

Chapter 1.

Actinobacillus pleuropneumoniae: A bacterial pathogen of swine and the virulence

factors required for infection and survival of oxidative stress .................................. 1
Introduction ................................................................................... 2
Epidemiology of A. pleuropneumoniae ................................................... 3
Prevention and treatment of disease ....................................................... 4
Pathology ..................................................................................... 5
Virulence factors ............................................................................ 6
Virulence factors associated with oxidative stress ..................................... 11
Bacterial response to oxygen radicals .................................................... 11
SoxRS ....................................................................................... l3
OxyR ......................................................................................... 14
Per ............................................................................................ 15
Oxidative regulators in A. pleuropneumoniae .......................................... l6
Scope of dissertation ....................................................................... 17
References ................................................................................... 19

Chapter 2.

Identification of virulence factors of A. pleuropneumoniae by the use of IVET (in vivo

expression technology) ............................................................................ 28
Abstract ..................................................................................... 29
Introduction ................................................................................. 31
Virulence factors of A. pleuropneumoniae ............................................. 31
Characterization of virulence genes ..................................................... 32
IVET background .......................................................................... 35
Materials and methods ..................................................................... 37
Results ....................................................................................... 47
Discussion ................................................................................... 63
References ................................................................................... 68

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Chapter 3.
ahr, encoding an organic hydroperoxide reductase is an in viva induced gene of

A. pleurapneumaniae ............................................................................... 74
Abstract ...................................................................................... 75
Introduction ................................................................................. 77
Materials and methods ..................................................................... 79
Results ....................................................................................... 89
Discussion ................................................................................. 106
References ................................................................................. 112

Chapter 4.

Genetic disruption of ahr and ile ............................................................... 117
Abstract .................................................................................... l 18
Introduction ................................................................................ 119
Materials and methods ................................................................... 121
Results and discussion ................................................................... 135
References ................................................................................. 141

Chapter 5.

Summary ............................................................................................ 143
References ................................................................................. 150

vii

Table .'
Table
Table
Table
Table

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Table 2-1.
Table 2-2.
Table 3-1.
Table 4-1.
Table 4-2.

Table 5-1.

LIST OF TABLES

Strains and plasmids used in this work. .......................................................... 38
Identification of M insert. ............................................................................... 50
Strains and plasmids used in this work. ........................................................... 80
Codon usage table for Actinabacillus pleurapneumam’ae ............................. J 23
Plasmids constructed in this study ................................................................. 124
Identification of 42 ivi clones. ....................................................................... .145

viii

 

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LIST OF FIGURES

Figure 2-1 Diagram of pTF86 .................................................................. 39
Figure 2-2 Diagram of A. pleurapneumaniae IVET construction and infection ......... 41
Figure 2-3 Southern blot of AP233 and AP233/piviE ....................................... 48
Figure 2-4 Photonic camera picture showing a pool of clones .............................. 49
Figure 2-5. Organization of the 25 unique in viva induced inserts ........................... 52
Figure 3-1. Pulmonary damage and in viva lux expression of a swine lung ............... 91
Figure 3-2. Protein sequence alignment of putative Ohr proteins ........................... 93
Figure 3-3. Correlation of ahr presence with resistance to organic hydroperoxides. . . ...95
Figure 3-4. Induction of the cloned ahr promoter in A. pleurapneumaniae serotype 1.96
Figure 3-5. Expression of the ahr promoter is induced by 125 M CHP ................ 100
Figure 3-6. Ohr activity .......................................................................... 102
Figure 3-7. Primer extension analysis ......................................................... 104
Figure 3-8. Purification of Ohr-hexahistidine protein ........................................ 105
Figure 4-1. Construction of ohr-kanamycin suicide vector ................................. 129
Figure 4-2. Construction of pilv15’3 ’ .......................................................... 130
Figure 4-3. Plasmid map of pC18KnadV ...................................................... 131
Figure 4-4. Plasmid map of pC18kanNadV .................................................. 133
Figure 4-5. Construction of pCl8ivianNad ................................................. 134

ix

Chapter 1

Actinabacillus pleurapneumaniae: A bacterial pathogen of swine and the virulence

factors required for infection and survival of oxidative stress

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Introduction

Actinabacillus pleurapneumaniae is a respiratory pathogen of swine that causes a
severe contagious hemorrhagic pleuropneumoniae. Several virulence factors have been
identified in A. pleurapneumaniae and have been shown to be required for infection.
While much is known about key virulence factors, a protective vaccine against all A.
pleurapneumam'ae serotypes is not available.

This chapter reviews the epidemiology and pathology of A. pleurapneumaniae, in
addition to discussing the known A. pleurapneumaniae virulence factors, these include
capsule, lipopolysaccharide, several exotoxins, pilin, and specific proteins. The
discussion of virulence factors includes those involved in survival in response to
oxidative stress. The discussion of the virulence factors involved in oxidative stress in A.
pleurapneumaniae includes those identified in this bacterium, as well as those found in
other bacteria and are hypothesized to function as virulence factors in A.
pleurapneumaniae.

The scope of this dissertation is to expand the knowledge of A. pleurapneumaniae
pathogenesis by identifying virulence factors required for infection. By identifying these
factors, potential subunit vaccines and attenuated mutants for use as vaccines can be

developed with the long term plan of creating an effective, heterologous vaccine.

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Epidemiology of Actinobacillus pleuropneumoniae

Since the initial characterization of Actinabacillus pleurapneumaniae in 1961, the
worldwide significance of this pathogen has grown (26, 49, 83). A. pleurapneumaniae is
the cause of porcine hemorrhagic pleuropneumonia, which in the acute disease causes
high morbidity and mortality. The severity of the clinical signs and the duration of the
infection vary with the immunological status of the pig, the degree of exposure, and the
environmental conditions (71). In the acute infection, the pigs are febrile and depressed
with respiratory signs, such as dypsnea and cough (26, 82). Pigs that recover from these
acute infections are often maintain the organism in the nasal passages and respiratory
discharges and can propagate the bacteria through aerosal or direct contact with na’r‘ve
pigs (71). The chronic infection is ofien missed, since the pigs exhibit no fever, and only
occasionally show respiratory distress (71). Often the chronic form is only detected at
slaughter by the high prevalence of pleuritis and fibrin deposition in the lungs of the
slaughtered swine (81). In either the acute or the chronic infection, the producer is
economically affected, either through the loss of individual animals or by a decrease in
weight gain in the chronically infected herd (71).

A. pleuropneumoniae is a gram negative pleomorphic member of the
Pasteurellaceae family, with two biotypes and twelve serotypes in biotype one. The
biotypes are based upon a requirement for nicotinamidc adenine dinucleotide (NAD),
whereas the serotypes are differentiated by capsular and LPS genes (74). In the United
States, the predominant serotypes are one, five and seven in the NAD dependent biotype

(74).

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Prevention and Treatment of Disease

A. pleurapneumam’ae infection can be treated with antimicrobials such as the
beta-lactams, chloramphenicol, cotn'moxazole, tetracyclins, and enrofloxacin (71).
However, since the lung damage is irreversible, the most effective treatment is
prevention. Preventative measures include vaccination with serotype-specific bacterins
and improved farm management to prevent exposure of naive pigs to chronically infected
animals. It has been shown that vaccination with a serotype-specific bacterin does not
provide heterologous protection against the other serotypes, whereas a natural infection
provides protection against subsequent challenge with other serotypes (15, 56, 72). This
observation has led to research in the development of vaccines that have the same
protective value as a natural infection.

Subunit vaccines composed of outer membrane antigens isolated from one
serotype have been proven to provide homologous protection against subsequent
challenge by the same serotype and have provided some protection against challenge
heterologous serotypes (10, 17, 24, 25). Vaccines consisting of exotoxins and other
extracellular proteins have been shown to provide good homologous protection and one
vaccine consisting of exotoxins and an outer membrane protein has shown homologous
and heterologous protection (22, 93). Attenuated live A. pleurapneumaniae vaccines are
currently in development with the hope of developing a vaccine that offers complete
heterologous protection (39, 40, 51). The use of an attenuated live A. pleurapneumaniae
that is unable to produce riboflavin as a vaccine provides complete protection against

subsequent homologous challenge and reasonable protection against challenge by a

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to provide the complete cross protection that occurs following natural infection.

Pathology

A. pleurapneumaniae causes a fulminating bronchopneumonia with an
accompanying pleuritis that can resolve to fibrin deposition and regions of necrotic lung
tissue in convalescent animals (82). The disease course can range from a subclinical
chronic infection to an acute infection in a herd naive to A. pleurapneumaniae. In these
immunologically na'r've herds, morbidity can be as high as 100 percent (47, 90). The lung
necrosis and fibrin deposition that are characteristic of this pneumonia are predominantly
seen in the apical and dorso-caudal lung lobes (82).

The diagnosis of porcine pleuropneumonia caused by A. pleurapneumam'ae can
be made initially based upon clinical signs and observation of the lung lesions. The lung
damage includes dilation of the alveolar capillaries with erythrocytes, thrombocytes and
fibrin. The lung parenchyma shows areas of coagulative necrosis and edema
accompanied by an influx of inflammatory cells, such as macrophages, lymphocytes, and
neutrophils (5, 63). Bacteria can be routinely cultured from the respiratory surfaces and
from the nasal discharge; with occasional isolation from the respiratory associated

lymphoid tissues (71).

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Virulence Factors

The disease symptoms are due in part to bacterial endotoxin, exotoxins, and
capsule, in addition to the activity of specific virulence proteins. Capsule and
lipopolysaccharide (LPS) contribute to bacterial survival within the host by protecting
against complement-mediated lysis and phagocytosis (16, 27, 52). The role of capsule in
preventing phagocytosis can be demonstrated by the addition of anti-capsular antibodies,
which when incubated with A. pleurapneumam'ae allow for effective opsonization of the
bacteria by polymorphoneutrophils (52, 79). However, the presence of anticapsular
antibodies only provides partial protection during infection, suggesting the presence of
other virulence factors required for disease (52, 79). Both LPS endotoxin and exotoxin

contribute to the lung damage by causing damage to the lung parenchyma (27, 28).

Lipopolysaccharide (LPS) LPS endotoxin is a common virulence factor in gram negative
bacteria and aids in bacterial survival within the host. LPS consists of a lipid portion
(lipid A) that is responsible for much of the toxicity associated with endotoxin and a
variable oligosaccharide region. The O-antigen oligosaccharides protect the bacteria
from complement-mediated lysis by sterically inhibiting insertion of the membrane attack
complex (15). In addition, surface-exposed LPS may act as an adhesin by binding
respiratory tract mucous and epithelial cells (3, 27). This binding may be a requisite
initial event in the disease process (3, 70).

In addition to these roles of LPS, it has been shown that purified endotoxin is
toxic to leukocytes, platelets, and vascular endothelium and can cause lung lesions, in a

dose-dependent manner (27, 28). These lung lesions are the result of an interstitial

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pneumonia but lack the hemorrhage typically seen during infection by A.
pleuropneumoniae. Perhaps the most important role of LPS in virulence is the effect of
LPS on the host immune system. It is believed that the immune cell influx associated
with gram-negative endotoxin and the release of proinflammatory mediators, such as IL-1
and TNF-or, is triggered by the presence of the LPS, in particular the lipid portion (18,
68). This results in a classical inflammatory reaction including edema and increased
capillary permeability, neutrophil infiltration, and deposition of fibrinogen. The presence
of antibodies directed against gram-negative LPS, while not preventing infection from
occurring, did cause a decrease in the morbidity and mortality associated with infection
by A. pleurapneumam'ae (29).

This evidence suggests that while LPS may be partially responsible for host
immune cell influx and a portion of the disease symptoms, other virulence factors are

required to produce the symptoms typically seen during infection.

Exotoxins. A key virulence factor of A. pleurapneumam'ae was hypothesized to be an
excreted toxin, since culture supematant was toxic for swine monocytes and respiratory
epithelium and induced lung lesions when endobronchially inoculated (4). The heat
labile protein Apx exotoxins found in A. pleurapneumaniae are of the RTX (glycine-rich
repeats in structural toxins) family. Four different exotoxins (ApxI-Aple), encoded by
distinct operons, have been described in A. pleuropneumoniae. Each serotype within
biotype one contains two or more of these different classes of exotoxins (32, 35, 80).
These toxins show both hemolytic and cytotoxic activity and can destroy alveolar

macrophages, thymic lymphocytes, erythrocytes, and neutrophils (77).

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The mechanism of action of the toxins can be extrapolated from work done with
Escherichia cali alpha hemolysin, HlyA, which is homologous to ApxI. This toxin
inserts into eukaryotic cell membranes, creating pores and leading to osmotic swelling
and subsequent cellular lysis (84). This lysis causes the release of oxygen radicals and
lysosomal enzymes from the damaged phagocytes, which in turn damage host tissue,
resulting in necrosis and hemorrhage (14, 23, 91). Even sublytic doses of toxin cause
abnormal phagocytosis and chemotaxis of macrophages and polymorphoneutrophils (23,
88, 91). The presence of the glycine rich repeats allows for efficient binding of calcium
and loss of the repeats results in decreases in both calcium binding and hemolytic ability
(6). Calcium binding is predicted to occur afler export from the bacterial cell (6). The
production of the pore occurs via two stages, the reversible adsorption followed by the
irreversible insertion of the toxin complex into the eukaryotic cell membrane. This
insertion requires proper protein folding to allow for the formation of hydrophobic alpha
helices flanked by amphipathic membrane-binding helices (84). A completely formed
pore alters the osmotic potential of the cell, in addition to allowing flux of ions across the
membrane. Calcium appears to be required for initial insertion, but does not appear to be
required for pore formation and may be involved in the proper folding of the protein and
exposure of hydrophobic regions (6). The acylation of the protoxin may serve to mediate
membrane association and the assembly of the multiple protein pore (84).

The first identified A. pleurapneumaniae RTX toxin, ApxI, was shown to be a
potent hemolysin and cytotoxin and was strongly immunogenic (35, 69). ApxI is induced
by calcium and requires calcium for activity (21, 94). Apr is a less potent hemolysin,

requires a higher concentration of calcium for hemolysis and is not induced by calcium

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(94). Apxl and Apr share greater than 50% identity at the protein level (35). Apxlll is
unable to cause erythrocyte lysis, but is able to cause lysis of alveolar macrophages (57,
64). In addition, Apxlll has a lower sequence homology to either Apxl or Apr (54).
Apr toxin, which shows sequence similarity to iron-regulated RTX toxins from
Neiserria meningitidis, is produced only during infection, with an unknown method of
regulation (80).

Each RTX toxin operon comprises four open reading frames encoding the
protoxin, an acetylase, and two secretion proteins. The protoxin is activated
posttranslationally through protein acylation by the acetylase. The toxin is secreted
through a type I secretion apparatus produced by the remaining two proteins within the
operon (34, 37). The ApxII protein, which is hemolytic and cytotoxic, is encoded by an
operon containing the two structural open reading frames, but without functional
secretion genes. Apr can be secreted by the secretion apparatus of either Apxl or
Apxlll (33, 55).

The importance of the RTX exotoxins has been demonstrated by several A.
pleurapneumaniae mutants that have been characterized. An A. pleurapneumaniae
serotype 1 mutant, which lacked Apxl and Apr was shown to have decreased virulence
in comparison to the parent strain (53). However, additional factors apart from the
exotoxins are required for disease, since piglets with high hemolysin neutralization titers
still show morbidity, albeit they have a lesser degree of lung damage than pigs with lower
titers (15).

While much has been leamed about the structure and the role in virulence of the

Apx toxins, the regulation of these genes during infection is still not complete. A gene

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has been identified in A. pleurapneumaniae that has been proposed to regulate the
expression of these toxins (36). The HlyX protein contains a [4Fe-4S] cluster and is
similar to the Escherichia cali FNR global regulator, which functions under anoxic
conditions (45). Additionally, the HlyX protein may not be the sole regulator of the
toxins. Aple, which is produced during infection, may be regulated by a complex

pathway or by more than one regulator (80).

F imbriae. F imbriae and pili are found on the surface of many prokaryotic cells and have
been shown to be essential for attachment to host cells and subsequent colonization. A.
pleurapneumam'ae has been shown to express fimbriae during infection, which are lost
upon growth on laboratory media (92). Type four fimbriae have recently been described
in A. pleurapneumaniae serotype one and have been shown to be expressed during
growth on a chemically defined media under microaerophilic conditions, suggesting that
there exist A. pleurapneumaniae genes that are induced during conditions such as the

nutrient limitation that occurs during infection of the porcine respiratory tract (96).

Iron scavenging proteins. One of the well characterized host responses to bacterial
infection is to limit the free iron concentration, thus limiting an essential element for
microbial grth and proliferation. Lactoferrin and transfen'in are secreted host factors
that are able to bind to free iron and sequester it. In order to obtain the iron necessary for
growth, some bacteria have evolved siderophores and iron-regulated proteins that are
capable of scavenging the free iron and host sequestered iron for microbial usage. A.

pleuropneumoniae expresses transfen'in-binding proteins (pr1 and pr2) that are

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specifically induced under iron limitation and are capable of scavenging the iron for use
by the bacterium (41, 42, 73). prl is a transmembrane protein capable of transporting
iron across the outer membrane (19), whereas pr2 has been shown to be an outer
membrane protein that contains an amino-terminal region capable of binding specifically
to porcine transferrin and not human or bovine transferrin (41-43, 86). A.
pleurapneumaniae mutants that lack either or both transferrin-binding proteins are unable
to survive within the host or cause disease (2). The pr proteins are cotranscribed with
two upstream genes, ebe and ebe (89). EbeD has been shown to be essential for the
utilization of iron from porcine transfenin (89). Three proteins (Aqu, AfuB, and AfirC)
have been identified in A. pleuropneumoniae and proposed to function in transport of the
iron from transferrin and into the bacterium (11). Aqu has homology to a periplasmic
iron-binding protein, whereas AfuB and AfuC are most likely membrane transport
systems (11). Together the pr and the Afu proteins are responsible for acquisition of

host iron in order to enable bacterial growth and survival.

Virulence Factors Associated with Oxidative Stress Response

Bacterial Response to Oxygen Radicals

To combat the lethal effect of reactive oxygen species, many bacteria have
adopted methods through which to detoxify these oxygen species. The oxygen species
are released by host immune cells as well as generated by the aerobic metabolic activities
of bacteria and bacterial enzymes (44, 67). Without the proteins necessary to combat

these reactive oxygen molecules, bacteria would be unable to survive and hence could not

11

cause infection. Several genes encoding proteins necessary for survival under oxidative
stress have been identified in A. pleurapneumam'ae. In addition, several proteins
necessary for detoxification of oxygen radicals have been identified in other bacteria and
could exist in A. pleurapneumaniae.

Major reactive oxygen species include the superoxides, peroxides, and
hypohalides. Superoxides are detoxified by the superoxide dismutases to form peroxides.
These peroxides are in turn degraded by peroxidases and catalases. The cellular damage
inflected by these oxygen radicals includes interacting with DNA and proteins to induce
DNA mutations and protein denaturation and inactivation, respectively. Additional
enzymatic damage results when these reactive oxygen species in turn oxidize the
disulfide bonds within proteins and oxidize metals causing protein denaturation and loss
of catalytic sites (67). Membrane damage to the cell can also result from autocatalytic
oxidation of lipid moities (67). Since the effect of oxygen radicals is so profound and if
lefl unchecked can lead to the destruction of the cell, continual vigilance is required to
detect the presence of oxidative damage and activate regulons capable of detoxifying
oxygen radicals and repairing the cellular damage. It is by monitoring the oxidative state
of the molecules, metals and sulfirr moities, that react so vigorously to oxygen radicals,
that bacteria can regulate the defense mechanisms involved in oxidative stress response
(78). The major global regulators of oxidative defense genes include SoxRS, OxyR and

PerR.

Virulence factors of A. pleuragneumaniae that respond to oxidative stress. One of the
key components in the host reaction to bacterial infection is the respiratory burst and

degranulation of host neutrophils and the subsequent release of hypoclorate, superoxides,

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and peroxides. These reactive oxygen species can cause cell membrane lysis,
inactivation of metabolic enzymes, oxidation of membrane lipids, and DNA damage (1,
7, 42, 50, 60). A. pleurapneumaniae contains superoxide dismutase genes, sadA and
sadC, that produce proteins capable of reducing superoxides. SodC, a copper-zinc
containing superoxide dismutase, is constitutively expressed, whereas SodA, a
manganese containing superoxide dismutase, has been shown to be produced under
anaerobic conditions of iron limitation (61). A. pleurapneumaniae has been shown to be
catalase positive; i.e., able to reduce hydrogen peroxide to oxygen and water, but the gene
responsible for this activity has not been identified (59).

A. pleurapneumaniae also contains Ohr, an organic hydroperoxide reductase that
is induced by exposure to organic hydroperoxides and may be responsible for the
reduction of organic peroxides that are encountered during exposure to neutrophil
contents or those generated during normal cellular metabolism. The identification and

characterization of ahr is the subject of chapter three of this dissertation.

SoxRS

SoxR, a major sensor of oxidative stress within the cell responds to the oxidative
state of a bound metal. SoxR contains two [2Fe-ZS] centers that upon oxidation of the
iron cause activation of SoxR (20). This activated protein then enhances transcription of
saxS, which encodes a transcription factor, resulting in increased SoxS production (78).

The SoxS protein in turn binds to a consensus site (AnnGCAY) overlapping the —35

13

regnn

reguk

SoxR
endor
inclui

slit

0“:

region and interacting with RNA polymerase to activate the SoxS regulon and negatively
regulate its own promoter (78).

The E. cali genes that are induced by superoxide stress and regulated by the
SoxRS system include genes that encode superoxide dismutases, a gene encoding
endonucleaselV, ahpC, and zwf (46, 58, 62, 95). The superoxide dismutases, which
include SodB, SodC, and SodA, are responsible for reducing superoxides to peroxides,
which can then be inactivated by catalase or by a different protein, such as
alkylhydroperoxide reductase (AhpC). The endonuclease, which is involved in DNA
repair, could be responsible for correcting DNA damaged by oxygen radicals (8). The
role of wa, a glucose-6-phosphate dehydrogenase, could be to supply reducing
equivalents in the form of NADH to help reduce oxidized proteins and metals. (46, 95)
(75). In addition it has been shown that both OxyR and SoxRS activate Fur, the ferric
iron uptake regulator, allowing for modulation of iron concentration (97). This regulation
in the concentration of iron is important, since the toxicity of both hydrogen peroxide and

superoxides are exacerbated by high concentrations of iron (38, 87, 97).

OxyR

OxyR is activated via the structural change that occurs when an intramolecular
disulfide bond between homodimers of OxyR is oxidized. This activated OxyR then
binds, with a consensus site of ATAG, to occupy four consecutive DNA major grooves,
which in turn results in increased RNA polymerase recruitment and transcription (12).

OxyR in turn represses its own transcription, allowing for a constant intracellular

14

COHCC

p€?0X'

cone:
They
peror
itht
liliOTi
nnra.
Uprn

redn.

Per

concentration of protein, which is important for a timely response to an influx of
peroxides.

OxyR regulates multiple genes, some of which are regulated by SoxRS as well.
The OxyR regulon was initially identified as the proteins that were responsible for
adaptation to hydrogen peroxide in Salmonella. The genes within the regulon allow
Salmonella to be grown in the presence of 10 mM hydrogen peroxide, a lethal
concentration, after the bacterium has been adapted in 60 uM hydrogen peroxide (13).
These genes encode proteins that include: a) those responsible for the reduction of the
peroxide to an alcohol, HPI catalase (KatG) and AhpCF; b) those responsible for
reducing oxidized enzymes, glutathione reductase (GorA), glutaredoxin I (erA), and
thioredoxin II (Ter); and c) those involved in modifying the concentration of
intracellular iron, Fur and an iron-sulfur containing protein (FhuF) that is involved in iron
uptake (13, 30, 76, 85, 97). In addition, zwf and sadA are regulated by OxyR to supply
reducing equivalents to the cell and protect against oxygen radicals, respectively. Other
genes induced by high concentrations of hydrogen peroxide encode proteins responsible
for disulfide bond formation (DsbG), formation of heme containing compounds (HemH),

and genes in an operon for sulfur mobilization (65, 98, 99).

Per

While OxyR and SoxRS have been found in many bacteria after their initial
characterization in Salmonella and E. cali, there exist additional oxidative stress
regulators in other bacteria. Per was initially characterized in Bacillus subtilis as a global

regulator that responded to iron, manganese, and hydrogen peroxide stress (9). Per has

15

K.“

been shon'r
functional 3
lentil. all
lCTAtnTT:
itself and fi

ind-nee 0le1

Oxidatii e

The
analyzed it
cell (31‘ be
i funetion;
l'dlE. a gel
that has in
Encoded b.
bten idem
da

Thus, [he

Gilliam.e S'

been shown to be a metalloregulatory protein that has sequence similarity to Fur and
functional analogy to OxyR (9). The genes in the Per regulon include ahpCF, katA, and
hemAH, all of which were shown to contain a consensus Per box in the promoter
(CTAtnTTAtAATNATTATAAattA) (9). In addition, Per has been shown to repress
itself and fur (38). The Per protein found in Staphylaccacus aureus has been shown to

induce ahpC, katA, and tpr (48).

Oxidative Regulators in Actinobacillus pleuropneumoniae

The genome of several Pasteurellaceae species have been sequenced and can be
analyzed for the presence of regulators that respond to the oxidative stress profile of the
cell (31, 66). Haemaphilus influenzae has been shown to contain an axyR homologue and
a functional OxyR consensus binding sequence has been identified in the promoter of
katE, a gene encoding a catalase (31, 78). In A. pleuropneumoniae serotype 5, a gene
that has homology to axyR has been identified, but a functional analysis of the protein
encoded by this gene has not been performed. However, saxRS homologues have not
been identified in H. influenzae or in the other Pasteurellaceae members sequenced to
date, including A. pleuropneumoniae serotype 5 or A. pleuropneumoniae serotype 1.
Thus, the search for the A. pleuropneumoniae regulators of genes that respond to

oxidative stress must be found by other mechanisms.

16

Scot

ple’ll.

lll'E

toil

dun}

(in i

redo
ln TC
Subs
and,
Chan

lllgj

Scope of Dissertation

The scope of this dissertation is to identify and characterize genes necessary for A.
pleuropneumoniae pathogenesis. To accomplish this goal, an in viva technology system
(IVET) developed in A. pleuropneumoniae was refined so that novel virulence factors
could be identified and characterized.

Chapter two of this dissertation discusses the twenty five unique in viva induced
(ivi) clones that were identified. These clones contained promoters that were induced
during infection of the pig (in viva) but had minimal expression on rich laboratory media
(in vitra). These genes that were upregulated during infection could be divided into four
categories: proteins previously identified as virulence factors, those required for
metabolism during rapid growth and colonization, proteins of known function that had
not been previously characterized as required for virulence, and unknown proteins.

Chapter three of this dissertation discusses Ohr, an organic hydroperoxide
reductase. Ohr was identified as an in viva induced gene and was shown to be regulated
in response to organic peroxides. Ohr functions during the oxidative stress that occurs
subsequent to host immune cell influx and degranulation to release oxygen radicals. Ohr
and its regulator are serotype specific, found only in three of the twelve serotypes. The
characterization of ahr shows the new information that can be gained from the use of
IVET screens to identify virulence genes.

Chapter four discusses the need to evaluate newly identified virulence genes as
potential vaccine candidates. The construction of the genetic tools to make attenuated

mutants is described for both ahr and for ile, a gene involved in isoleucine-leucine-

17

 

raline bios)
modification

C liar
experiments

virulence lat

valine biosynthesis. While the mutants were unable to be constructed, future
modifications for successfully obtaining the mutants is discussed.

Chapter five summarizes this dissertation, in order to be able to suggest future
experiments to expand upon the current knowledge of A. pleuropneumoniae and its

virulence factors.

18

.flu‘

Refe

J

10.

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Esr‘.
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Hig
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Heir
Mic

H01
Elli”)
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Ple’r

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Clol
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25

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1. Bl

Tari
FUni
SUbl
Atth

Ton
Actr
lian‘
boll

78.

79.

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81.

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83.

84.

85.

86.

87.

88.

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Schaller, A., R. Kuhn, P. Kuhnert, J. Nicolet, T. J. Anderson, J. I. MacInnes,
R. P. Segers, and J. Frey. 1999. Characterization of apxIV A, a new RTX
determinant of Actinobacillus pleuropneumoniae. Microbiol. 145:2105-2116.

Sebunya, T. N. K., and J. R. Saunders. 1983. Haemaphilus pleuropneumoniae
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Shope, R. E. 1964. Porcine Contagious Pleuropneumonia 1. Experimental
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Strutzberg, K., L. von Olleschik, B. Franz, C. Pyne, M. A. Schmidt, and G. F.
Gerlach. 1995. Mapping of functional regions on the transferrin-binding protein
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Tarigan, S., R. F. Slocombe, G. F. Browning, and W. Kimpton. 1994.
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26

89.

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95.

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97.

98.

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27

Chapter 2

Identification of virulence factors of Actinobacillus pleuropneumoniae by the use of

IVET (in viva expression technology)

A portion of this chapter has been published previously.
Fuller, T. B., R. J. Shea, B. J. Thacker, and M. H. Mulks. 1999. Identification of in viva
induced genes of Actinobacillus pleuropneumoniae. Microb. Pathog. 27: 311-327.

28

Abstract

Actinobacillus pleuropneumoniae promoters that are induced during infection but
have minimal expression on rich laboratory media have been identified using an m
gxpression technology (IVET) system. This system is composed of an avirulent
riboflavin-requiring A. pleuropneumoniae mutant (AP233), a promoter-trap vector
(pTF86), and an infection model, the natural swine host. The pTF86 vector contains a T4
terminator, a unique BamHI cloning site, and promoterless copies of the luxAB reporter
genes and Bacillus subtilis ribBAH genes in an Escherichia cali-A. pleuropneumoniae
shuttle vector. Partially digested A. pleuropneumoniae genomic DNA was cloned into
pTF86 and electroporated into AP233. Pigs were infected with pools of approximately
600 transformants by endobronchial inoculation. The pigs were euthanized at twelve to
sixteen hours after initial infection and surviving bacteria were isolated from the swine
lungs. This infection strongly selected for transforrnants containing cloned promoters
that allowed expression of the ribBAH genes and complementation and survival of the
attenuated mutant. The clones isolated from the pig lungs were assayed for luciferase
expression to identify those that had minimal expression in vitra. The clones that
survived in viva, but which had minimal luciferase expression in vitra, are hypothesized
to contain cloned promoters that are specifically induced during infection. Twenty-five
clones were isolated which contain promoters that are induced during infection. Thirteen
clones showed strong homology to known genes. The remaining twelve clones contained
either no identifiable open reading frame or not enough sequence information to identify

the gene driven by the in viva expressed promoter. Nine of these twelve clones have had

29

the in \‘i‘
database
identitiea

caused by

the in vivo induced gene identified by inverse PCR or by sequence comparison to a
database of A. pleuropneumoniae serotype 5 genomic DNA sequences. The
identification of in viva induced genes has contributed a new understanding to disease

caused by A. pleuropneumoniae.

30

lntrodi

\irulei

pleura;
pneuni
hemor
rendts
decree

infeeti

to cor
lysis

leuko
htmo
RITK
iiVeli

and

Introduction

Virulence factors of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is the causative agent of porcine
pleuropneumonia, a contagious disease with an acute onset of dyspnea, hyperemia, and
pneumonia. This bacterium causes significant lung lesions that are composed of
hemorrhage, fibrin deposition, and pleural adhesions. The economic toll of this disease
results from both the rapid death of naive swine in peracute infection and from a
decreased growth and increased production costs that occur in subclinical chronic
infections (23, 48).

Currently identified virulence factors of A. pleuropneumoniae include toxins,
lipopolysaccharide, and capsule. Capsule and lipopolysaccharide (LPS) have been shown
to contribute to bacterial survival in the host by protecting against complement mediated
lysis and phagocytosis (6, 10, 24). Purified LPS has been shown to be toxic to
leukocytes, platelets, and vascular endothelium. This cellular damage results in non-
hemorrhagic pulmonary lesions, often seen during an acute infection (6, 10, 24). The
RTX exotoxins (glycine-rich [epeats in structural toains) have been identified in all
twelve serotypes of A. pleuropneumoniae and have been shown to have both hemolytic
and cytotoxic activity (12, 13). The toxins are capable of destroying alveolar
macrophages and lymphocytes by creating pores in the eukaryotic cell membranes and
causing cell lysis (6, 46, 50). This lysis results in the release of oxygen radicals and
lysosomal enzymes from the damaged phagocytes, which in turn damages host tissue by

causing necrosis and hemorrhage (4, 8).

31

The
factors but
RTX toxir
factors all

maintainer

Characte

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“Elfin
are expre
media tin
during at
Immoter
39d an in
random E
alld the l
coniahler
"a“SCrib
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distase I
The W
etpmm

mmtae

The identification and characterization of the toxins and other known virulence
factors have contributed to only a partial understanding of the disease pathogenesis. The
RTX toxins and LPS contribute to the disease lesions, but the presence of these virulence
factors alone does not explain how the infection spreads through the host and is

maintained in the chronically infected herd.

Characterization of virulence genes

In an effort to further understand the pathogenesis of A. pleuropneumoniae, an
IVET (in yiva gxpression technology) system has been developed to identify genes that
are expressed during infection (in viva), but show minimal expression on laboratory
media (in vitra). The IVET method selects for the promoters of genes that are expressed
during an infection. The tools required are an attenuated strain of the pathogen, a
promoter trap plasmid that is able to complement this attenuation and restore virulence,
and an infection model. The plasmid contains a unique BamHI restriction site into which
random genomic DNA fragments can be cloned upstream of a promoterless reporter gene
and the promoterless gene that complements the attenuating mutation. If a promoter is
contained within the cloned DNA, the promoter-reporter-biosynthetic gene fusion is
transcribed during infection, the biosynthetic gene complements the mutation, and the
bacteria are able to survive and cause disease in viva. Clones selected in viva by the
disease process are expressed during the infection, thus allowing for complementation.
The reporter gene is used to quantitate expression to determine the degree of in vitra
expression. Those that are only induced in viva, but not in vitra generally fall into one of

four categories of virulence genes. These categories include genes that are known to be

32

involved in pathogenesis or regulators of these genes, biosynthetic genes that are needed
during the rapid growth in viva, genes of known function that have not yet been identified

as virulence factors, and unknown genes.

Methods for identification of in viva expressedJenes Multiple methods, including IVET,
have been developed to allow for the identification of genes that are expressed in viva.
One method to identify genes that are induced during infection has been done using in
vitro conditions designed to mimic specific environmental conditions encountered in
viva, such as nutrient limitation or exposure to oxygen radicals, and then screen for genes
that respond to these conditions. This system will miss many of the virulence factors that
respond to a more complicated series of environmental stimuli prior to expression (35).
Another method to identify virulence genes is by homology to known virulence
factors from other bacteria. This technique has been applied with limited success in
identifying possible virulence factors in A. pleuropneumoniae. The sadC locus (Cu, Zn
superoxide dismutase) was identified by constructing PCR primers from regions of high
homology to other superoxide dismutases and amplifying sadC from A.
pleuropneumoniae (26). The transferrin binding proteins were isolated by screening for
genes that complemented an E. cali mutation (2). However, these methods are limited to
genes hypothesized to exist in one bacteria based upon the knowledge of another related
bacteria. Since little is known about the virulence mechanisms in A. pleuropneumoniae
or related respiratory pathogens, a different approach is needed for a more thorough
screen. The use of genetic tools has replaced the need for screening for individual genes

with methods to perform a complete screen for virulence factors.

33

I
display:
that ider
sunive 1
clones tc
present i
the baet
eompari
unique 5
by SIM

lSland at

Conditir

The CD‘

These genetic tools include signature-tagged mutagenesis (STM), differential
display, and in viva expression technology (IVET). STM is a negative selection process
that identifies genes that, when interrupted by a transposon, result in an inability to
survive in viva. The process compares an in vitra grown pool of transposon-insertion
clones to the pool recovered from an infection to identify which transposon mutants were
present in the in vitra pool but were lost during infection. This in viva loss occurs when
the bacteria could not survive without an intact copy of the interrupted gene. This
comparison is accomplished by PCR amplification and subsequent hybridization to
unique signature sequence tags that are found flanking each transposon. Genes identified
by STM have included a secretion apparatus in a Salmonella typhimurium pathogenicity
island and genes for adhesin maintenance in Staphylococcus aureus (34, 42).

Differential display involves comparing the mRNA produced under different
conditions. The total mRN A is isolated is isolated from bacteria grown in vitra and those
recovered after infection. From this mRNA, cDNA is obtained by reverse transcription.
The cDNA can then be amplified with polymerase chain reaction (PCR) primers that are
specific for a gene of interest, to determine if that gene is transcribed during infection.
Another method using differential display involves comparing both pools of cDNA, using
gel electrophoresis or Southern blots to determine which transcripts are found under only
one condition (9). This comparison can be used to isolate genes which are regulated such

that they are only expressed during infection.

34

IVET ba

infection
promote
The pla-
promote
compler
strain.

rounder
All €101
for Con
l'ltro 0
detem
in \‘im
int-01Ve
four C2
Paihgg‘
knOm-l

genes_

IV ET background

The IVET method selects for the promoters of genes that are expressed during an
infection. The tools required are an attenuated mutant of the bacteria of interest and a
promoter trap plasmid that is able to complement this attenuation and restore virulence.
The plasmid consists of a randomly cloned genomic fragment containing a potential
promoter fused upstream of a promoterless reporter gene and the promoterless gene that
complements the attenuation. The plasmid is transformed into the attenuated bacterial
strain. If the promoter fusion is transcribed during infection, the biosynthetic gene
complements the mutation and allows the bacteria to survive and cause disease in viva.
All clones selected in viva by the disease process are expressed at a high level to allow
for complementation. The bacteria are harvested from the infected host and plated in
vitra on laboratory media. The reporter gene is used to quantitate expression to
determine the degree of in vitra expression. Those that are also expressed at a high level
in vitra are likely to be constitutively expressed housekeeping genes, such as those
involved in metabolism. Those that are only induced in viva generally fall into one of
four categories. These categories include genes that are known to be involved in
pathogenesis, biosynthetic genes that are needed during the rapid grth in viva, genes of
known function that have not yet been identified as virulence factors, and unknown
genes.

IVET was initially developed to identify virulence genes in a Salmonella
typhimurium BALB/c mouse spleen model. The IVET vector was constructed as a single

copy suicide plasmid with the lacZ Y gene as a reporter gene and purA to complement the

35

purine auxotrophy (32, 44). The single copy recombines into the host chromosome to
produce two functional promoters, one promoter to drive the expression of the lacZ Y and
purA genes, while the other promoter remains uninterrupted to continue to regulate the
gene of interest. Since this initial work, IVET systems have been continually developed
by using different animal models and pathogenic bacteria and by modifying the vector.
Some of the modifications have included using different auxotrophic or antibiotic
markers to complement the bacterial host, and eliminating or modifying the reporter gene
to include a recombinase protein or different colorimetric markers (9, 22, 30, 32, 33, 53,

57).

36

Materials and methods

Bacterial strains and media. The bacterial strains and plasmids propagated in these
strains are listed in Table 1. A. pleuropneumoniae were cultured at 35 ‘C in either brain
heart infusion (BHI) or heart infusion (HI) (Difco Laboratories, Detroit, Mich.)
containing 10 ug/ml B-NAD (V-factor) (Sigma Chemical Company, St. Louis, MO).
Riboflavin (Sigma) was added to a final concentration of 200 ug/ml when needed for the
propagation of AP233. Ampicillin was added at 50 11ng for plasmid maintenance and
at 20 ug/ml for selection after electroporation and lung culture. Escherichia cali strain
XLl-Blue mRF’ (Stratagene, LaJolla, Calif.) was cultured in Luria-Bertani media with

100 ug/ml ampicillin added when needed for plasmid selection.

DNA manipulations. DNA modifying enzymes were obtained from Boehringer-
Mannheim Biochemicals (Indianapolis, Ind.) and used according to the manufacturer’s
specifications. PCR was performed using Gibco Taq polymerase (Life Technologies,
Rockville, MD). PCR products were separated with a 2% agarose gel containing three
parts Gibco (Life Technologies) agarose and one part NuSieve GTG agarose (FMC
Bioproducts, Rockland, ME). Plasmid DNA was isolated from bacterial strains using the
Qiagen miniprep kit (Qiagen Inc., Valencia, Calif). E. cali transformation was

performed by the method of Hanahan (20).

37

APZBI

XIII

Phnn

pTFSt

PGZF

Table 2-1. Strains and plasmids used in this work.

 

 

Strain or plasmid Characteristics Source
Strains
APlOO A. pleuropneumoniae ATCC 27088, serotype 1A, ATCCa
passaged through pigs
AP233 A genetically defined riboflavin auxotroph of APIOO (17)
XLl-Blue mRF’ E. cali cloning strain Promegab
Plasmids
pTF86 A. pleuropneumoniae IVET vector containing (15)
promoterless luxAB and ribBAH genes
downstream of BamHI cloning site
(Figure l)
pGZRSlS Apr A. pleuropneumoniae-E. cali shuttle vector (55)

 

a American Type Culture Collection, Rockville, Md.

b Promega Corporation, Madison, Wis

38

Flgur
Slit. 31

Copy
methc
With 1
Schue
lBOel
de\‘el
Man,
film

 
   
 
 

’be‘m

ribA

   
   
       

 
 

quA
pTF86

’in 9.6 kb 1'4 C}

Q

BamHI

Figure 2-1 Diagram of pTF86, showing the T4 terminator, the unique BamHI cloning
site, and the orientation of the promoterless luxAB and ribBAH genes.

Copy number determination. The copy number of pTF 86 was determined by the

method of Xia et al. (56). Briefly, total DNA from AP233 containing piviE was digested

with SalI, separated on an agarose gel, and blotted onto a nylon membrane (Schleicher &

Schuell, Keene, NH.) The insert from piviE was labeled with digoxigenin-tagged dUTP

(Boehringer-Mannheim Biochemicals). This insert was used as a probe and the blot was

developed with the CDP-Star chemiluminescent substrate protocol (Boehringer-

Mannheim Biochemicals). The luminescent bands were visualized on X-ray film and the

film scanned into an image analysis system (Ambis Inc., San Diego, Calif.). Band

densities were quantified using Ambis QuantiProbe version 3.01 software. Copy number

39

1135 C3

chrome

Constr
clones
lysis p:
and Se
electro
EXIIZICT
from s

Prepar

was calculated as the density of the plasmid band divided by the density of the

chromosomal band.

Construction of IVET clones. The protocol for construction and passage of the IVET
clones is diagrammed in Figure 2. Genomic DNA from AP233 was prepared using the
lysis/proteinase K method of the Gene Fusion Manual (43) and incubated with RNaseA
and Sau3A to create partially digested fragments. The partially digested DNA was
electrophoresed on an 0.8% agarose gel and the region from 400 to 1000 bp was
extracted using the Qiaex gel extraction kit (Qiagen). The plasmid, pTF86, was prepared
from stationary phase APlOO using Qiagen midi-columns. AP233 competent cells were
prepared as previously described and used within three weeks of competency treatment
(15). Approximately 15 ug of pTF 86 was digested with 30 units of BamHI followed by
an ethanol precipitation. This digested DNA was then incubated with 2 units of calf
alkaline phosphatase (Boehringer-Mannheim Biochemicals) for forty minutes at 37°C,
followed by a phenol-chloroform extraction and ethanol precipitation. A 100 pl ligation
consisting of 1 pg of AP233 genomic DNA fragments and 5 ug of cut and phosphatase-
treated pTF86 was incubated for 12-16 hours at 4°C with 8 units of T4 ligase
(Boehringer-Mannheim Biochemicals). Two ligation mixtures were combined and
extracted with phenol-chloroform, followed by an ethanol precipitation and resuspension
in 20 ul of water. AP233 was electroporated using 10 ul of the resuspended ligation
mixture (16). Transformants were allowed to recover for four hours in BHIV with 200
ug/ ml riboflavin and then plated on BHIV plates with 200 ug/ml riboflavin and 20 rig/ml

ampicillin. Approximately 600 IVET clones were pooled directly from the

40

Figure 2-2. Diagram of A. pleuropneumoniae IVET construction and infection. This
flow chart shows the method by which ivi clones were constructed using pTF86 and the
riboflavin mutant. The clones were then used to infect the swine respiratory model.

41

Plasmid pTF86 APP-1A chromosomal DNA

     
 

Di est with Sau 3A

Digest estwlth .
PurIfy .4- 1.0 kb fragments

BamHl- CIP

Ligate and electroporate into AP233 (Rib-)

Select Ap resistant colonies on media containing riboflavin

l

Pool in groups of 100 and freeze at -80c
Grow pools individually to an ODof 0.8

Combine cultures, centrifu e and wash with sterile PBS, dilute appropriately
lnoculate 8 x 0 cfu endobronchially

Infect pigs and
monitor

l

Euthanize at 16 - 24 hours p.i.

1

Culture strains from lun s as optically by swab or bronchoalveolar lavage
on o BHI + riboflavin + Ap

l

Screen for Lux expression
and select Lux - colonies

l

Isolate plasmid DNA and sequence into chromosomal insert

Figure 2-2

42

electroporated plates without further subculture. Colonies were washed off the plates
with HIV broth containing 200 ug/ml riboflavin, 50 ug/ml ampicillin and 5 mM calcium
chloride. Filter sterilized glycerol was added to a final concentration of 20%. The
mixture was aliquoted into freezer vials and stored at -80°C. In order to estimate the total
cell density of the mixture, a portion was diluted ten-fold and an optical density (ODszo)

was obtained.

Preparation of challenge inocula. Each pool of 600 transfonnants was thawed and
diluted into prewarmed HIV broth containing 5 mM calcium chloride, 200 ug/ml
riboflavin, and 50 rig/ml ampicillin, to produce an initial starting volume of 25 ml and an
initial ODszo of 0.2. Pools were grown in sidearm flasks at 35°C with shaking at 160 rpm
to an ODszo of 0.8. If needed, cultures were diluted once to prevent overgrowth. An
aliquot of the culture was taken for luminometric assays and bacterial counts, and 10 ml
was harvested by centrifugation at room temperature and washed once with sterile PBS
containing 5 rig/ml riboflavin (PBS-Rib). The bacterial pellet was resuspended in 10 m1

of PBS-Rib and diluted if necessary to obtain a desired challenge inoculum of 3 X 108

cfu.

Experimental infections. Eight week old specific pathogen-free castrated male pigs
from Whiteshire Hamroc, Inc. (Albion, Ind.) were housed in separate cages in BioSafety
Level 2 isolation rooms at the Michigan State University Research Containment Facility.
For the challenge procedure, pigs were anesthetized and the challenge inoculum was

placed into the lungs by a shallow endobronchial injection. Clinical signs of

43

pleuropneumonia, including increased respiratory rate, elevated temperature, dypsnea and
depression, were monitored and scored as previously described (25). Animals were
euthanized at 12 to 16 hours post infection and necropsied. The lungs were examined
macroscopically for A. pleuropneumoniae lesions, including edema, congestion,
hemorrhage, necrosis, fibrosis, and pleuritis.

A. pleuropneumoniae isolates were recovered from aseptic culture of the lung
tissue or from bronchoalveolar lavage (BAL). BAL was performed by inserting sterile
tubing through the trachea and into the lungs and instilling 200-300 ml of PBS-Rib. The
recovered fluid, typically 200-150 ml, was centrifuged to pellet bacteria. The pelleted
bacteria were resuspended in PBS-Rib and used for luminometric assay, plate counts, and
recovery of isolated colonies. All isolates recovered from the lungs, by either BAL or
direct plating, were cultured on BHIV agar plates containing 200 jig/ml riboflavin and 20
pg/ml ampicillin.

The All University Committee on Animal Use and Care at Michigan State
University reviewed and approved all protocols for animal experiments. All procedures

conformed to university and USDA regulations and guidelines.

Lux analysis. Qualitative screening of A. pleuropneumoniae for expression of the luxAB
genes was performed using a Hamamatsu C1966 Photonic Camera Microscope System.
Colonies plated on BHIV plates with 200 ug/ ml riboflavin and 50 ug/ ml ampicillin
were exposed to N-decyl aldehyde distributed evenly on a glass petri dish lid. The plate

was analyzed with an aperture setting of 11, a digital gain of 2 and photon counting for

44

lumin
by so
dissol
water
subst
then '
and a
relati

deter

11 seconds. Removal of strongly expressing colonies from the plate was performed as
needed to evaluate colonies with lower expression levels.

Quantitative Lux assays were performed using a Turner Model 20c single cell
luminometer (Turner Designs, Sunnyvale, Calif). N—decyl aldehyde substrate was made
by sonicating 20 mg/ml Essentially Fatty Acid Free Bovine Serum Albumin (Sigma)
dissolved in 1 ml of water with l til/ml N-decyl aldehyde (Sigma) using a sonicating
water bath. For analysis, 20 pl of culture was mixed for three seconds with 20 ul of
substrate in a polypropylene luminometer cuvette (Turner Designs). The mixture was
then read in full integral, autoranging mode without a predelay, a delay of 10 seconds,
and an integration time of 30 seconds. Luminometric values were normalized to micro-
relative light units per colony forming unit (uRLU/cfu) with the number of bacteria

determined by plate counts or by normalizing to the OD520 when in pure broth culture.

DNA sequencing. For sequencing of the DNA inserts, a primer complementary to the 5’
end of the luxA gene (5’~GCTGCCTCCATCCATGGGGTTCCTC-3’) and a primer (5’-
GGCAACCGTTTCTGCCGGGGAATCC-3’) corresponding to the 3’ end of the T4
terminator were used to obtain the initial sequence, after which custom internal primers
were designed to obtain additional intervening sequence. Nucleotide sequencing plasmid
DNA isolated from E. cali was performed using the Sequenase 2.0 kit (U .S.
Biochemicals, Cleveland, Ohio) and 35S-dATP (Amersham Corp., Arlington Heights,

111.).

45

Inverse PCR. An inverse PCR (iPCR) technique was designed to clone DNA regions
flanking the ivi sequence (39). 2.5 ug of A. pleuropneumoniae serotype 1 genomic DNA
was digested to completion with a restriction enzyme, followed by a self-ligation using
T4 DNA ligase (Roche) to form closed circular fragments. PCR amplification with
AmpliTaq (Roche) was performed with 0.04 ug of ligated DNA using primers that bound
within the ivi coding region and were oriented to PCR amplify the flanking regions. The
resulting PCR fragments were isolated from an agarose gel using the Qiaex II kit
(Qiagen) and cloned into the pGEM-T vector (Promega, Madison, Wisc.). The clones
were sequenced using an ABIlOO Model 377 automated sequencer (Applied Biosystems,
Foster City, Calif). Complete open reading frames (ORF) were identified by comparison
of the sequence obtained from the ivi clone and the sequence that resulted from the iPCR

clones.

Nucleotide and protein sequence analysis. Nucleotide and amino acid sequences were
compared with GenBank sequence databases using both the F ASTA/T FASTA functions
of the GCG Programs and the BLAST suite of programs. All alignments and calculated

percent identity and similarity were made using either the GCG Programs or BLAST (19,

47).

46

Results

AP233/pTF86 lacking an in viva induced promoter is unable to be recovered from
infected pigs. Two pigs were each inoculated with AP233/pTF86 at a dose of 2.5 X 108
cfir, a dosage identical to that used for the IVET pools. Neither pig showed clinical signs
of pleuropneumonia and no visible lesions were present in the lungs upon necropsy, eight
to twelve hours post infection. Upon culturing the bronchoalveolar lavage (BAL) fluid
and the lungs of the pig that was necropsied after eight hours, less than 10 cfu/ml was
recovered from the BAL fluid and a total of 147 colonies were recovered from the culture
of the lung sections. In the pig that was euthanized twelve hours post infection, we were
unable to isolate bacteria from either the BAL fluid or the lung sections. This is in
contrast to the greater than 106 cfu/ml BAL fluid and confluent lawns from lung culture

that were generally obtained from infection with an IVET pool.

Copy number of IVET vector. The copy number of the IVET plasmid was ascertained
in order to determine if the plasmid was being maintained in a high copy number that
might permit clones with minimal expression to survive the infection. The copy number
of pTF86 was calculated using piviE, since this plasmid contained an insert of 643 bp,
which was close in length to the average insert size of 475 bp (16). The copy number,
determined to be 8-10 copies per bacterial cell, was calculated by normalizing the
intensity of the plasmid DNA to the chromosomal DNA as determined by a Southern blot

of AP233/piviE using the piviE insert as the probe (Figure 3).

47

Flgu
“llh
lC I

now

in

1m,

De

Ur

 

Figure 2-3 Southern blot of AP233 (lane 1) and AP233/piviE (lane 2) when examined
with a probe corresponding to piviE. The relative intensities of the chromosomal band
(C) and the plasmid band (P) from AP233/piviE allowed for the calculation of copy
number of piviE.

In viva screening of IVET clones. A total of 11,000 clones’were constructed by
inserting random genomic DNA fragments in pTF86 and electroporating these plasmids
into AP233. A control ligation of alkaline-phosphatase treated vector without insert was
performed and indicated that approximately 10% of the total transformants contained no
insert. Approximately 5,000 of these clones were used to infect eight pigs, each with

575-750 clones. The remainder of the 11,000 clones was stored at -80°C for future

48

experiments. The dosage was approximately 3 X 108 cfu per pig with a dosage of 5 X
105 cfu of each individual clone. This dosage resulted in mild to moderate disease within
8-16 hours, allowing necropsy at 12-16 hours post infection, which minimized the chance
of recovering clones lacking an in viva induced promoter (16).

Clones containing in viva induced promoters were able to complement the
riboflavin deficiency of AP233 and survive greater than twelve hours after inoculation.
Screening of a subset of clones prior to inoculation demonstrated that approximately 85%
of the strains had minimal Lux expression, indicative of clones with inserts that lacked a
promoter or contained a promoter that was not expressed in vitra. The remaining 15% of
the clones contained a promoter that was highly expressed prior to passage through the
pig. This was in comparison to the Lux expression of the clones isolated after infection, a

process that selected for clones with promoters. (Figure 4).

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Pre—infection Post-infection

Figure 2-4 Photonic camera picture showing a pool of 100 clones before infection and
a pool of 50 clones afler infection.

49

In vitra screening of IVET clones. Clones recovered from BAL dilution plates or from
lung cultures were plated in groups of 50 and examined using a photonic camera for
qualitative identification of clones with low in vitra Lux expression. It was often
necessary to remove clones that had high Lux expression from the plates during this
initial screen in order to identify those with low expression. Clones that had low
expression levels were individually frozen and PCR performed from the frozen cultures
using primers that annealed to the lux and T4 terminator regions to determine the size and
to group clones that contained inserts of similar sizes. Several clones of each identically
sized subgroup were then assayed quantitatively to determine the degree of Lux
expression. Clones that had Lux expression levels less than 200 RLU/ODszo were
partially sequenced using the lux primer to further cohort each subgroup. This
sequencing was performed using plasmid DNA obtained from E. cali transformed with
each of the in viva induced plasmids. This strategy led to the identification of 25 unique

genetic loci (Table 2, Figure 5).

Table 2-2. Identification of ivi insert.

 

 

Clone Database homology (% similarity) Function of homologue Span

iviG A. pleuropneumoniae CpslB (100%) Capsule biosynthesis 14 aa

iviH no identifiable ORFs NA NA

iviI Haemaphilus influenzae Ilvl (95%) Synthesis of branched chain 81 aa
amino acids

iviJ H. influenzae PrfC (99%) Peptide release factor 3 134 aa

50

Table 2-2 (cont’d).

 

 

Clone Database homology (% similarity) Function of homologue Span
iviK Vibria chalerae Ohr (58%) Organic hydroperoxidase 97 aa
iviP Pasteurella multacida AmpD (84%) Amidase 60 aa
iviR H. influenzae ZnuA (90%) zinc transport 66 aa
iviS Shigellaflexneri miaA (68%) tRNA 352 nt
iviT H. influenzae Gnd(97%) 6-phophogluconate 58 aa
dehydrogenase
iviU P. multacida MioC (72%) Chromosomal replication 67 aa
iviW Escherichia cali YfiC (72%) Hypothetical orf 41 aa
iviX no identifiable ORFs NA NA
iviY E. cali FtsY (57%) Cell division/secretion 105 aa
ivi17A H. influenzae PyrG (95%) CTP synthetase 86 aa
ivil7B P. multacida TexA (92%) Toxin expression 86 aa
ivi17C P. multacida EonI (94%) Acetyl-D-glucosaminadase 60 aa
ivi17D P. multacida MioC (72%) Chromosomal replication 20 aa
ivi17E P.multacida FolD (78%) Methylenetetrahydrofolate 43 aa
dehydrogenase/cyclohydrolase
ivi17F no identifiable ORFs NA NA
ivi17G no identifiable ORFs NA NA
ivi19A A. pleuropneumoniae Asd (100%) Aspartate semialdehyde 69 aa
dehydrogenase
ivil9B H. influenzae TehB (80%) Tellurite resistance 148 aa
iviZOA H. influenzae GlpC (91%) G1ycerol-3-Phosphate 62 aa
dehydrogenase
iviZOB Erwinia chrysanthemi Rec] (87%) DNA specific exonuclease 65 aa
iviZOC no identifiable ORFs NA NA

Figure 2-5 Organization of the 25 unique in viva induced inserts (ivi clones). Shaded
boxes indicate open reading frames. P- indicates a potential promoter. All ORFs fused
to luxA were preceded by potential ribosome binding site.

52

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53

Verification of in viva survival. To confirm that the clones identified as in viva induced
could be isolated again from an infected pig, groups of five to ten clones were pooled at 5
X 105 cfu of each clone and used to infect individual pigs. All pigs developed classical
pleuropneumonia symptoms and were euthanized and necropsied 12 to 16 hours post
infection. Necropsy and bacterial isolation and characterization was performed as before,
with the exception that Lux expression of the recovered clones was not assayed.
Sequencing of recovered clones proved that all clones could be recovered from the repeat

infection, strengthening the classification of these clones as in viva induced.

Sequence analysis. The complete inserts of the 25 clones were sequenced and both the
nucleotide and the predicted amino acid sequences were used to search GenBank
databases and a database consisting of sequence information from A. pleuropneumoniae
serotype 5 (John Nash, personal communication). Based on the initial analysis of the ivi
clone, thirteen clones showed strong homology to identified genes, and twelve clones
contained either no identifiable open reading frame or not enough sequence information
to identify the gene driven by the in viva expressed promoter without determining the
downstream sequence through inverse pcr or database analysis (Table 2, Figure 5).

IviI, iviK, iviW, iviY, ivi17b, ivil7c, ivil7c, ivi19a, and ivi19b each contained a
region containing a potential promoter, preceding a known partial ORF, oriented
upstream of the luxAB genes and in the proper orientation to allow expression of the lux
and rib genes. While iviJ, iviR, ivi17a, ivi20a, and ivi20b encode a recognizable protein,
the orientation or region within the protein are such that the known gene may not be the

in viva induced gene. The recognizable gene is described, but with all of these ivi clones,

54

there is the possibility that the promoter is responsible for the expression of a downstream
gene of unknown sequence.

The remaining eleven clones did not have enough sequence information to
determine the identity of the in vivo induced gene. Both inverse PCR and sequence
comparison to the genome of A. pleuropneumoniae serotype 5 was used to determine the
sequence flanking the promoter within the ivi insert. Through inverse PCR, completed
by Trevor Wagner, iviS was identified as qu (host integration factor), iviX was
identified as VapBC, iviP was confirmed as AmpD, and ivil7g was identified as Can.

The remaining seven clones contain inserts having short unidentifiable ORFS,
sequences lacking homology to identified proteins, or sequences containing known genes
without identifiable promoters to drive expression of the luxAB genes. In order to
identify the gene expressed by the ivi promoter, a database containing A.
pleuropneumoniae DNA sequence was required to evaluate the flanking DNA.
Information for two of the seven clones (iviG and ivil7f) was not able to be found in the
A. pleuropneumoniae serotype 5 database. IviG was not found in the database and ivil7f
was found at the 3’ end of a contig and the downstream sequence was unavailable.
Thus, the five clones; iviH, iviT, iviU, ivi17D, and ivi20C were used to search the A.
pleuropneumoniae serotype 5 database.

Thus the information gained from a variety of sources has been assimilated to
give the following information about the 25 ivi clones that were identified as a result of
this IVET study. IviG contains the first 14 amino acids of cps-[B (AF518558), one of
several proteins involved in capsule biosynthesis. IviH contains no recognizable ORFs

in either the ivi insert or in the A. pleuropneumoniae serotype 5 database.

55

 

Ivil was identified as 1le by analysis of the ivi clone sequence, which contained
81 a of the 5’ end of Ile. The full gene was cloned by inverse PCR and is 1689 bp (563
aa) in length. This A. pleurapneumaniae protein is 81 % homologous to P. multacida Ile
(AAK02954) and 98 % identical to the homologue in A. pleuropneumoniae serotype 5.
Ile is acetohydroxy acid synthase III, which is necessary for biosynthesis of isoleucine
and valine (45).
IviJ was identified as PrfC by analysis of the ivi clone sequence, which contained
134 a of the 5’ end of PrfC. The full gene has not yet been cloned. This A.
pleuropneumoniae protein is 96 % homologous to H. influenzae PrfC (HIl735) and 99 %
identical to the homologue in A. pleuropneumoniae serotype 5. IviJ contains an antisense
ORF with sequence similarity to PrfC, required for protein release from the ribosome
(36). PrfC plays a role in protein synthesis and may have increased expression during
times of rapid growth and cell division. An antisense RNA to per might help to regulate
the production of new proteins and might transiently limit cell growth during times of
nutrient limitation
IviK was identified as Ohr by analysis of the ivi clone sequence, which
contained 97 aa of the 5’ end of Ohr. The full gene was cloned by inverse PCR and is
429 bp (143 a) in length. This A. pleuropneumoniae protein is 62 % homologous to
Pseudamanas aeruginasa Ohr (AAG06238) and does not have a homologue in A.
pleuropneumoniae serotype 5. Ohr (Qrganic hydroperoxide geductase), which has been
implicated in survival under conditions of oxidative stress (38). The full analysis of Ohr

is the subject of Chapter 3 of this dissertation.

56

IviP was identified as AmpD by analysis of the ivi clone sequence, which
contained 58 a of the 5’ end of AmpD. The full gene has not been cloned. This A.
pleuropneumoniae protein is 84 % homologous to P. multocida AmpD (AAK02167) and
98 % identical to the homologue in A. pleuropneumoniae serotype 5. AmpD, a N-
acetylmuramyl-L-alanine amidase, might function in protection against B- lactam
antimicrobials or in intracellular recycling of valuable cell wall components in an energy
deficient environment (51).

IviR was identified as ZnuA by analysis of the ivi clone sequence, which
contained 66 a of the 5’ end of ZnuA. The hall gene has not been cloned. This A.
pleuropneumoniae protein is 92 % homologous to H. ducreyi ZnuA (AAFOOOI 15 .1) and
90 % identical to the homologue in A. pleuropneumoniae serotype 5. IviR contains an
ORF with sequence similarity to the middle of ZnuA, a periplasmic zinc transport protein
(27). This protein is responsible for regulation of zinc levels within the cell and has been
shown to be required for virulence in H. ducreyi (27). ZnuA has not been fully
characterized and the location of the start codon and the active site have not been
mapped. It is therefore possible that iviR contains the promoter responsible for
expression of a truncated, but functional protein. This hypothesis must be tested by
determining if the protein encoded by iviR is able to complement a H. influenze mutant
that lacks a functional ZnuA (31).

IviS was identified as qu cloning the flanking sequence by inverse PCR and
finding an ORF with homology to qu. This A. pleuropneumoniae protein is 84 %

homologous to H. influenzae qu(H104l 1) and 99 % identical to the homologue in A.

57

pleuropneumoniae serotype 5. qu has been shown to be a RNA binding protein that is
required for efficient translation of several key virulence factors, including oxyS (58).

IviT and iviU both contain recognizable proteins that are upstream of regions that
contain promoters that are required for expression of downstream hypothetical ORFs.
The ORFs identified do not have significant homology to hypothetical ORFs from other
microbial genomes. IviT is 91% identical to the A. pleuropneumoniae serotype 5
genome, and iviU is 98% identical to the A. pleuropneumoniae serotype 5 genome.

IviW was identified as YfiC by analysis of the ivi clone sequence, which
contained 41 aa of the 5’ end of YfiC. The full gene has not been cloned. This A.
pleuropneumoniae protein is 72 % homologous to E. cali YfiC (b2575) and 96 %
identical to the homologue in A. pleuropneumoniae serotype 5. IviW has strong
homology to yfiC, a gene, encoding a hypothetical protein identified initially during
sequencing of the E. coli genome that has since been found in the genomes of multiple
organisms (1).

IviX was identified as VapB by cloning the flanking sequence using inverse PCR.
and is 627 bp (209 aa) in length. This A. pleuropneumoniae protein is 89 % homologous
to H. influenzae VapB (H10321) and 96% identical to the homologue in A.
pleuropneumoniae serotype 5. VapBC was shown to be required in Salmonella dublin
for plasmid stability during nutrient limitation and may play a role in maintaining key
virulence genes during rapid growth (35).

IviY was identified as FtsY by analysis of the ivi clone sequence, which contained
105 a of the 5’ end of FtsYI. The full gene has not yet been cloned. This A.

pleuropneumoniae protein is 57 % homologous to E. coli FtsY (b3464) and 98 %

58

identical to the homologue in A. pleuropneumoniae serotype 5. IviY was similar to F tsY,
which is a required component in bacterial cell division (18, 41).

Ivi17a contains an internal fragment, beginning 140 nucleotides after the start
codon, of pyrG. The full gene has not yet been cloned. This A. pleuropneumoniae
protein is 95 % homologous to H. influenzae PyrG (HIlO77) and 100 % identical to the
homologue in A. pleuropneumoniae serotype 5. PyrG is a CTP synthetase, that is
responsible for the formation of CTP from UTP and ATP (54). CTP is necessary for
synthesis of DNA and mRNA, both of which are necessary for cellular growth and
replication. The transcriptional start site has been mapped in E. coli to a region upstream
of the sequence contained in ivi17a, but the intact protein from A. pleurpopneumaniae
has not been studied (54). It is possible that ivi17a contains the promoter region for a
truncated, but functional protein.

Ivi17b was identified as TexA by analysis of the ivi clone sequence, which
contained 86 aa of the 5’ end of TexA. The full gene has not yet been cloned. This A.
pleuropneumoniae protein is 92 % homologous to P. multacida TexA (PM1549) and 98
% identical to the homologue in A. pleuropneumoniae serotype 5. Ivi17b encodes an
ORF with similarity to TexA, which has been implicated in regulation of toxin expression
in Bordetella pertussis (14).

Ivil7c was identified as Eonl by analysis of the ivi clone sequence, which
contained 60 aa of the 5’ end of EonI. The full gene has not been cloned. This A.
pleuropneumoniae protein is 94 % homologous to P. multacida EonI (PM0081) and

97% identical to the homologue in A. pleuropneumoniae serotype 5. Ivil7c encodes an

59

ORF with sequence homology to EonI, a B-N-acetylglucosaminidase that may be
involved in antigenic variation (3).

Ivil7d was identified as SelA by comparison to the A. pleuropneumoniae
serotype 5 database. The insert was 97 % identical to the A. pleuropneumoniae serotype
5 genome and the downstream gene was selA. SelA is 75% homologous to H. influenzae
SelA (HIO708). Ivil7d contains a promoter that regulates expression of selA, which
encodes selenocysteine synthase (1 l). SelA is responsible for the formation of
selenocysteine tRNA which replaces a serine amino acid with a selenocysteine instead,
allowing for variation in the protein sequence and quartenary structure. This may be
valuable during times of antigenic variation.

Ivil7c was identified as FolD by analysis of the ivi clone sequence, which
contained 43 aa of the 5’ end of FolD. The full gene has not been cloned. This A.
pleuropneumoniae protein is 78 % homologous to P. multacida FolD (PM2085) and 100
% identical to the homologue in A. pleuropneumoniae serotype 5. Ivil7c encodes FolD,
which is required for synthesis of methyltetrahydrofolate, a precursor of pyrimidines (7).

Ivil7f was unable to be identified. The homologous region in A.
pleuropneumoniae serotype 5 was at the end of a contig and lacked downstream
sequences. There were no ORFs in the insert that had homology to proteins in the
microbial genomes.

Ivi17g was identified as Can by cloning the flanking regions using inverse PCR
and finding an ORF that was 75% homologous to E. coli Can (ECsOlOS). This region is
98% identical to the homologue in A. pleuropneumoniae serotype 5. Can,

dephosphocoenzyme A kinase, is a key component, and one of the final steps, in the

60

biosynthesis of coenzyme A (37). This enzyme may be required for the increased growth
and metabolic requirements that occur during infection.

Ivi19a was identified as SodC by analysis of the ivi clone sequence, which 69 aa
of the 5’ end of Asd. This region has been sequenced in A. pleuropneumoniae serotype 1
and the downstream gene, divergently transcribed, is sadC (26). The sadC gene encodes
a superoxide dismutase, capable of degrading superoxides to peroxides which can then be
further dexotified by other proteins.

Ivi19b was identified as TehB by analysis of the ivi clone sequence, which
contained 148 a of the 5’ end of TehB. The full gene has not been cloned. This A.
pleuropneumoniae protein is 80 % homologous to H. influenzae TehB (H11275) and 90%
identical to the homologue in A. pleuropneumoniae serotype 5. Ivi19b has homology to
TehB, which has been implicated in resistance to the heavy metal tellurite and as a
potential multi-drug efflux pump (49, 52).

IviZOa contains an internal fragment, beginning 132 bp from the start codon, of
GlpC. The insert contains 62 a of GlpC. This A. pleuropneumoniae protein is 91 %
homologous to H. influenzae GlpC (HIO683) and 98 % identical to the homologue in A.
pleuropneumoniae serotype 5. GlpC has been shown to be a membrane bound protein,
but its exact function has not been identified and its role in the function of GlpAB as
electron transporters has not been established (5). By mapping glpC to the A.
pleuropneumoniae serotype 5 genome, it was discovered that glpC is part of a potential
operon that encodes an unknown ORF and RibGBAH. This operon structure is not seen
downstream of the E. coli glpC (5). It is possible that the promoter for this ORF and the

ribGBAH genes are found internal to glpC and that the gene that ivi20a contains the

61

promoter for is the riboflavin biosynthetic operon and a hypothetical ORF that is a
regulator of this operon.

Ivi20b contains an antisense ORF with sequence similarity to RecJ. This insert
contains 65 a of the protein. This A. pleuropneumoniae protein is 90 % homologous to
Erwinia chrysanthemi RecJ (U57963) and 98 % identical to the homologue in A.
pleuropneumoniae serotype 5. RecJ is a single stranded DNA nuclease that is required
for recombination and DNA excision and repair (28, 29). An antisense mRNA might
limit recombination during times that DNA repair is not desired, such as when a
recombinatorial event might result in antigenic variation.

Ivi20c was identified as TrnS by sequence comparison to the A.
pleuropneumoniae serotype 5 genome database. This insert is 97 % identical to the
homologous region in A. pleuropneumoniae serotype 5. Ivi20c contains sequence
upstream of an ORF that has been identified as TrnS, a putative transport protein, which
is 77 % homologous to TrnS from Salmonella enterica (STY3536). Transport proteins
could be involved in uptake of needed nutrients or in export of proteins required for

infection such as adhesins or toxins.

62

Discussion

This study is a continuation of the initial development of an IVET (in viva
expression technology) system in Actinobacillus pleuropneumoniae (16). Approximately
5000 clones were used to infect swine to select for clones containing promoters that were
strongly expressed during infection. Of these 5000, 25 clones contained inserts that were
expressed during infection, in viva, and had minimal expression on laboratory media, in
vitra. While these 5000 clones are an initial beginning of the 30,000 needed for full
coverage of the genome, the identification of 25 in viva induced genes has already
identified candidate virulence genes. Among these 25 clones are newly identified
virulence factors that might be possible targets for vaccine production or metabolic
pathways capable of being targeted by antibiotics. This study has also provided new
information about the conditions that occur in the pig respiratory tract during infection as
select amino acids and metabolic precursors become limiting and the host immune
response produces inhospitable conditions.

The in viva induction of ile and falD, suggests that during infection there exists
limited nutrients and the invading pathogen must synthesize the precursors needed to
produce pyrimidines and branch-chained amino acids by upregulating falD and ile,
respectively. This might in turn slow down bacterial growth due to an increased
metabolic demand or halt growth entirely if the microbe is unable to synthesize the
necessary metabolic components. On rich media, an excess of metabolites potentially
exists and thus there is no need to express the genes required for biosynthesis of

metabolic precursors. Other clones that contain genes that are responsible for

63

metabolism include ivil7g, which encodes dephosphokinase coenzymeA, and ivil7a,
which encodes CTP synthetase. Dephosphokinase coenzymeA is necessary for the
production of coenzymeA, which is a necessary cellular cofactor. CTP synthetase is
necessary for production of DNA and mRNA, both of which are required for cellular
replication. All of these enzymes are necessary for survival during infection, when the
host limits nutrients as part of the immune reaction to bacterial invasion and so could be 9
considered virulence factors.

Another host immune response is the production of oxygen radicals from
macrophages. In order to survive this assault, bacteria have acquired enzymes, such as
Ohr and SodC, MK and ivi19a respectively, which might detoxify these oxygen radicals.

Novel A. pleuropneumoniae virulence factors have also been identified through
the use of this IVET screen. Several proteins have been identified that might alter the
antigenic profile of the outer membrane, allowing escape from antibody mediated
opsonization. EonI, a glucosaminidase, could potentially modify bacterial glycoproteins
and in doing so, alter the antigenic profile of outer membrane proteins (3). Another
potential role for EonI is to breakdown environmental or host produced glycosides to
generate precursor molecules for bacterial metabolism. In addition to EonI, AmpD and
SelA could participate in antigenic variation of membrane proteins. AmpD, a N-
acetylmuramyl-L-alanine amidase, could modify the cell wall, making it less
antigenically sensitive. AmpD has also been implicated in the resistance to the B—lactam
antibiotics, allowing the bacterium to survive in the presence of antimicrobials that are

used to treat and prevent infection by A. pleuropneumoniae and other bacteria. SelA, by

allowing for the production of a unique amino acid, could alter the quartenary structure of
proteins through the formation of a disulfide bond or interaction of the selenium group.
Another category of genes identified through the use of IVET, have been
transport proteins. IviR, iviY, ivi19b, and ivi20c have sequence similarity to known or
hypothesized transport proteins and efflux pumps. The role of transport proteins is to
regulate and maintain the required levels of nutrients in order for cell growth and
replication. IviR encodes ZnuA, a zinc transporter that has been implicated in
maintaining a nontoxic level of zinc within the cell. In addition, a H. ducreyi znuA
mutant has been shown to have reduced virulence, perhaps because zinc is required for
other virulence factors. IviY encodes FtsY, which has been shown to be a component of
SRP (signal recognition particle) a cotranslational protein targeting to the plasma
membrane in conjunction with the SecE pathway (18, 41). SecE has been previously
identified as an A. pleuropneumoniae in viva induced gene, suggesting that protein export
to the bacterial membrane is an important feature of the disease, perhaps in the
production of required adhesins and recognition particles (16). FtsY has also been shown
to be involved in cell division, required during the growth of the bacteria (18). ftsY has
been identified as an in viva induced gene during a Pseudamanas aeruginasa IV ET
screen, suggesting that FtsY may be a universally important protein and that an antibiotic
targeting this enzyme might be useful in multiple bacterial infections (21). Ivil9b
encodes TehB. The overexpression of tehB increases the resistance to several quaternary
ammonium compounds and causes an increase in the efflux of ethidium bromide (49).
This efflux pump might play a role in A. pleuropneumoniae survival during infection

when it is surrounded by toxic molecules generated by macrophage lysosomes.

65

Another category of genes are regulators of virulence factors. This includes iviS,
iviX, and ivi17b. IviS encodes qu, a RNA binding protein that has been shown to
regulate expression of virulence factors such as axyS. IviX encodes VapBC, a newly
identified complex that regulates maintenance of virulence factors on plasmids such that
during times of rapid growth, the daughter cells are as virulent as the parent cell. Ivi17b
encodes TexA. TexA is a potential regulator of virulence factors, having first been
identified as an essential gene in Bardetella pertussis, required for the regulation of
several toxins. This putative RNA binding factor might be a global regulator of virulence
factors, or function in a manner similar to that in B. pertussis where it acts in the
differential expression of virulence factors (14). In addition, the gene expressed by the
promoter in iv120a might be a regulator of virulence. The ORF found in this clone has
homology to GlpC, a protein of unknown function that might help in association of
GlpAB to the bacterial membrane. The other role for the potential promoter of ivi20a
might be for expression of the downstream ribGBAH operon.

An additional category of ivi clones are those that potentially encode antisense
RNA. These clones contain DNA that has homology to the middle of a known gene. IviJ
contains prfC and ivi20b contains recJ, both in an antisense direction. It has yet to be
tested to determine if antisense mRNA for these genes are actually present within A.
pleuropneumoniae. The function of antisense RNA is to limit the expression of proteins
that might need to be transiently regulated during times of energy limitation or nutrient
stress.

A final category of ivi clones are those that encode hypothetical proteins that are

highly conserved through many databases and other unknown genes. Three of the 25 ivi

66

clones contain promoters of ORFs that have no known function. An additional three
clones were unable to be characterized, even in the presence of a database containing
DNA sequence from A. pleuropneumoniae serotype 5. This is due to the fact that one of
the clones was not found in serotype 5, another clone was at the end of a contig and
downstream genes could not be characterized, and the final clone was found in the
middle of a region of multiple short ORFs that lacked sequence similarity to known
proteins.

Thus this IVET study has contributed to the understanding of A.
pleuropneumoniae pathogenesis through the proposal of several new virulence factors
and pathogenically important metabolic pathways. This increase in the understanding of
A. pleuropneumoniae induced disease, and potentially of similar respiratory infections in
other mammalian hosts, might lead to important antibiotic targets or to enhanced vaccine
production through the incorporation of important antigens. With the further
characterization of the currently identified in viva induced genes from this study and the
isolation of more clones from A. pleuropneumoniae IVET studies, the understanding of
the this disease process will increase dramatically. With the sequencing of the A.
pleuropneumoniae serotype 1 genome, the flanking regions to these promoters can be
determined, easing the characterization steps and allowing further insight into the

pathogenesis of A. pleuropneumoniae infection.

67

References

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

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73

Chapter 3

0hr, Encoding an Organic Hydroperoxide Reductase,

Is an In Vivo Induced Gene in Actinobacillus pleuropneumoniae

This manuscript has been previously published in Infection and Immunity.
Shea, R. J. and M. H. Mulks. 2002. ahr, encoding an organic hydroperoxide reductase,
is an in viva-induced gene in Actinobacillus pleuropneumoniae. 70: 794-802.

74

Abstract

Actinobacillus pleuropneumoniae is the causative agent of porcine
pleuropneumonia, a disease characterized by pulmonary necrosis and hemorrhage caused
in part by neutrophil degranulation. In order to survive within this toxic environment,
bacteria have developed enzymes to detoxify the molecules released during
degranulation. Many of these enzymes can be considered virulence factors that are only
required during infection and are regulated in a manner such that they are only expressed
during infection. In an effort to understand the pathogenesis of this disease, we have
developed an IV ET (in yivo gxpression technology) system to identify genes that are
specifically up-regulated during infection. One of the genes that we have identified as
being in vivo induced is ahr, encoding Qrganic hydroperoxide reductase, an enzyme that
could play a role in detoxification of organic hydroperoxides generated during infection.
This identification was based upon protein homology to previously characterized Ohr
proteins.

Among the twelve serotypes of A. pleuropneumoniae, ahr was only found in
serotypes 1, 9, and 11. This distribution correlated with increased resistance to cumene
hydroperoxide, an organic hydroperoxide, but not to hydrogen peroxide or to paraquat, a
superoxide generator. Functional assays of Ohr activity demonstrated that A.
pleuropneumoniae serotype 1 cultures, but not serotype 5 cultures, were able to degrade
cumene hydroperoxide. In A. pleuropneumoniae serotype 1, expression of ahr was
induced by cumene hydroperoxide, but not by either hydrogen peroxide or paraquat. In

contrast, an ahr gene from serotype 1 cloned into A. pleuropneumoniae serotype five was

75

not induced by cumene hydroperoxide or by other forms of oxidative stress, suggesting
the presence of a serotype-specific regulator of ahr in A. pleuropneumoniae serotype 1.

The search for a novel regulator of ahr is ongoing.

76

Introduction

Actinobacillus pleuropneumoniae is the causative agent of porcine
pleuropneumonia, a disease characterized by massive lung necrosis and pulmonary
hemorrhage. This necrosis is due in part to the influx of host immune cells and the
release of neutrophil lysosomal contents that include oxygen radicals, which can destroy
the invading bacteria as well as the host tissue (28). In an effort to understand the
pathogenesis of this respiratory disease and the effect of the host immune response on A.
pleuropneumoniae, we have developed an in y_ivo expression technology (IVET) system
to identify genes that are expressed during infection but not during growth in vitro on
laboratory media (1 3). One of the genes that we have identified with this selection shows
homology to ohr (Qrganic hydroperoxide geductase), which has been described in
Xanthamanas campestris (25), Pseudomonas aeruginasa (30), Enterococcus faecalis
(34), and Bacillus subtilis (l2). Ohr has been implicated in resistance to and
detoxification of the organic peroxides, such as cumene hydroperoxide (25, 30).

Bacteria are frequently exposed to reactive oxygen species during the course of
infection. Oxygen radicals in the form of superoxides, hydrogen peroxides, and organic
hydroperoxides can result from release of lysosomal contents within inflammatory cells
or can be generated by bacterial cellular metabolism (3, 39). During infection of the
porcine lung, A. pleuropneumoniae is exposed to oxygen radicals in the form of
superoxides and peroxides generated by the neutrophil oxidative burst (28). A third class
of oxygen radicals, organic hydroperoxides, can be generated either directly within the

phagosome or as a consequence of oxygen radicals interacting with the bacterial cell

77

membrane [reviewed in Miller and Britigan (24)]. To survive and protect its cellular
metabolism within this dangerous milieu, A. pleuropneumoniae may require enzymes
capable of inactivating these oxygen species (21).

A. pleuropneumoniae has been shown previously to contain catalase and two
distinct superoxide dismutases, SodA and SodC, which can relieve a portion of the
oxidative stress that occurs during infection (22). Enzymes that could detoxify the third
category of oxidative stress reagents, the organic hydroperoxides, have not been
previously identified in A. pleuropneumoniae. In this work, we identify another
potentially protective gene, ahr, which is specifically induced during infection and
produces a protein that is capable of detoxifying organic peroxides encountered by A.

pleuropneumoniae during infection of the porcine lung.

78

Materials and methods

Bacterial strains. The A. pleuropneumoniae strains that were used in this study and the
plasmids propagated in these strains are listed in Table 1. A. pleuropneumoniae strains

were cultured at 35°C on either brain heart infusion (BHI) (Difco, Detroit, Mich.) or heart
infusion (HI) (Difco) media supplemented with V factor (B-NAD) added at 10 ug/ml
(BHIV or HIV, respectively). Riboflavin, when needed for maintenance of APP233, was

added at 200 pig/ml. Media was supplemented with ampicillin at 50 ug/ml to propagate

plasmids or at 20 ug/ml to recover A. pleuropneumoniae from porcine lungs and to select
for transfonnants after electroporation. Escherichia coli strain XLI-Blue mRF’
(Stratagene, LaJolla, Calif.) was used for construction and propagation of plasmids. E.
cali was cultured on Luria-Bertani (LB) medium (Difco) supplemented with ampicillin at

100 pig/ml.

Molecular manipulations. Genomic DNA from A. pleuropneumoniae was prepared
according to the lysis/proteinase K method of the Gene Fusion Manual (37). Plasmid
DNA was purified using Qiagen spin columns (Qiagen Inc., Valencia, Calif). DNA
modifying enzymes were obtained from Roche (Roche Molecular Biochemicals,
Indianapolis, Ind.) and used according to the manufacturer’s specifications.

Preparation of electrocompetent A. pleuropneumoniae serotype 1 and
electroporation of these cells with plasmid DNA prepared from either E. cali or A.
pleuropneumoniae serotype 1 was performed as previously described (15). A.

pleuropneumoniae serotype 5 (14) was made competent by the method of Ward et al (43)

79

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and electroporated using the same electroporation conditions as for serotype 1, but using
plasmid prepared from E. coli. E. coli transformation was performed by the Hanahan

method (17).

Southern blotting. Genomic DNA from all 12 A. pleuropneumoniae serotypes (Table 1)
was digested to completion using EcoRI and separated on a 0.8% agarose gel. DNA
fragments were transferred to a Nytran (Schleicher & Schuell, Keene, NH.) membrane
by the method of Southern (36). The membrane was hybridized with a digoxigenin-
labeled probe generated by PCR amplification of the complete 0hr gene using primers
(MMISO: 5’-GACAAGAATTCAACAAGGACAATATTATG-3’ and MMISI: 5-
CCTAAATCGTCCCAGATCTGGTAGG-3’) that flank the open reading frame (Roche
PCR DIG synthesis Kit). For high stringency hybridization, blots were incubated for 16
hours at 42°C with the probe diluted in a hybridization buffer that contained 50%
formamide, 5 X SSC (sodium chloride, sodium citrate), and 2% blocking reagent
(Roche). High stringency washes were performed at 68°C with 0.1 X SSC, 0.1% SDS
(sodium dodecyl sulfate). For low stringency hybridization, blots were incubated for 16
hours at 42°C with the probe diluted in an aqueous solution containing 6 X SSC and 1%
blocking reagent, with no forrnamide added. Low stringency blots were washed for 10
minutes at room temperature with 2 X SSC, 0.1% SDS. Hybridizing bands were
detected using alkaline-phosphatase tagged anti-digoxigenin and the CDP“

Chemiluminescent substrate (Roche).

82

Infection model. The infection of pigs with either APP233/piviK or APP233/pTF86 was
performed as previously described (13). Briefly, each clone was inoculated into HIV
broth containing 200 ug/ml riboflavin, 50 pg/ml ampicillin, and 5 mM calcium chloride
and grown at 35°C to an ODszo of 0.8. The bacteria were washed with saline and
resuspended in 10 ml of saline containing 200 ug/ml riboflavin at a final concentration of
7 X 108 CPU. The bacterial suspension was inoculated, via an endotracheal catheter, into
the lungs of six week old pigs. The pigs were monitored for development of clinical
signs of pleuropneumonia as previously described (19) and in conjunction with animal
use approval. Sterile lung samples were collected for bacterial isolation and photography
following necropsy. Lung samples were examined for in situ luciferase activity using a
Hamamatsu C1966 Photonic Microscope System (13). All experimental protocols for
animal experiments were reviewed by the Michigan State University All University
Committee on Animal Use and Care, and all procedures conformed to university and US.

Department of Agriculture regulations and guidelines.

Cloning of the intact 0hr gene. An inverse PCR (iPCR) technique was designed to
clone the intact 0hr gene (29). 2.5 pg of A. pleuropneumoniae serotype 1 genomic DNA
was digested to completion with EcoRI, followed by a self-ligation using T4 DNA ligase
(Roche) to form closed circular fragments. PCR amplification with AmpliTaq (Roche)
was performed with 0.04 pg of ligated DNA using primers (MM139: 5’-
AACCAAGTGAACCGTCATCTACTC-3’ and MM140: 5’-GTGGCAAAGTCGGCAC
AAACC-B’) designed from the known sequence of the MK clone, which contained the

Promoter region and partial open reading frame of the 0hr gene. These primers bound

83

within the coding region of 0hr and were oriented to PCR amplify the flanking regions.
The resulting 2.5 kb PCR fragment was isolated from an agarose gel using the Qiaex H
kit (Qiagen) and was cloned into the pGEM-T vector (Promega, Madison, Wisc.) to form
pinKE. This clone was sequenced using an ABIlOO Model 377 automated sequencer
(Applied Biosystems, Foster City, Calif.) and a primer internal (MM141: 5’-
CTGTAGGCGTGGGAATCGGTC-3 ’) to the known 0hr sequence. A complete open
reading frame (ORF) was identified by comparison of the sequence obtained from the
MK clone and the sequence that resulted from the iPCR clone pinKE. The complete 0hr
ORF with the upstream promoter region was amplified from A. pleuropneumoniae
serotype 1 genomic DNA using Pfix polymerase (Promega) and a primer pair (MMl38:
5’-GGCTACGAAATATTGGACACG—3’ and MM 151) that binds 360 bp upstream of
the start codon and 50 bp downstream of the stop codon. The PCR product was cloned
into SmaI cut pGZRSl8 to form pGeohr (44). This plasmid was transformed into both A.

pleuropneumoniae serotype 1 and A. pleuropneumoniae serotype 5.

Oxidative stress growth inhibition assay. Hydrogen peroxide (Sigma, St. Louis, Mo),
cumene hydroperoxide (CHP, Sigma) and the superoxide generator paraquat (Sigma)
were used as oxidative stress reagents. Disk inhibition assays were used to analyze
bacterial sensitivity to these reagents (39). Briefly, 100 u] of an overnight bacterial liquid
culture was added to 3 ml of BHIV top agar (0.7%) and poured onto BHIV plates. Filter
paper disks (10mm, Whatman #1, Whatman Paper Ltd., Maidstone, England) saturated
with 10 ul of 0.88 M hydrogen peroxide, 200 mM CHP, or 0.074 M paraquat were placed

onto the hardened top agar (39). Diameters of the zones of growth inhibition were

84

recorded after 22 hours of incubation at 35° under 5% C02. Statistical analysis of zone
diameter significance within each treatment group for serotypes containing 0hr as
compared to serotypes lacking the gene was evaluated by a two-tailed Student’s t-test.
The Analysis ToolPak in Microsofi Excel (2000) was used to perform the t-test under

homoscedastic and heteroscedastic conditions.

Oxidative stress and measurement of tax expression. Induction of oxidative stress in
broth cultures was performed as follows. A. pleuropneumoniae strains were grown in 25
ml of BHIV broth containing 50pg/ml ampicillin, at 35° C and shaking at 150 rpm, to an
ODszo of 0.8, and were then dispensed into a 96 well microtiter plate in 200 pL aliquots.
This was followed by a 30 minute incubation period at 35°C under 5% C02 prior to
addition of the stress reagent to allow for acclimation. For stress induction, stress
reagents were added to a final sublethal concentration of 125 pM, 300 pM, or 1 mM
CHP; 56 pM hydrogen peroxide; or 50 pM paraquat (39). Aliquots were taken for
luminometric assays and primer extension at designated intervals.

Quantitative luciferase assays were performed using a Turner Model 20c
luminometer as previously described (13). Briefly, 20 pl of culture was mixed with 20 pl
of N-decyl aldehyde in a polypropylene luminometer cuvette. The sample was read in
full integral, autoranging mode with a predelay of 0 seconds, a delay of 10 seconds, and
an integration time of 30 seconds. The N-decyl aldehyde substrate was made by
sonicating 20 mg/ml of Essentially Fatty Acid Free Bovine Serum Albumin (Sigma) with

l pl/ml of N-decyl aldehyde in a sonicating water bath at room temperature.

85

Lurninometer readings were normalized to relative light units per optical density

(RLU/ODszo).

Functional analysis of Ohr activity. An assay to evaluate the degradation of CHP was
adapted from procedures developed by Dringen et al. (9) and Ochsner et al. (30). A.
pleuropneumoniae was grown in BHIV broth to early stationary phase (OD520 = 0.8-1.0)
and diluted with fresh prewarmed media. CHP was added to a final concentration of
either 0 pM, 125 pM, 300 pM or 600 pM CHP. Residual CHP concentrations were
determined at five minute intervals using a xylenol orange- iron reaction. At each time
point, 100 pl of the culture was pelleted and 20 p1 of the cell- free supernatant was added
to 80 pl of 25 mM sulfuric acid in a 96 well microtiter plate. When all samples had been
collected, 100 pl of freshly prepared reaction buffer containing 200 pM xylenol orange
(Sigma), 200 pM ammonium ferrous sulfate (Sigma), and 25 mM sulfuric acid was added
to each sample. After 10 minutes of incubation at room temperature, absorbance was
read at 540 nm using a Bio-Tek ELISA plate reader Model EL310 (Bio-Tek Instruments,
Inc., Winooski, VT). Concentrations of CHP in each sample were determined by
comparison to a CHP standard curve performed at the time of each assay. Ohr activity
was measured as pmoles CHP degraded/ minute.

To evaluate the induction of Ohr activity by CHP, CHP was added to 1 ml of the
freshly diluted culture at a final concentration of 0 pM, 125 pM, or 300 pM, and this
mixture was held without shaking for 30 minutes at 35° C. A sample was collected to
determine the residual CHP concentration. Fresh CHP was added and Ohr activity was

assayed as described above.

86

Primer extension. Primer extension of the 0hr gene was performed as previously
described (8) using a primer (MM220: 5’-CGAGTATGACCATCACGACCGCCACT
GC-3’) that bound 30 bp into the 0hr coding region. Bacteria were incubated for 30
minutes in a 96 well microtiter plate under inducing conditions with 1 mM CHP, and
without CHP as an non-induced control. The mRNA was isolated using a hot phenol
extraction method (45). For reverse transcription, 10 pg of RNA was incubated with 1
pM of 32P-ATP(Amersham Pharrnacia Biotech, Piscataway, N.J.)—labeled primer and
AMV-Reverse transcriptase (Promega) for 1 hour at 42°C. The samples were separated
on an 8% denaturing polyacrylamide gel along with a 35S-dATP (Amersham) sequencing
ladder. The sequencing ladder was prepared using the Sequenase v 2.01 kit (Amersham)

and the same primer that was used for primer extension (8).

Nucleotide sequence accession number. The sequence reported in this paper has been

submitted to GenBank and assigned accession number AF 395877.

Protein expression of Ohr. The complete sequence of 0hr was used to design primers to
fuse the gene to a hexa-histidine tag in order to purify Ohr protein. Plasmid pRS97b was
constructed by PCR amplifying the majority of the 0hr ORF using primers MM 194 (5’-
GCACCATGGAAATTTTTTACAAAACATCAGCAACAGC-3’) and MM206 (5’-
TACGGATCCATGTAGACGTACGTCCACGTTACC-3’) and cloning this 400 bp
fragment into pQE60 (Qiagen). The pRS97b plasmid contained the first 134 amino acids

of Ohr, including the putative functional region, fused in frame to a C-terminal six

87

histidine tag. pRS97b was transformed into XLl-Blue mRF’ and expression of His-
tagged Ohr protein was induced as follows: First, a 50 ml broth culture in LBAloo was
grown to an ODszo of about 0.4 shaking at 150 rpm in 37°C waterbath. IPTG was added
to a final concentration of lmM and the culture was maintained under the same
incubation conditions for six hours. After six hours, the 50 ml culture was diluted into
250 ml of fresh LBA100 broth containing 1 mM IPTG, and the culture incubated for an
additional two hours to allow for a high cell density. Protein could be isolated from this
cell culture using the protocol for denaturing conditions. This involved lysing the cells
by vortexing with 8M urea. The lysate was then incubated with Ni-NTA resin in a batch
format and the mixture was loaded into an empty column. The flow-throughs were
collected an analyzed as the resin was washed with increasing elution buffers, of

decreasing pH, all of which contained 8M urea. (4th edition QIAexpressionist, Qiagen).

88

Results

Identification of 0hr as an in vivo induced gene. We previously developed an IV ET (in
yivo expression technology) screen to identify genes from A. pleuropneumoniae that
were induced during infection of the porcine lung but had minimal expression during in
vitro growth on laboratory media (13). This IV ET system utilized a promoter trap vector
(pTF 86) with a cloning site for genomic DNA fragments upstream of promoterless copies
of the luxAB and ribBAH genes. A library of random genomic DNA fragments fiom A.
pleuropneumoniae serotype 1 was cloned into the pTF86 vector and subsequently
electroporated into APP233, a virulent A. pleuropneumoniae serotype 1 strain that is
unable to produce riboflavin due to a directed mutation within the ribGBAH operon (15).
When a functional promoter was placed in the cloning site of pTF86, riboflavin was
produced and complemented the riboflavin deficiency of the host strain, APP233,
restoring full virulence. Without a functional promoter, riboflavin was not produced and
APP233 failed to survive and cause disease (15). Thus the initial portion of the IVET
procedure utilized infection of the natural host to select clones containing promoters that
were expressed during infection. Clones containing functional promoters were isolated
fi'om the pig lung and examined for in vitro expression of the luxAB genes both
quantitatively and qualitatively. Clones that had in vitro expression levels in APP233
that were less than or equal to 200 RLU/ODszo were identified as ivi (in yivo induced)
clones.

Forty-two unique ivi clones were identified during this selection. One of these

clones, iviK, contained an 801 bp insert that included a partial ORF of 98 amino acids

89

fused to luxAB, as well as 507 bp of upstream noncoding sequence. When this ORF,
which contained a start codon but lacked a stop codon, was used to search microbial
sequence databases, it showed 56% similarity to Ohr fiom X. campestris, an enzyme
responsible for protection against organic peroxides (25).

To confirm the in vivo induction of the MK promoter, a pig was infected
intratracheally with 7 x 108 CFU of APP233/piviK to monitor development and
progression of the puhnonary disease. We have previously demonstrated that APP233
alone is avirulent at doses as high as 5 X 109 CFU in this animal infection model (13, 15).
Within 6 hours, the infected pig developed an increased respiratory rate and fever and
showed depression and anorexia. The disease progressed to severe dypsnea by 9 hours
post infection. At necropsy, 90-100% of the right lung lobes and the accessory lobe
showed edema, hemorrhage, congestion, and regions of necrosis. These symptoms are
consistent with peracute pleuropneumonia. A portion of the right caudal lung lobe from
this pig was photographed by visible light camera and by photonic camera (Figure 1).

The visible light picture shows regions of severe necrosis and hemorrhage. The
photonic camera picture of this same region of lung shows qu expression at the edges of
this necrotic tissue, which is the region of active infection. In contrast to infection by this
iviK clone, we have previously shown that infection with a 5 X 109 CF U of a clone
containing the pTF86 vector only does not result in disease symptoms in the pig (13).
When lung tissue isolated from a pig infected with APP233/pTF86 was examined, there

were no regions of necrosis or of lux expression (Figure l).

90

 

 

pTF86

 

 

 

 

 

WH<

 

Figure 3—1 Pulmonary damage and in vivo qu expression resulting from infection of a
swine lung with either APP233/pTF86 (row 1) or APP233/piviK (row 2). For each
sample, on the left is an incidental light photograph of the lung specimen. On the right is
a corresponding photograph, taken by photonic camera, showing bioluminescence of the
same lung specimen. In the absence of a promoter, APP233/pTF86 showed no lung
damage and no lux expression. In the presence of the piviK plasmid, significant lung
damage was apparent and bioluminescence was seen.
Cloning and characterization of full length 0hr. The nucleotide sequence of iviK was
used to design inverse PCR primers to clone the full length 0hr. A 2.5 Kb PCR product
was obtained and cloned into pGEM-T to form pinKE. This insert was sequenced, and
alignment of this sequence with that of iviK demonstrated a contiguous ORF of 432 bp
encoding 143 amino acids. This ORF was identified based on sequence homology as 0hr
(Figure 2). Primers were designed to amplify the entire 0hr gene plus 360 bp of upstream
sequence. This PCR product was cloned into the pGZRSl8 vector to form pGeohr.

A potential ribosome binding site (AAGGA) at 12 bp upstream of the start codon

was identified. Potential transcriptional terminators flanking the ORF were identified

91

using the GCG Stemloop program (16). The predicted protein sequence was compared to
finished and partially finished bacterial genomes deposited in the Microbial Genomes
BLAST Databases using the BLAST programs (National Center for Biotechnology,
NCBI). The ORF had highest homology to Ohr from P. aeruginasa with 62% similarity
and 48% identity when examined using BLAST pairwise homology (40) (30). Proteins
with strong homology to Ohr were identified from 14 species of bacteria and were
aligned to identify regions of identity and similarity (Figure 2). This alignment shows
two regions of high conservation that center around conserved cysteines, with the
sequence outside of these regions lacking extensive similarity. Analysis of the protein
using PSORT to predict cellular localization suggested that Ohr was a cytoplasmic

protein (27).

Distribution of 0hr among serotypes correlates with organic peroxide resistance. To
determine if 0hr was present in all 12 serotypes of A. pleuropneumoniae, a Southern blot
was performed using EcoRI digested genomic DNA from all 12 serotypes as the target
and the full length 0hr from A. pleuropneumoniae serotype 1 to construct the probe.
Genomic DNA fragments that hybridized to 0hr were seen only in A. pleuropneumoniae
serotypes 1, 9, and 11 (Figure 3A), and not found in the other serotypes, under conditions
of either high or low stringency.

Sensitivity to oxidative stress reagents for each of the 12 A. pleuropneumoniae
serotypes was examined using a disk inhibition assay. Overnight cultures were added to
top agar and overlaid with disks saturated with hydrogen peroxide, CHP, or the

superoxide generator, paraquat. After 22 hours, the zones of growth inhibition were

92

Figure 3-2. Protein sequence alignment of putative Ohr proteins from 14 bacterial
species aligned using Boxshade v3.31 (http://biophysics.med.jhu.edu/prog/boxshade/
PC_and_MAC/win16.zip) and ClustalX (41). Black shaded regions indicate residues that
are identical in the majority of species. Gray shaded regions indicate residues that are
functionally conserved in the majority of species. This alignment highlights the highly
conserved regions surrounding the conserved cysteine residues (*). Species
abbreviations used are as follows: App: Actinobacillus pleuropneumoniae; Pa:
Pseudomonas aeruginasa; Vc: Vibrio cholerae; Sp: Shewenella putrefaciens; Lp:
Legionella pneumophila; Xc: Xanthamanas campestris; Cc: Caulobacter crescentus;
le: Bacillus subtilis YklA (OhrA); Ba: Bacillus anthracis; B32: B. subtilis Ysz
(OhrB); Dr: Deinococcus radiodurans; Sa: Staphylococcus aureus; Ef: Enterococcus
faecalis; Mp: Mycoplasma pneumoniae.

93

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Figure 3-3. Correlation of 0hr presence with resistance to organic hydroperoxides. The
presence of the gene was determined by Southern blot of genomic DNA using the intact
0hr gene as a probe (3A). Molecular weight standards (in kb) are shown on the left. The
resistance of each of the 12 A. pleuropneumoniae serotypes to oxidative stress agents was
determined by measuring the diameter of the zone of growth inhibition due to CHP (3B),
hydrogen peroxide (3C) and paraquat (3D). The data presented is the average of three
experiments with the standard deviation shown by error bars. The differences in the zone
diameters, after incubation with CHP, seen with serotype 1, 9, and 11, in comparison to
the remaining serotypes, are statistically significant (P < 0.0001).

95

 

 

 

 

 

 

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96

10 11 12

recorded as a measure of the sensitivity of each serotype to the oxidative stress imposed.
Eleven of the twelve serotypes showed equivalent sensitivity to hydrogen peroxide and
paraquat (Figure 3C, 3D). The exception was serotype 6, which showed a significantly
larger zone diameter (P < 0.01) with all three forms of oxidative stress, suggesting a
decreased resistance to oxidative stress reagents in general for this serotype. Serotypes 2-
8, 10, and 12 showed sensitivity to CHP similar to that seen with hydrogen peroxide and
paraquat (Figure 3B, 3C, 3D). In contrast, serotypes 1, 9, and 11 showed a significantly
reduced zone of growth inhibition upon incubation with CHP, reflecting an increased
resistance to oxidative stress caused by organic peroxides (Figure 3B). This increased
resistance of serotypes 1, 9, and 11 to CHP was highly statistically significant (P <
0.0001), and no statistically significant difference was seen for these serotypes in
response to incubation with hydrogen peroxide or paraquat. This increased resistance to
CHP, but not to hydrogen peroxide or paraquat, correlated with the presence of the 0hr

gene as shown by Southern blot (Figure 3A).

Induction of 0hr in response to oxidative stress. To characterize the expression of 0hr
in response to different oxidative stress reagents, induction studies were performed in
wild type A. pleuropneumoniae serotype 1. The analysis was performed in the wild type
strain in order to decrease the oxidative stress imposed on the cultures due to the presence
of riboflavin in the media that is necessary for grth of the APP233 strain.
APP225/piviK, which contained the 0hr promoter-luxAB fusion, was induced in
microtiter plates with paraquat, CHP, or hydrogen peroxide. Addition of CHP resulted in

a rapid increase in lux expression in comparison to the noninduced control (Figure 4).

97

 

 

 

Lux activity (103 RLU/ooszo)

 

 

Time (minutes)

Figure 3-4. Induction of the cloned 0hr promoter in A. pleuropneumoniae serotype 1
(APP225/piviK) by various oxidative stress reagents. 1 mM CHP (I), 50 M paraquat

(o), and 56 M hydrogen peroxide (A) were added as oxidative stress reagents and qu
activity, expressed as RLU/ODszo, was measured. Lux activity was also measured in

control cultures to which no inducing agent was added ( D ). Data presented are from a
representative experiment. Trends were identical in all experiments.

Neither paraquat nor hydrogen peroxide caused any induction, and the level of lux

expression was equivalent to that seen in the absence of oxidative stress (Figure 4).

Comparison of induction of 0hr in A. pleuropneumoniae serotype 1 and A.
pleuropneumoniae serotype 5. We compared the expression of the 0hr:lux fusion using
APP225/piviK (serotype 1) and APP227/piviK (serotype 5) during normal grth in

culture and under induction by CHP. A growth curve was performed for each serotype to

98

evaluate the expression level of 0hr during growth in an aerated broth culture. The
expression of 0hr for both A. pleuropneumoniae serotype 1 and A. pleuropneumoniae
serotype 5, as measured by luciferase activity, remained constant during normal growth,
with the increase over time directly correlated to the increase in total cell number (data
not shown). The expression level was independent of serotype, with both serotypes
showing low but equal expression levels during normal grth in the absence of
inducers. This expression level of luciferase in the wild type strains of A.
pleuropneumoniae serotype 1 and A. pleuropneumoniae serotype 5, which averaged 1000
RLU/OD, is greater than that seen in the A. pleuropneumoniae serotype 1 riboflavin
mutant (APP233), which was never higher than 200 RLU/OD. Expression of luciferase
from the pTF86 vector alone, with no insert, showed minimal expression of <50
RLU/OD in both serotypes (data not shown).

Expression of 0hr under inducing conditions was examined in both A.
pleuropneumoniae serotype 1 and A. pleuropneumoniae serotype 5. Induction of 0hr in
response to incubation with CHP was seen only in A. pleuropneumoniae serotype 1 and
not in A. pleuropneumoniae serotype 5 (Figure 5). APP225/piviK showed rapid
induction of expression as measured by Lux assay with a two-fold increase within 10
minutes post exposure to CHP. Lux activity increased over time with maximal levels
detected between 30 and 60 minutes after induction. In contrast, APP227/piviK showed
no increase in lux expression in response to CHP and maintained a level of expression
slightly greater than that of the vector only control. This data suggests that incubation

with CHP does not cause induction of 0hr in A. pleuropneumoniae serotype 5.

99

 

   
  
    

 

 

 

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Figure 3-5. Expression of the 0hr promoter is induced by 125 M CHP in A.
pleuropneumoniae serotype 1 and not in serotype 5. Lux expression driven by the 0hr
promoter was measured over time in A. pleuropneumoniae serotype 1 (APP225/piviK)
(I) and in A. pleuropneumoniae serotype 5 (APP227/piviK) (A), in comparison to a
vector only control, APP225/pTF86 ( D ). Data presented are from a representative
experiment. Trends were identical in all experiments.

This induction data was confirmed through a functional assay of Ohr enzymatic
activity. A xylenol orange colorimetric assay was used to determine the concentration of
CHP after incubation of CHP with bacteria. Each serotype was grown in broth and then
diluted into fresh media containing 125 M CHP or 300 M CHP followed by incubation
for 30 minutes to allow for induction. After this induction period, 125 M CHP, 300 uM
CHP, or 600 M CHP was added and the rate of CHP degradation was measured.
APP225, A. pleuropneumoniae serotype 1, showed significant Ohr activity, as measured

by the rate of CHP degradation, in the absence of induction. Induction of A.

pleuropneumoniae serotype 1 for 30 minutes with either 125 uM CHP or 300 M CHP

100

resulted in an approximately two-fold increase in enzymatic activity, which correlated
well with the increase in 0hr expression as measured by Lux activity (Figure 5, Figure 6).

In contrast, neither APP227 (A. pleuropneumoniae serotype 5),
APP227/pGZRSl8, nor APP227/pGeohr showed significant Ohr activity in the absence
of induction, and no increase in activity was evident under inducing conditions. These
results correlate with and confirm the lack of expression of 0hr in APP227 as measured
by Lux activity (Figure 5, Figure 6). The assays performed with APP227 showed that

concentrations of CHP greater than or equal to 300 uM CHP were lethal to the cells and

thus induction and assays were performed at 125 pM CHP.

Evaluation of the mRNA start site in serotype 1 under CHP induction. Primer
extension was performed using mRNA isolated from APP225/piviK that had been
induced with CHP. The major transcriptional start site was located 31 bp upstream of the
0hr start codon (Figure 7). A —10 region (TAAAAT) was identified 6 bp upstream of the

transcription start site. However, no —35 region similar to that found in A.
pleuropneumoniae housekeeping genes (TTRAA, where R is A or G) could be identified

(8). In the region in which a —35 site would be expected to exist, a SoxS binding motif
(ACCGCAT) was found (3 5). Primer extension under non-inducing conditions was also
performed using six-fold more RNA. A primer extension product could not be detected

under these non-inducing conditions (data not shown).

101

Figure 3-6. A) Ohr activity, expressed as umoles CHP degraded per minute, is induced
by CHP in A. pleuropneumoniae serotype 1 (APP225) but not in serotype 5 containing
the intact 0hr gene plus promoter region (APP227/pGeohr). Controls include APP227
with no plasmid and APP227 containing the pGZRSl8 shuttle vector only. For
induction, CHP was added at final concentrations of 0, 125, or 300 uM to 1 ml broth
cultures, which were held for 30 minutes at 35°C. Ohr activity was measured as
degradation of CHP. B) Lux activity, measured as RLU/OD, is similarly induced in
APP225/piviK but not in APP227/piviK. Induction conditions for these assays were
identical to those in panel A. Data presented are from a representative experiment.
Trends were identical in all experiments.

102

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Figure 3-7. Primer extension analysis of APP225/piviK induced with 1 mM CHP. The
sequencing reactions and the primer extension reaction were performed with an identical
primer. The lane on the right contains the primer extension product. The transcriptional
start site is labeled in bold (C). The -10 region (TAAAAT) and the potential SoxS
binding motif (CCGCAT) are indicated on the lefi. An intervening region between the
sequencing ladder and the primer extension product was removed, without alteration of
alignment.

Evaluation of the Ohr-hexahistidine protein. When XLl-Blue mRF’/pRS97b was
induced with 1 mM IPTG, the cell pellet from 50 pl of culture was examined on a 15%
SDS-PAGE gel, a heavily stained band of approximately 14 kDa was seen. No similar
protein band was seen in 50 pl of uninduced cells grown under the same conditions

(Figure 8, lane 5). In initial attempts to purify the protein, minimal binding to the nickel-

104

NTA column was obtained with non-denaturing, native, conditions (data not shown).
Using denaturing conditions (8 M urea), the protein was bound to the column and
relatively pure protein was obtained after a series of wash steps (Figure 8, lane 2). This
observation suggested that the induction protocol developed led to the formation of
inclusion bodies that prevented the histidine tag from binding to the column in the native,
nondenatured state. The production of pure protein may prove useful in Ohr-antibody
production, crystallization of the Ohr-histidine protein, or in enzymatic assays of pure

protein.

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14 kDa a.” - «I-

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Figure 3-8. Purification of Ohr-hexahistidine protein. A 15% SDS-PAGE denaturing
gel was loaded with molecular weight markers (M) and various purification steps of the
Ohr protein. Lane 1 contains purified protein, lane 2 contains protein purified from
uninduced cells. Lane 3 contains uninduced cell lysate and lane 4 contains whole cell
lysate from cells induced with IPTG.

105

Discussion

Using the IVET system we developed to facilitate identification of Actinobacillus
pleuropneumoniae genes involved in the pathogenesis of disease, we identified 42 unique
promoter clones that were specifically induced during infection of the natural swine host.
In this study, we report the identification and characterization of one of these in vivo
induced clones, 0hr, which encodes an organic hydroperoxide reductase that we
hypothesize is involved in protection from oxidative stress encountered during infection.

Enzymes responsible for conferring enhanced resistance to oxidative stress
encountered during infection of the respiratory tract are potentially important virulence
factors for organisms that cause pneumonia, such as A. pleuropneumoniae (6, 46). The
mechanisms by which A. pleuropneumoniae causes disease lead to an environment filled
with oxygen radicals (21, 28). Upon infection of the porcine lung by A.
pleuropneumoniae, the host immune response to bacterial cell components, such as
lipopolysaccharide, triggers an influx of inflammatory cells, particularly neutrophils,
which limit the bacterial infection. Within this environment, A. pleuropneumoniae
produces hemolysins and cytotoxins, in the form of RTX toxins (repeats in toxin). These
pore-forming RTX toxins secreted by A. pleuropneumoniae insert into eukaryotic cell
membranes and cause lysis and cell death of neutrophils and macrophages, which in turn
releases phagocyte contents such as oxygen radicals in the form of peroxides and
superoxides (7, ll, 35). To survive in this environment, A. pleuropneumoniae likely
requires enzymes that allow the bacteria to evade or detoxify these oxygen radicals. A.

pleuropneumoniae has previously been shown to produce several enzymes involved in

106

response to oxidative stress, including catalase and two separate types of superoxide
dismutase (20, 22). This is the first report that this pulmonary pathogen produces an
additional oxidative stress protectant, an organic hydroperoxide reductase, and .the first
demonstration that this type of enzyme can be specifically induced in vivo during the
course of infection.

Ohr enzymes have been recently described in Xanthamanas campestris,
Pseudomonas aeruginasa, Enterococcus faecalis, and Bacillus subtilis and have been
shown to be important in the survival of these bacteria when exposed to oxidative stress
in vitro, although Ohr has not been previously implicated in virulence, (12, 25, 30, 34).
The 0hr gene from these organisms exhibits a similar pattern of expression to that of A.
pleuropneumoniae 0hr. In each of these organisms, 0hr is induced specifically in
response to organic hydroperoxides, with little or no induction in response to hydrogen
peroxide and superoxide (12, 25, 30, 34). This pattern of induction is distinct from that
seen with ahpC, which encodes Ahp (alkyl hydroperoxide reductase), a second class of
organic hydroperoxide reductase found in many bacterial species, including E. coli,
Salmonella typhimurium, B. subtilis, and P. aeruginasa (1, 4, 31, 38). Ahp enzymes are
induced by both hydrogen peroxide and organic peroxides but not superoxides (31, 35).
We identified putative Ohr sequences, based on homology to these five identified Ohr
proteins, from nine additional species (Figure 2), although not from any members of the
Enterobacteriaceae. The predicted Ohr proteins from these fourteen species share two
regions of strong homology that flank conserved cysteines, which may be responsible for

the reduction of peroxides to the corresponding alcohol (10). Similar conserved

107

cysteines are seen in Ahp enzymes, which are functionally similar to Ohr enzymes but
not closely related at the DNA level or protein level.

When we examined the type strains of the 12 known serotypes of A.
pleuropneumoniae for the presence of 0hr by Southern blot, we were able to detect an
0hr homologue only in A. pleuropneumoniae serotypes l, 9, and 11 and not in serotypes
2-8, 10, and 12. This distribution correlates with what is known about the relatedness of
A. pleuropneumoniae serotypes. Serotypes 1, 9, and 11 are closely related to one
another, having essentially the same LPS O-antigen chain, the same complement of RTX
toxins produced, and an identical genotype for one of these toxins, apxIA (18, 32). A.
pleuropneumoniae serotypes 1 and 9 have also been shown to be closely related to one
another and distinct from serotypes 2-8 using multilocus enzyme electrophoresis (26).
The differential distribution of 0hr among the serotypes may reflect both an evolutionary
relatedness of these serotypes and a need for detoxification of organic hydroperoxides
encountered during the course of infection.

The presence of 0hr among the 12 serotypes of A. pleuropneumoniae correlates
with resistance to oxidative stress reagents. Serotypes 2-8, 10, and 12, which do not
contain an 0hr gene, were equally sensitive to all types of oxidative stress agents tested,
as judged by the zone of grth inhibition upon exposure to cumene hydroperoxide
(CHP), paraquat, and hydrogen peroxide (Figure 3). In contrast, serotypes 1, 9, and 11
were significantly less sensitive to growth inhibition by CHP than the other serotypes, but
were similar to the other serotypes in sensitivity to hydrogen peroxide and paraquat. A.

pleuropneumoniae serotypes 1, 9, and 11 showed an increased resistance to CHP, but not

108

to hydrogen peroxide or superoxide, that correlates with the presence of the 0hr gene
(Figure 3).

The increased resistance to organic peroxides but not to other forms of oxidative
stress seen in A. pleuropneumoniae serotypes 1, 9, and 11 correlates well with data on the
induction of the 0hr promoter by various stress reagents in A. pleuropneumoniae serotype
1. Induction of 0hr was measured by luciferase assays using the 0hr promoter fused to
luxAB reporter genes and by assay of Ohr enzymatic activity via colorimetric detection of
CHP degradation. With both of these methods, 0hr expression in A. pleuropneumoniae
serotype 1 was induced by CHP but not by either hydrogen peroxide or paraquat (Figure
4, Figure 6).

We cloned both the intact serotype 1 0hr gene plus promoter region and an
ohr:lu.xAB gene fusion into A. pleuropneumoniae serotype 5, which lacks 0hr. During
growth in broth under non-inducing conditions, serotype 1 and serotype 5 showed low
but equivalent expression as assayed by hot expression. However, while A.
pleumpneumoniae serotype 1 is rapidly induced upon exposure to CHP, this induction is
not seen in A. pleuropneumoniae serotype 5, either as increased expression of luciferase
or as increased Ohr enzymatic activity (Figure 5, Figure 6). We conclude that A.
pleuropneumoniae serotype 5 not only does not contain a WT 0hr gene but also is unable
respond to exposure to CHP by induction of the cloned serotype 1 0hr gene. This
suggests that A. pleuropneumoniae serotype 5 may lack not only the 0hr gene itself but
also additional gene(s) necessary to increase the expression of 0hr in A.

pleuropneumoniae serotype 1.

109

Multiple regulators that respond to oxidative stress have been identified in other
prokaryotes, but none have as yet been identified in A. pleuropneumoniae. Three of the
most well studied regulators of oxidative stress responses in bacteria are OxyR, PerR, and
SoxR (2, 35). OxyR, which has been identified in many gram-negative bacteria, is
activated by exposure to peroxide, induces expression of ahpC (alkyl hydroperoxide
reductase) and catalase, and also represses its own expression (30, 42). In both X.
campestris and P. aeruginasa, 0hr expression was not altered by lack of OxyR (25, 30).
In many gram-positive organisms, ahpC and catalase are regulated by PerR, a homologue
of the ferric uptake regulator Fur, which is functionally analogous to OxyR (2, 5, 35).
Both of these regulators have known binding motifs that were not found in the A.
pleuropneumoniae 0hr promoter region (5, 42). SoxR, a transcription factor that is
activated by superoxide, induces expression of a second transcription factor, SoxS, which
in turn induces expression of superoxide-regulated genes such as sodA. SoxRS has also
been shown to regulate ahpC in some organisms, but has not been demonstrated to be
activated by peroxides (23, 33, 35),. In P. aeruginasa, 0hr induction was not affected by
mutations in SoxR (30).

When we examined the promoter region of A. pleuropneumoniae 0hr for potential
regulatory sequences, we identified a potential SoxS box but no PerR or OxyR binding
sequences. 80x8 in other organisms does not respond to organic peroxides, and the A.
pleuropneumoniae 0hr gene was not induced by superoxide generators. Our results
suggest that a novel regulator or regulatory sequence is responsible for induction of 0hr
in A. pleuropneumoniae, and that this novel regulator exists in A. pleuropneumoniae

serotype 1 and not in serotype 5. Further studies are in progress to identify this

110

regulatory molecule and to evaluate the role of Ohr in pulmonary infection caused by A.

pleuropneumoniae.

111

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116

Chapter 4

Genetic disruption of 0hr and ile

117

Abstract

Actinobacillus pleuropneumoniae is a respiratory pathogen of swine that causes a
hemorrhagic pleuropneumonia that results in high morbidity and mortality. A study has
been completed to identify in vivo induced genes in order to further study the
pathogenesis and virulence genes of A. pleuropneumoniae. 0hr and ile have been
previously identified as two in vivo induced genes. These genes were selected in order to
further understand how the bacteria adapts to host attempts to limit infection and as a
possible antibiotic target, respectively. Ohr is an organic hydroperoxide reductase that
could detoxify oxygen radicals that result from the influx of host immune cells (Chapter
3, and reference 15). 1le encodes acetohydroxy acid synthase 111, one of several proteins
responsible for fulfilling the metabolic requirement for isoleucine and valine (19). In
order to assess the role in virulence of these two genes, clones were constructed for gene
disruption through allelic replacement and insertion of either a cassette for kanamycin
resistance or NAD independence. Multiple matings and electroporations failed to yield a
stable double recombinant through homologous recombination. Several methods for

improving the selection of double recombinants are discussed.

118

Introduction

Actinobacillus pleuropneumoniae is the causative agent of porcine
pleuropneumonia, a disease characterized by massive lung necrosis and pulmonary
hemorrhage. This necrosis is due in part to the influx of host immune cells and the
release of neutrophil lysosomal contents that include oxygen radicals, which can destroy
the invading bacteria as well as the host tissue (10). In addition, the host is able to limit
bacterial growth and tissue destruction through prevention of bacterial acquisition
essential nutrients.

During infection of the porcine lung, A. pleuropneumoniae is exposed to oxygen
radicals generated by the neutrophil oxidative burst (10). One class of oxygen radicals,
organic hydroperoxides, can be generated either directly within the phagosome or as a
consequence of oxygen radicals interacting with the bacterial cell membrane [reviewed in
Miller and Britigan (6)]. To survive and protect its cellular metabolism within this
dangerous milieu, A. pleuropneumoniae contains enzymes capable of inactivating
peroxides and superoxides (6). The role of Ohr in the inactivation of organic
hydroperoxides has been previously described (Chapter 3 and reference 15) . In order to
further the study of this protein and its role in virulence, an A. pleuropneumoniae ohr-
mutant was required to determine if Ohr was essential for virulence and if this mutant
was more susceptible to oxidative stress.

Another gene upregulated during infection was ile. Ile, encoded by the ileH
operon, is homologous to several enzymes involved in the production of the hydrophobic

branch chained amino acids, isoleucine, leucine, and valine (13, 18, 19). 1le encodes

119

acetohydroxy acid synthase III (AHASIII), one of three isozymes that have been
characterized in both Escherichia coli and Salmonella typhimurium, that can catalyze the
condensation of pyruvate with either pyruvate or a—ketobutyrate to form valine or
isoleucine, respectively (19). The three isozymes appear to have arisen through gene
duplication and can be biochemically differentiated by their inhibition by the branch
chained amino acids (17). In A. pleuropneumoniae serotype 5, genes encoding
homologues of all three isozymes are present, but the function of the proteins encoded by
these genes has not yet been analyzed (John Nash, personal communication). AHASIII is
inhibited by valine, with the valine inhibition requiring IlvH. It has been proposed that
the two proteins encoded by the ileH operon encode a large catalytic subunit (ile) and a
regulatory small subunit (ilvH) (18). Ille is also regulated by LRP (leucine responsive
protein) in response to the concentration of leucine. For ileH, bound LRP increases the
transcription of this operon and high concentrations of leucine reduce expression of the
operon by reducing the activation of ileH by LRP (12, 21).

The role in virulence of ile is not yet clear. Ile might be a survival factor
directly, since in a hostile, nutrient limiting environment, increased biosynthesis of amino
acids would be required. The identification of ile as an in vivo induced gene might also
be due to a requirement for this gene in virulence, but rather because LRP and LRP-
regulated genes are required for virulence and increased transcription of ile is a

secondary effect due to increased production of LRP.

120

Materials and Methods

Bacterial strains. A. pleuropneumoniae strains were cultured at 35°C on brain heart
infusion (BHI) (Difco, Detroit, Mich.) media supplemented with V factor (B-NAD) added
at 10 ug/ml (BHIV). Media was supplemented with ampicillin at 50 ug/ml to propagate
plasmids or at 20 ug/ml to select for transformants after electroporation. The A.
pleuropneumoniae strains used were APP225, a virulent nalidixic-acid resistant
derivative of A. pleuropneumoniae ATCC 27088 (serotype 1A); and APP227, a virulent
naladixic-acid resistant derivative of A. pleuropneumoniae ISU178 (serotype 5 field
isolate) (1, 2). Escherichia coli strain XLI-Blue mRF’ (Stratagene, LaJolla, Calif.) was
used for construction and propagation of pUC and pGZRS-based plasmids. E. coli S-l7-
1 (7t pir) was used for the propagation and mating for all plasmids based on pGP704. E.
coli was cultured on Luria-Bertani (LB) medium (Difco) supplemented with ampicillin at
100 ug/ml. Isoleucine, leucine, and valine, when required for the construction of A.
pleuropneumoniae ile-., were supplemented at the same concentrations as that found in

chemically defined media for the growth of A. pleuropneumoniae (5).

Construction of codon usage table. In order to construct degenerate primers to amplify
the 3’ end of ile, a codon usage table was required. This was constructed using the
codon usage program of GCG (Genetics Computer Group, Madison, WI) with the
sequence inputs derived from A. pleuropneumoniae gene sequences deposited in
GenBank, prior to 1998. The selection criteria for inclusion of a sequence were: the

entire protein coding region must be present and must start with a methionine and the

121

protein must be deemed essential and not required solely for virulence. A total of 28
proteins were included in the codon usage profile. The program generated a table (Table

4-1) for each amino acid and the codon preference determined by percent representation.

Molecular manipulations. A description of the plasmids constructed for this study is
listed in Table 4-2. Genomic DNA from A. pleuropneumoniae was prepared according to
the lysis/proteinase K method of the Gene Fusion Manual (16). Plasmid DNA was
purified using Qiagen spin columns (Qiagen Inc., Valencia, Calif). DNA modifying
enzymes were obtained from Roche (Roche Molecular Biochemicals, Indianapolis, Ind.)
and used according to the manufacturer’s specifications. All PCR amplification was
performed with Pfi: polymerase (Stratagene, LaJolla, CA). All plasmids constructed
were sequenced using commercially available pUC18 primers or primers designed based
upon known gene sequence. Sequencing was performed using an ABIlOO Model 377
automated sequencer (Applied Biosystems, Foster City, Calif.)

Preparation of electrocompetent A. pleuropneumoniae serotype 1 and
electroporation of these cells with plasmid DNA prepared from either E. coli or A.
pleuropneumoniae serotype 1 was performed as previously described (2, 22). A.
pleuropneumoniae serotype 5 was made competent by the method of Ward et a1 (22) and
electroporated using the same electroporation conditions as for serotype 1, but using
plasmid prepared from E. coli. E. coli transformation was performed by the Hanahan

method (3).

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Construction of mutants by filter mating. Bacterial cultures were grown overnight in
BHIV broth, with antibiotics as required, at 35°C. All subsequent steps were performed
at room temperature. The total CFUs (colony forming units), as determined by ODszo,
was calculated. Both donor (E. coli) and recipient (A. pleuropneumoniae) cells, 100 pl or
less of each, were added to 5 ml of 10 mM MgSO.. to obtain set proportions of donor to
recipient. Cells were pelleted by centrifiigation and resuspended in 0.1 ml of 10 mM
MgSO4. The suspension was placed on a sterile filter that was placed on a prewarmed
BHIV plate and incubated for four to six hours at 35°C under 5% C02. Cells were
washed from the filter into sterile phosphate buffered saline (PBS) and centrifuged. The
pelleted cells were resuspended in 0.5 ml PBS and plated on BHIV plates containing 125
pg/ml of kanamycin. After incubation for 24-48 hours at 35°C under 5% C02, colonies
were selected from filter mating plates and screened by PCR and Southern blot for

transconjugants.

Southern blotting. Genomic DNA was digested to completion using various restriction
enzymes and separated on a 0.8% agarose gel. DNA fragments were transferred to a
Nytran (Schleicher & Schuell, Keene, NH.) membrane by the method of Southern (14).
The membrane was hybridized with a digoxigenin-labeled probe generated by PCR
amplification of the gene or gene fragment of interest (Roche PCR DIG synthesis Kit).
For high stringency hybridization, blots were incubated for 16 hours at 42°C with the
probe diluted in a hybridization buffer that contained 50% formamide, 5 X SSC (sodium
chloride, sodium citrate), and 2% blocking reagent (Roche). High stringency washes

were performed at 68°C with 0.1 X SSC, 0.1% SDS (sodium dodecyl sulfate).

126

Hybridizing bands were detected using alkaline-phosphatase tagged anti-digoxigenin and

the CDP“ Chemiluminescent substrate (Roche).

Cloning of the intact 1'1le and 0hr genes. The 0hr promoter and 291 nt of coding
region was encoded in the insert of piviK. The downstream sequence of 0hr was cloned
by using inverse polymerase chain reaction (iPCR) and is described in Chapter 3 of this
dissertation. The promoter and 243 nt of coding sequence of ile was encoded by piviI.
The remainder of ileH, that which was not contained in iviI, was cloned through the use
of degenerate primers based upon IleH sequence from other prokaryotes and the A.
pleuropneumoniae codon usage table. Primer MM219 (5’-CAGHGCRC
CRCCNCGWACTTGCATYGG-3’) was designed based upon a C-terminus region that
was highly conserved among various bacteria, including Haemophilus influenzae,
Pasteurella multacida, Pseudomonas aeruginasa, Escherichia coli, and Buchnera sp.
APS. Primer MM219, used in conjunction with primer MM197 (5’-
GGACGGTTAAGCTTGCTCAACCGG-3’), was based upon known A.
pleuropneumoniae iIvI sequence generated from the in vivo induced clone containing the
5’ end of 1'le (piviI). These primers were used in a PCR reaction to generate a 1.5 kb
fragment containing the entire ile gene, lacking the last approximately 30 nucleotides.
This fragment was cloned into pUC18 to form pIdeg.

To obtain the sequence of the C-terminus of Ile, degenerate primers based upon
IlvH N-terminus sequence were constructed using IlvH sequence from the same bacteria
as were used to design the MM219 ile primer. Primer MM324 (5’-GGGYTTRTGYA

AYTGYTTYTCRATYTGYTCGGG-3’) was used to PCR amplify the 3’ end of ile in

127

conjunction with a primer internal to ile MM29O (5’-CCCGGATCCGG
CTAATATTCCGA'I'TGTGG-3’). The resulting PCR fragment of approximately 800 bp
was cloned into pUC18 to generate pILVHdeg. This clone was then sequenced to

determine the sequence of the 3’ end of the ile gene.

Construction of ohr-kanamycin suicide vector. The construction of the ohr-
kanamycin suicide vector is outlined in Figure 4-1. Primers (MMISO: 5’-
GACAAGAA'I‘TCAACAAGGACAATA’I'I’ATG-3’ and MM151: 5’-CCTAAATCG
TCCCAGATCTGGTAGG-3’) were designed from known A. pleuropneumoniae 0hr
sequence in order to amplify the entire coding region. This fragment, which included the
ribosome binding site and the start codon, was cloned into pUC19 to generate pRS9l.
This plasmid was then digested with EcoRV to insert the kanamycin cassette from
pUC4K. The resulting plasmid was designated pRS93a, with the kanamycin gene in
opposite orientation to 0hr, or pRS93b, with the kanamycin gene in same orientation as
0hr. The ahr-kanamycin fragment from EcoRI and XbaI digested pRS93b was then
inserted into pGP704 that had been digested with EcoRI and XbaI to form pRS94b.
Plasmids pRS93a, pRS93b, and pRS94b were used during attempts to generate double
recombinants using electroporation. Plasmid pRS94b was used for matings.

Additional constructs of ahr-kanamycin were made by digesting pRS91 with AvaI
and Xbal to generate an 0hr clone (pRS91C) that lacked the ribosome binding site and
the start codon. This plasmid was then used to insert a blunt-ended kanamycin gene to

form pRS93KanF and pRS93KanR, based upon the kanamycin gene in the same

128

  

 

 

Sphl Xbol Baml-ll
EcoRl -' 1L San Pad
Dlgoot with BamHI
and Klonow flll-ln

Km'

 

AaflllBglll 4-

Figure 4-1. Construction of ahr-kanamycin suicide vector. The kanamycin resistance
gene was cloned into the middle of the 0hr gene. This was then inserted into the suicide
vector, pGP704.

orientation and opposite orientation to ohr, respectively. pRS93KanF was then digested

with EcoRI and XbaI to insert into pGP704 to form pRS94KanF.

Construction of ile-kanamycin suicide vector. The construction of a plasmid in
whichan internal coding region of 0.3 kb of ile had been replaced with the kanamycin
gene is detailed in Figure 4-2. This clone was constructed by PCR amplifying the 3’ end
of 2'le from A. pleuropneumoniae genomic DNA using primers MM290 and MM219.
This 0.7 kb fragment was inserted into SmaI digested pUC18 to form pile3’. Next the 5’

end of ile was PCR amplified using primers MM286 (5’-CGTTGGAGGATTGCATG

129

 

     
  

pllvl5’3’Kan

Dlgeot wlth Baml-ll 5500 bp

 
   
   
 

   

and "gate 5’ llvl
Sphl / EcoR ,
Sect
Hm 5- "V. Baml-ll Dlgofl wlth Baml-ll KP’"
Klenow flll-ln
ngato In Kan
cassette

pllvl5’3’ 3' llvl
4200 bp

 
 
   

Apt EcoRBacKpnl

Figure 4-2. Construction of pilv15’3’Kan. This construct contained the 5’ end and the 3’
end of ile and had the kanamycin resistance gene inserted in place of an internal 300 bp.

CAAAAACTTTCCG-3’) and MM288 (5’-GAGAGGATCCCTCCACCAATGTAT
AAAACCG -3’). This 0.7 kb fragment was then digested with BamHI and SphI and
inserted into BamHI and SphI digested pilv13’ to generate pilv15’3’. The kanamycin
cassette, from BamHI digested pUC4K was then cloned into the BamHI restriction site of

pilv15’3’ to form pilv15’3Kan.

130

EcoRl

Bar“
at!
Apr KanP
[$18!th
4300:»
Now
Hm Sphl Pat!

Figure 4-3. Plasmid map of pC18KnadV. This construct contained a promoter upstream
of the nad V gene that allowed for nad V expression independent of orientation within the
vector.
Construction of nadV-kanamycin cassette. From previous studies, it was shown that
the expression of the nad V gene was dependent upon the orientation of the gene in the
shuttle vector (5). This suggests that the expression of nad V was from a promoter within
the vector rather than its own promoter. The promoter from the kanamycin cassette from
the pUC4K vector was cloned in front of the nadV gene to allow for expression of the
gene independent of orientation (Figure 4-3).

The promoter region of the kanamycin gene was cloned by PCR amplification

using primers MM185 (5’—GTGAGCGGATAACAA'I"I“I'AAAACAGG-3’) and MM207

(5’-CCCGTTGAATATGGCCCATGGCACCCCTTG -3’). Primer MM185 was located

131

upstream of the nested restriction sites and primer MM207, which contained a NcoI
restriction site, was centered over the start codon of the open reading frame of this gene.
This PCR product was then digested with PstI and NcoI and inserted upstream of the
nadV gene. The nad V gene was prepared by PCR amplification with primers MM208
(5’-CGACCATGGATAACCTATTAAATTATAGTAGTCG-B’) and MM191 (5’-
GCGTATI‘AACTGCAGAT ATCATAGCGTAGTGCG-3’). MM208 was designed to
incorporate a NcoI site centered over the start codon and MM191 allowed retention of a
PstI site immediately downstream of the stop codon. The nad V PCR product was
digested with NcoI. A three way ligation consisting of the Kan promoter region, the
nad V gene, and PstI digested pUC18 vector allowed for preparation of the nadV gene
downstream of a constitutively expressed promoter. This plasmid was designated
pC18KnadV (Figure 4-3). The 1.6 kb insert from pC18KnadV was cloned into both
pGZRSlS and pGZRSl9 to form GZlSKnadV and pGZ19KnadV, respectively. To
provide a double selection cassette, the kanamycin cassette from pUC4K was cloned into
the pC18KnadV plasmid. The kanamycin resistance gene was isolated from BamHI
digested pUC4K and cloned into BamHl digested pC18KnadV to form pC18KanNad

(Figure 4-4).

132

BamHl
Psfl

  
     
       
   

Kmr

KanP

Ba Ncol

ECOR pC18KanNadV

5700 bp
NadV
Ap'

Hindll Sphl Psfl

Figure 4-4. Plasmid map of pC18KanNadV. This plasmid contained the double
selection cassette of a kanamycin resistance gene and the nad V gene allowing for NAD
independence. This 2.9 kb insert can be used as a selectable marker in the construction of
mutants.

Construction of ile-nadV-kanamycin suicide vector. The construction of this clone is
detailed in Figure 4-5. The pilv15’3’ construct was digested with BamHI and the single
stranded ends were filled in using Klenow polymerase to form blunt ends. The nadV-
KanR cassette was PCR amplified from pC18KanNad using the pUC18 forward and
reverse primers. The nadV-KanR cassette was ligated into the center of pilv15’3’,

replacing the 0.3 kb internal fragment of ile, to form pCl8ivianNad.

133

BamHl

     

pllv3’ 3' llvl

   

coRl Sad Kpnl BamHl

Na
Kan'
Baml-ll KanP Ncol

EcoRl

4 BamHl

Klenow fill-In
ngato KanNad PCR
product

 
   
 
 

pC18|vianNad
5’Ilvl 7100 DP

    

Digest with BamHI
and "gate 5’ Ilvl

  

NadV

  
 

Ap'

 
 

5’ llvl

pllvl5’3’ 3’ Ilv

4200 bp

 

Figure 4-5. Construction of pC18ivianNad. This vector was identical to pilv5’3’Kan,
but with a double selection (kanamycin resistance and NAD independence) used in place

of the selectable kanamycin cassette.

134

Results and Discussion

Attempts to construct an ohr- mutant. Attempts were made to construct a double
recombinant A. pleuropneumoniae ohr- mutant. These involved mating AP225 with
varied concentrations of the donor 817-1 (3. pir)/pRS94b. The mating procedure had
already been optimized using aopA (9). Fifteen attempts at mating were performed and a
total of 250 transconjugates were isolated on BHIV with 125 ug/ml kanamycin. 75% of
these were wild type and lacked the kanamycin gene, as determined by lack of a PCR
fragment when primers specific to the kanamycin gene were used, and had only one wild
type copy of the 0hr gene, as shown by a 500 bp product when 0hr specific primers were
used for PCR. The remainder of the transconjugates were single crossovers containing
two copies of 0hr, the kanamycin gene, and the pGP704 vector. These single crossovers
were confirmed by Southern analysis with a pGP704 specific probe. In addition, many of
these single crossovers contained intact pGP704 plasmid that could be isolated with a
genomic DNA isolation fi'om the transconjugate and transformed into fresh Sl7-1 (kpir)
to confer kanamycin resistance.

Since the attempts at mating were unsuccessful, methods of DNA uptake by
electroporation were tried. Plasmid (pRS93a, pRS93b, and pRS94b) was isolated from E.
coli and purified using Qiagen columns followed by phenol-chloroform cleanup and
ethanol precipitation. A total of 3 electroporations were performed and 80 colonies
resulted. Of these 80 colonies, 90% lacked the kanamycin gene, as determined by lack of
a PCR fragment when primers specific to the kanamycin gene were used, and had only

one wild type copy of the 0hr gene, as shown by a 500 bp product when 0hr specific

135

primers were used for PCR. Six colonies were single crossovers as determined by
Southern. No double recombinants were found by Southern analysis.

Since the initial 0hr constructs contained sequence upstream of the start codon, it
was thought that the generation of two functional copies, the wild type and the copy from
the suicide vector which contained a ribosome binding site and start codon, might be the
cause for failure to obtain double crossovers. Therefore, pRS91C and its derivatives
were constructed. These plasmids lacked the first 60 nucleotides, including the ribosome
binding site and start codon. Six matings with these constructs resulted in the generation
of 14 wild type colonies and 1 single crossover.

At the time these experiments were being performed, a functional assay for the
presence of Ohr was not yet in place. The subsequent development of the iron-xylenol
orange assay (15) and Chapter 3) might allow for evaluation of the single crossovers for
stability of the mutation and attenuation of the Ohr protein. While the disk inhibition
assay (Chapter 3) did not allow for differentiation between single crossovers and
wildtype cells, the xylenol orange assay might be more consistent and sensitive.

The lack of double crossovers may have resulted from either the essential nature
of Ohr or a conditional requirement for Ohr in the presence of oxygen radicals.
Pretreating all media by incubating the media with peroxidases might decrease the
oxidative stress and allow for propagation of stable double crossovers that were selected
against due to the presence of reactive oxygen species and the requirement for enzymes
capable of detoxifying oxygen radicals. Another recommendation is that all experiments
be conducted anaerobically to decrease the production of oxygen radicals, such that the

requirement for detoxifying enzymes are reduced. The final possibility for the lack of

136

double crossovers is that Ohr is an essential enzyme in A. pleuropneumoniae. If Ohr is
an essential enzyme, this will be unique among bacteria, since Ohr has been successfully
disrupted and mutated via homologous recombination in Xanthamanas campestris and
Bacillus subtilis (8, 11).

Since it was impossible to differentiate between the inability to construct a
knockout due to technical difficulties and that due to the essential nature of the gene, an
attempt to perfect the mating and electroporation protocols via construction of a knockout
of a different gene was performed. For this, an A. pleuropneumoniae ile- mutant was

attempted.

Attempts to construct an ile- mutant using a kanamycin cassette. Construction of an
iIvI- mutant was attempted, since it was believed that the conditions for mutant
construction, i.e. the requirement for supplementation of isoleucine, leucine, and valine,
were known. In addition, it was believed that the larger size and the ability to construct a
mutant that had 600-800 bp of sequence flanking the kanamycin cassette would facilitate
insertion into the chromosome. Thus, an 1'le cassette was prepared for double
recombination into the chromosome. This cassette contained iIvI sequence in which an
internal 300 bp fragment had been removed and the kanamycin resistance gene had been
inserted in its place. This construct was electroporated into the A. pleuropneumoniae
serotype 1 recipient strain. From four electroporations, 104 colonies resulted. Of these,
80 % were wild type and the remainder were single crossovers. Since double crossovers
were unable to be generated and all of the transformants were spontaneously kanamycin

resistant, the need for a better selectable marker became obvious. It was decided that a

137

nonantibiotic resistance marker might decrease the percentage of wild type colonies
resulting from matings or electroporations. Thus, a gene that would confer NAD

independence was a logical choice for replacement of the kanamycin resistance gene.

Construction of a nad V expression cassette. From previous studies , it was shown that
the Haemophilus ducreyi nad V gene apparently lacked a promoter that functioned in A.
pleuropneumoniae (5). Therefore, to construct a nad V expression cassette, a promoter
that functions in A. pleuropneumoniae was needed upstream to allow expression of the
nadV promoter independent of orientation within the vector.

The pUC4K vector contains a kanamycin expression cassette that has previously
been shown to be constitutively expressed in A. pleuropneumoniae independent of
orientation in the vector. The promoter for the kanamycin resistance gene when cloned
in front of the nadV gene, allowed for expression independent of orientation within the
vector. When either pGZl8KnadV or pGZl9KnadV were electroporated into A.
pleuropneumoniae serotype 1, both allowed for growth on BHI agar in the absence of
exogenous NAD. These results confirm that nad V was expressed from the kanamycin

promoter, which is functional in A. pleuropneumoniae.

Attempts to construct an ile- mutant using a kanamycin-nadV cassette. The
plasmid pC18ivianNad was purified from E. coli and used to electroporate competent
A. pleuropneumoniae serotype 1. Transformants were plated on BHI without NAD but

containing isoleucine, leucine, and valine (BHI+ILV), in addition to BHI+ILV with

kanamycin (100 ug/ml). No grth was seen on plates containing kanamycin,

138

suggesting too rigorous a selection process. After 48 hours, 52 transfonnants from
BHI+ILV were transferred to BHI+ILV containing kanamycin (100 ug/ ml). Over 96%
of the colonies that grew on BHI also grew on BHI with kanamycin.

Colonies were subcultured onto BHI plates lacking either NAD or ILV. Four
colonies were selected that failed to grow in the absence of exogenous ILV. Southern
analysis of these colonies showed that they were single crossovers. Neither double
crossovers nor wildtype were identified in this experiment. These results demonstrate
that the nad V gene can be expressed efficiently in a single copy in the bacterial
chromosome and that the NAD independence phenotype conferred by the presence of
nad V can be used successfirlly to select mutants in A. pleuropneumoniae.

This experiment did not yield any recombinants that were double crossovers.
There are several changes that can be made to improve the yield for double recombinants.
Additional attempts at repeating the electroporation might yield more transformants and
if a double crossover is a rare event, these additional recombinants might be required to
be screened prior to discovery of a double crossover. An additional attempt at isolating
plasmid DNA for electroporation that was digested such that the DNA contained only the
insert without the vector backbone, thereby preventing insertion of the plasmid into the
chromosome might yield double crossovers. Additionally, higher concentrations of
isoleucine, leucine, or valine, might be required for supplementation of the mutants.

Another tool that is required is a functional screen for lack of AHASIII in the A.
pleuropneumoniae ile- mutant. While assays exist for screening of AHAS isozymes in
E. coli (4), none have been developed yet for use in A. pleuropneumoniae. The

perfection of an AHASIII assay in A. pleuropneumoniae will permit determination of the

139

stability of the single crossovers generated. If stable single crossovers exist and inhibit
the production of Ilvl, then it is possible that these single crossovers can be used for

further studies.

The construction of double crossovers of in vivo induced genes has proven to be
an elusive goal. The inability to construct these double crossovers may be due to the
intractability of the system and A. pleuropneumoniae genetics, the essential nature of the
gene, or to conditions for mutant survivability that have not yet been determined. As
more mutants are constructed using the current system (1, 9) (personal communication, S.
Doree), the system will become more refined and easier to generate mutants.
Additionally, as knowledge is gained about the function of these in vivo induced genes in

A. pleuropneumoniae, the conditions for mutant construction will become more obvious.

140

References

10.

ll.

Fuller, T. E., B. J. Thacker, C. O. Duran, and M. H. Mulks. 2000. A
genetically-defined riboflavin auxotroph of Actinobacillus pleuropneumoniae as a
live attenuated vaccine. Vaccine 18: 2867-2877.

Fuller, T. E., B. J. Thacker, and M. H. Mulks. 1996. A riboflavin auxotroph of
Actinobacillus pleuropneumoniae is attenuated in swine. Infect. Immun. 64:4659-
4664.

Hanahan, D. 1983. Studies on Transformation of Escherchia coli with plasmids.
J. Mol. Biol. 166:557.

Jackson, J. H. 1988. Rapid assay of acetolactate synthase in permeabilized
bacteria. Methods Enzym. 166:230-233.

Martin, P. R., R. J. Shea, and M. H. Mulks. 2001. Identification of a plasmid-
encoded gene from Haemophilus ducreyi that confers NAD independence. J.
Bacteriol. 183: 1168-1174.

Miller, R. A., and B. E. Britigan. 1997. Role of oxidants in microbial
pathophysiology. Clin. Micro. Rev. 10:1-18.

Miller, V. L., and J. J. Mekelanos. 1988. A novel suicide vector and its use in
construction of insertion mutants: osmoregulation of outer membrane proteins and

virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170:2575-
2583.

Mongkolsuk, S., W. Praituan, S. Loprasert, M. Fuangthong, and S.
Chamnongpol. 1998. Identification and Characterization of a New Organic
Hydroperoxide Resistence (0hr) Gene with a Novel Pattern of Oxidative Stress

Regulation from Xanthamanas campestris pv. phaseoli. J. Bacteriol. 180:2636-
2643.

Mulks, M. H., and J. M. Buysse. 1995. A targeted mutagenesis system for
Actinobacillus pleuropneumoniae. Gene 165:61-66.

Nicolet, J. 1992. Actinobacillus pleuropneumoniae, p. 401-408. In A. D Leman.
et al. (ed.), Diseases of Swine, 7th ed. Iowa State University Press, Ames, IA.

Ochsner, U. A., D. J. Hassett, and M. L. Vasil. 2001. Genetic and physiological

characterization of 0hr, encoding a protein involved in organic hydroperoxide
resistance in Pseudomonas aeruginasa. J. Bacteriol. 183:773-778.

141

12.

13.

14.

15.

16.

l7.

18.

19.

20.

21.

22.

Platko, J. V., D. A. Willins, and J. M. Calvo. 1990. The ileH operon of
Escherichia coli is positively regulated. J. Bacteriol. 172:4563-4570.

Ricca, E., D. Limauro, C. Lago, and M. De Felice. 1988. Enhanced
acetohydroxy acid synthase [II activity in an ilvH mutant of Escherichia coli K-
12. J. Bacteriol. 170:5197-5199.

Sambrook, J., E. F. Fritsch, and T. Maiatis. 1989. Molecular Cloning: A
Labratory Manual., 2 ed. Cold Spring Harbor Labratory, Cold Spring Harbor, NY.

Shea, R. J., and M. H. Mulks. 2002. 0hr, encoding an organic hydroperoxide
reductase, is an in viva-induced gene in Actinobacillus pleuropneumoniae. J.
Bacteriol. 70:794-802.

Silhavy, T. J. 1984. DNA extraction from bacterial cells, p. 137-139,
Experiments with Gene Fusions. Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY.

Squires, C. H., M. De Felice, J. Devereux, and J. M. Calvo. 1983. Molecular
structure of ileH and its evolutionary relationship to ilvG in Escherichia coli K-
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Squires, C. H., M. De Felice, S. R. Wessler, and J. M. Calvo. 1981. Physical
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Umbarger, H. E. 1996. Biosynthesis of branched-chain amino acids, p. 442-457.
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Vieiro, J., and J. Messing. 1982. The pUC plasmids, a Ml3amp7 derived system
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Wang, Q., and J. M. Calvo. 1993. Lrp, a global regulatory protein of
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Ward, C. K., M. L. Lawrence, H. P. Veit, and T. J. Inzana. 1998. Cloning and
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142

Chapter 5

Summary

143

Actinobacillus pleuropneumoniae is the causative agent of porcine
pleuropneumonia, an economically devastating disease that results in high morbidity and
mortality in naive herds. This bacterium was first identified in 1961 and the agent
classified as Haemaphilus pleuropneumoniae (5, 7). In the thirty-five years following the
discovery of this pathogenic bacteria, the study of H. pleuropneumoniae has led to its
reclassification as A. pleuropneumoniae and identification of multiple virulence factors
associated with disease (3). These factors include capsule, lipopolysaccharide, exotoxins,
iron acquisition proteins, and many metabolic enzymes (riboflavin biosynthesis, two
superoxide dismutases, urease genes, amino acid and purine biosynthesis genes).

However, a genetic system for this bacterium was lacking and a genome wide
surveillance of virulence factors was not able to be performed. It was only in 1992 that a
plasmid shuttle vector was constructed that facilitated horizontal transfer of DNA from
Escherichia coli to A. pleuropneumoniae (1, 8, 9). A protocol for construction of
genetically defined mutants of A. pleuropneumoniae was developed in 1995 (4). With
these tools, it was possible to develop an IVET (in viva expression technology) system
that would allow a genome wide study of virulence factors.

This IVET system required an attenuated mutant, an expression vector that
contained genes to complement this mutation and genes to evaluate the degree of
expression, and an animal infection model. The first experiment using this IVET system
occurred in 1996 and the system was perfected within two years (2). Over 20,000 clones
have been constructed and used to infect pigs. From these 20,000 clones, 42 unique ivi
clones have been identified and are presented in Table 1. This dissertation focused on 25

of these clones that were discussed in Chapter 2. The 25 clones contain in vivo induced

144

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genes identified can be classified into four categories: known virulence genes and
regulators of these genes, previously identified genes that have not been previously
identified as virulence factors, genes encoding highly conserved hypothetical proteins
that are found in the genomes of many pathogens, and previously unidentified genes.
Within this group of 25, known virulence factors such as SodC, TehB, FtsY Tex, and
VapBC have been identified. These proteins help A. pleuropneumoniae escape oxidative
stress (SodC), export toxic molecules and proteins (TehB and FtsY), and regulate
virulence genes (TexA and VapBC).

Previously identified metabolic genes such as folD, ile, 0hr, ampD, eonI, can,
and selA have taken on a role as virulence factors that are required for growth and
pathogenesis during infection. The identification of these genes as in vivo induced genes
have allowed knowledge about the host response to infection by A. pleuropneumoniae.
The increased expression of £le folD, and can suggest that nutrient limitation occurs
during infection and requires a subsequent increase in enzyme biosynthesis to supply
necessary amino acids, nucleotide precursors, and enzymatic cofactors. The expression
of ampD, eonI, and selA suggests that infection of the porcine host requires modification
of proteins and cell walls in order to escape immune detection.

In addition to these ivi clones that contain proteins known to function as virulence
factors or metabolic survival proteins, several clones contain hypothetical ORFs or
proteins that have been previously identified, but not yet characterized as virulence
factors. The identification of 0hr as an in viva induced gene and as a prominent virulence
factor is a key contribution to the scientific literature (6). This gene has been shown to be

induced in response to organic peroxides alone and not other oxygen radicals. In

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addition, the gene is found only in three of the twelve serotypes, and it has been shown
that 0hr is regulated by a serotype specific regulator that is not found in A.
pleuropneumoniae serotype 5, which lacks 0hr. The identification of 0hr as an in vivo
induced gene suggests that in order to escape the destructive potential of the swine
immune cells, proteins such as Ohr and SodC are required to detoxify the oxygen
radicals. The rapid immune destruction of both bacteria and host tissue by the swine
immune system is a key component to porcine pleuropneumonia. In order to survive this
dangerous environment, A. pleuropneumoniae requires proteins that are unique and have
not been previously identified as virulence factors in many of the enteric pathogens. The
identification of in vivo induced genes in A. pleuropneumoniae will aid in the
understanding of other respiratory pathogens.

The generation and characterization of these clones has led to new discoveries
about A. pleuropneumoniae. It has also generated questions that were not able to be
asked previously. These questions include why certain genes (iviG and iviK/ohr) are
only found in A. pleuropneumoniae serotype 1 and not in A. pleuropneumoniae serotype
5, for which a whole genome sequence has recently become available. Is it possible that
one or more pathogenecity islands exist that contain these genes? If so, from whence did
these islands arise? Why are they only in certain serotypes and how does the presence of
these genes correlate with pathogenesis? Perhaps some of these questions can be
answered as more microbial genomes are sequenced and the sequencing of A.
pleuropneumoniae serotype 1 is completed.

Another set of questions centers around the regulation of these virulence genes.

Are sensors present that detect the host immune response and coordinately regulate

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groups of genes? What previously unknown regulons will be identified using this
system? Can these in vivo induced clones be clustered into regulons? If so, what
regulator is present? Perhaps these questions can be addressed through the development
and use of A. pleuropneumoniae genome chips and mimicking of conditions that occur
during infection, such as exposure to hydrogen peroxide or superoxides or amino acid
limitation.

The final set of questions that arise from these studies concern the significant
number of genes that encode proteins of unknown function. What are the functions of
these genes? Do they encode virulence factors that are specific to certain pathogens that
share unique niches? What is the best way to address the function and classification of
these genes? Will classifying by regulator or response to a subset of stressors help to
identify the protein’s role in virulence? If not, what is the best way to cohort these
unknown proteins? Will the characterization of these genes lead to new antibiotic targets

or insights into pathways common to multiple pathogens?

This dissertation was begun with the hope that by identifying and characterizing
virulence genes, the disease of porcine pleuropneumonia might be abated as new, more
effective vaccines were produced and new antibiotic targets were identified. Instead of
simplifying this disease, the identification of in vivo induced genes, by the use of IVET,
has instead presented more questions, which need to be addressed in order to develop
more effective vaccines and identify new antibiotic targets. It is the hope that by raising

these questions, the devastating disease of porcine pleuropneumonia might be further

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studied with the eventual goal of reducing the morbidity and mortality associated with

this disease.

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