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This is to certify that the
dissertation entitled
PROTEINS OF TYPE II SECRETION SYSTEM IN VIBRIO CHOLERAE:

INTERACTION OF EPSC PROTEIN WITH
THE EPSD OUTER MEMBRANE PROTEIN

presented by

Harry Sung Lee

has been accepted towards fulfillment
of the requirements for

Ph-D- degree in Microbiology

 

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Ma' professor

Date December 12 #2002

MS U i: an Afflrmatiw Action/Equal Opportunity Institution 0-12771

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Michigan State

University

 

 

 

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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 cJCIRC/DaIeDue.p65~p.15

 

PROTEINS OF TYPE II SECRETION SYSTEM IN VIBRIO CHOLERAE:
INTERACTION OF EPSC PROTEIN WITH
THE EPSD OUTER MEMBRANE PROTEIN

By

Harry Sung Lee

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

ABSTRACT

PROTEINS OF TYPE ll SECRETION SYSTEM IN VIBRIO CHOLERAE:
INTERACTION OF EPSC PROTEIN WITH
THE EPSD OUTER MEMBRANE PROTEIN

BY

Harry Sung Lee

Protein EpsD is the only protein of the type II secretion system located in
the outer membrane of Vibrio cholerae. Strong evidence from other bacterial
systems indicates that EpsD homologues constitute the pore through which
exoproteins are transferred from the periplasm into extracellular medium. All
other proteins essential for the secretion are located in the cytoplasmic
membrane. In this thesis, I present experimental results indicating the interaction
between EpsC protein and EpsD: (i) EpsD multimer is unstable in the absence of
EpsC; (ii) only coexpression of epsC and epsD in cis can complement the epsC
mutant to restore the secretion; (iii) in addition to epsCD genes, an open reading
frame homologous to Hsp15 of Escherichia coli is required for this

complementation.

Dedicated to my parents, Sungchoon Lee and Youngae Choi.

iii

ACKNOWLEGEMENT

I would like to express my sincere appreciation to my mentor, Dr. Michael
Bagdasarian for the opportunity to learn in his laboratory and excellent guidance
throughout the years. It has been the most wonderful learning experience in my
life. I also thank Dr. Mira Bagdasarian for the great help in many ways.

I thank my committee members, Drs. Robert Brubaker, Sheng Yang He,
Martha Mulks and Pat Oriel for their time, advice, and . discussion. Their
reactions were very encouraging and stimulating in every meeting.

It was a great pleasure getting to know all members of Korean Bioscience
Student Association at MSU. Although I have known them not so long, they have
been really great colleagues not only socially, but also scientifically. Best wishes
to all of the members.

I would like to dedicate special acknowledgment to my parents for their
lifelong support and continuous faith in me. | feel very blessed for having such
wonderful parents.

Many thanks go to my family, wife Miri Kim and two lovely daughters,
Sharon and Arin. Especially, without my wife's sacrifice, it would have been
impossible to finish the school successfully. I love you all with all my heart and

thanks again for waiting for so long with patience.

iv

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. vi
LIST OF FIGURES ............................................................................... vii
LIST OF ABBREVIATIONS .................................................................... viii
CHAPTER 1
GENERAL INTRODUCTION .................................................................... 1
Mechanisms of Protein Secretion in Gram-negative Bacteria ...................... 2
Mechanism of Type II Secretion in Vibrio cholerae and Its
Significance in Cholera Pathogenesis ................................................ 17
Components of Type II Secretion System .............................................. 21

Assembly and Polar Localization of Eps Complex in
V. cholerae cells 38

References ..................................................................................... 41

CHAPTER 2

COMPLEMENTATION OF THE epsC MUTANT INDICATES

INTERACTIONS BETWEEN EPSC AND EPSD ........................................ 71

Abstract ......................................................................................... 72
Introduction ..................................................................................... 73
Materials and Methods ...................................................................... 77
Results .............................................................................................. 82
Discussion ...................................................................................... 98
References ................................................................................... 100

CHAPTER 3

A NEW GENE REQUIRED FOR TYPE II SECRETION IN

VIBRIO CHOLERAE .......................................................................... 104

Abstract ....................................................................................... 105
Introduction ................................................................................... 106
Materials and Methods ..................................................................... 108
Results ......................................................................................... 111
Discussion ..................................................................................... 1 19
References .................................................................................... 122

CHAPTER 4

CONCLUSION ................................................................................... 124
References ................................................................................... 127

LIST OF TABLES

TABLE 1-1 Similarities between proteins essential for extracellular
protein secretion and subcellular location of each

gene product ........................................................................ 6
TABLE 2-1 Strains and plasmids used in this study ................................... 78
TABLE 2-2 Complementation of the Bah-2 epsD::Km mutant strain 9.........6
TABLE 3-1 Strains and plasmids used in this study ................................. 108

vi

LIST OF FIGURES
FIGURE 1-1 Assembly and secretion of cholera toxin shown with
a schematic hypothetical model of Eps type II secretion
multiprotein complex in V. cholerae ............................................... 39
FIGURE 2-1 Purified EpsC and detection of EpsC by anti-EpsC antibodies.....83
FIGURE 2-2 Stabilization of EpsD by EpsC in trans ..................................... 84

FIGURE 2-3 Stability of EpsD is affected by absence of epsC in the
cosmid (epsC-epsN) when expressed in E. coli ......................... 85

FIGURE 24 Effect of the epsC mutation on steady-state level of EpsD
protein in V. cholerae ............................................................ 87

FIGURE 2-5 Effect of various eps mutants on the stability of EpsD.................88

FIGURE 2-6 Complementation of epsC mutant by a plasmid expressing
epsCD .............................................................................. 90

FIGURE 2-7 V. cholerae epsC mutant cannot be complemented by the
clones of epsC and epsD supplied in trans .............................. 92

FIGURE 3-1 Upstream sequence of epsC, including an ORF with
amino acid sequence ......................................................... 112

FIGURE 3-2 Expression of epsC and epsD from pMMBBOO lacking the
ORF is much stronger than that of pMMB799 with the ORF113

FIGURE 3-3 A putative Hsp15 ORF is required in a plasmid expressing
epsCD to restore secretion of the epsC mutant ....................... 114

FIGURE 34 Expression levels of epsC in various plasmids 116

FIGURE 3-5 Expression of putative Hsp15 homologue ORF of V. cholerae
under the control of the bacteriophage T7 CD10 promoter ........... 118

vii

KEY TO ABBREVIATIONS

AAA .................................. ATPases associated with different cellular activities
ABC ........................................................................ ATP-Binding Cassette
ADP ............................................................................... Adenosine 5’-Diphosphate
ApR .......................................................................... Ampicillin Resistance
ATP .................................................................. Adenosine 5’-Triphosphate
ATPase ............................................................................. ATP Hydrolase
BFP ............................................................................ Bundle-forming Pili
bp ......................................................................................... base pair(s)
C-terminus ..................................................................... Carboxyl terminus
CmR ................................................................ Chloramphenicol Resistance
CT ..................................................................................... Cholera Toxin
EDTA ......................................................... Ethylenediaminetetraacetic Acid
EH EC ................................................... Enterohemorrhagic Escherichia coli

ELISA ................................................ Enzyme-Linked lmmunosorbent Assay
EM ........................................................................................... Electron Microscopy
EPEC ...................................................... Enteropathogenic Escherichia coli

eps ................................................................ extracellular protein secretion
ETEC ........................................................ Enterotoxigenic Escherichia coli

FRT ..................................................................... FLP Recognition Target

GEP ..................................................................... General Export Pathway
GFP .................................................................. Green Fluorescent Protein
GM1 .............................................................................. GM1 Ganglioside
GSP .................................................................. Genera Secretion Pathway
HAP ................................................................................... Hemagglutinin/Protease
HgR .................................................................... Hygromycin B Resistance
(His)6 ..................................................................................................... 6X Histidine
lgA ................................................................................ lmmunoglobulin A
IgG ............................................................................... lmmunoglobulin G
IM ............................................... Inner Membrane or Cytoplasmic Membrane
IPTG ................................................... lsopropyl-B-D-ThioGalactopyranoside
kb .......................................................................................... kilobase(s)

kDaR ...................................................................................... kilodalton(s)

KmR ......................................................................... Kanamycin resistance
LB ............................................................................ Luria-Bertani medium
LT .................................................... Escherichia coli Heat Labile Enterotoxin
MFP ................................................................... Membrane Fusion Protein
MTB ........................................................................ Main Terminal Branch

MW ................................................................................ Molecular Weight
N-terminus ......................................................................... Amino terminus
NTA ................................................................................ Nitriloacetic Acid
NTP .................................................................. Nucleoside-5’ Triphosphate
NTPase ............................................................................. NTP Hydrolase

viii

ODsso ................................................................... Optical Density at 650nm

ORF ......................................................................... Open Reading Frame
PAGE .................................................... Polyacrylamide Gel Electrophoresis
PAI ............................................................................ Pathogenicity Island
PBS .......................................... 10mM Phosphate Buffer oH7.4/136 mM NaCl
PBS-T ..................................................................... PBS + 0.5% Tween 20
PCR ................................................................ Polymerase Chain Reaction
PDZ ............................. Post-synaptic density protein, Disc large and Zo-1 proteins
me ........................................................................... Polymyxin B Sulfate
PT .................................................................................... Pertussis Toxin
SDS ...................................................................... Sodium Dodecyl Sulfate
PAGE ............................................................. Poly Acrylamide Gel Electrophoresis
SmR ..................................................................... Streptomycin Resistance
TCP ........................................................................ Toxin-coregulated Pili
TcR ......................................................................... Tetracyclin Resistance
Tfp ......................................................................................... Type IV Pili
TMD .................................................................... Trans-Membrane Domain
Tn5 ..................................................................................... Transposon 5
TPS ........................................................................ Two Partner Secretion
T‘I'SS .................................................................. Type III Secretion System
TZSS ................................................................... Type II Secretion System
TFSS .................................................................. Type IV secretion System
VPI ....................................................... Vibrio cholerae Pathogenicity Island
WT .......................................................................................... Wild Type

ix

CHAPTER 1

GENERAL INTRODUCTION

Mechanisms of Protein Secretion in Gram-negative Bacteria

Bacteria produce numerous kinds of proteins and some of them are
secreted to the extracellular environment. Oftentimes these secreted proteins
are critical for the virulence of bacterial pathogens or for survival of bacteria in
different environments (40, 56, 57, 64, 98, 105, 106, 114, 121, 132, 197, 275,
277, 286, 301, 302, 307, 343). In Gram—negative bacteria, these extracellular
proteins have to pass through the cytoplasmic and outer membranes (IM and
OM, respectively) and the periplasmic space between the membranes that has
been estimated to be 7-25nm wide (118). This distance is too large to allow the
direct interaction of most IM and OM proteins During evolution, several distinct
mechanisms have been developed to accommodate transfer processes of
structurally and functionally diverse extracellular proteins (101, 142, 182, 208,
276, 318, 334, 335). In the past two decades five main pathways of extracellular
protein secretion have been identified. The current knowledge of these secretion

mechanisms in Gram-negative bacteria is summarized below.

Type I Secretion Pathway

Type I secretion system or ABC exporter is composed of three protein
components, an ATP-binding cassette (ABC) protein, a membrane fusion protein
(MFP) that spans the periplasm, and a porin-like OM protein (11, 22, 35, 82, 97,
177, 290). The secretion is independent of the Sec machinery and the proteins
are transported across the two membranes in one step without generating any

periplasmic intermediate (178). This pathway is exemplified by the secretion of

a-hemolysin (HlyA, 110kDa) in Escherichia coli, and the complex consists of
HlyB (ATPase), HIyD (MFP) and OM protein TolC (145, 336).

The synthesis, activation and secretion of HIyA are determined by the
thCABD operon, which is transcribed from a promoter located upstream of thC
(145). TolC is not encoded in this gene cluster in E. coli, but several homologues
of this OM component, widespread in Gram-negative bacteria, are found in the
same gene cluster with two other components (1, 201, 336). HIyC, which is a
fatty acid acyltransferase and required for hemolytic activity, is not involved in the
secretion of HIyA. HlyB is inserted in the IM via hydrophobic a-helical
transmembrane domains (TMD) (337). HIyD is anchored in the IM via a single
TMD and possesses a large periplasmic domain within the carboxy-terminal 100
amino acids, which are highly conserved among MFPs. Thanabalu et al. (317)
have shown interactions between the components by cross-linking experiments
in the presence or absence of the substrate and protease accessibility
experiments indicated conformational changes in each of the three exporter
proteins HlyB, HIyD and TolC. HIyD and HlyB can be assembled into a stable IM
complex in the absence of HIyA and TolC. Engagement of translocating
substrate HIyA induces the IM complex to make a bridge, via a HIyD trimer, to
the OM exit pore TolC, allowing direct passage of substrates across two
membranes and the intervening periplasmic space through the ToIC channel
tunnel (11). This continuous conduit transiently connects the cell cytosol to the
external environment through completely proteinaceous machinery. After the

passage of hemolysin, the bridged components of the export channel revert to IM

and OM states (317). The crystal structure of TolC was determined to 2.1 A
resolution and the TolC homotrimer was seen as a tapered cylinder 140 A long,
extending from the extracellular side of the OM, through most of the periplasmic
space, and ending in close proximity to the periplasmic side of the IM (177, 180).
Recently, gating of the TolC tunnel has been shown to be achieved by an iris-like
alignment of helices at the periplasmic tunnel entrance (12). Whereas the IM
proteins HIyB and HIyD are specific components of the transport apparatus of a-
hemolysin, TolC is a multifunctional protein, which also appears to be involved in
the efflux of antibiotics, heavy metal ions, detergents, and solvent (242, 350).
Very interestingly, TolC is also responsible for secretion of E. coli heat-stable
enterotoxin l across the OM, which is translocated across the IM by the general
export pathway (GEP) consisting of Sec proteins (107, 346, 347).

Other proteins secreted by the type I mechanism include proteins such as
Bordetella pertussis adenylate cyclase, Pasteurella haemolytica leukotoxin,
Erwinia chrysanthemi metalloprotease, Pseudomonas fluorescens lipase and
proteases of Serratia marcescens and Pseudomonas aeruginosa (3, 11, 75, 88,
90, 117, 120, 242). Analysis of secretion via hybrid exporters in S. marcescens,
which possesses multiple ABC exporter systems for secretion of several
proteins, showed that specific interactions between the ABC protein and the MFP
are required for the formation of active exporters (3). The secreted proteins have
uncleavable C-terminal secretion signal specifically recognized by the ABC
protein and it is believed that the secondary structure of this region is more

important for function than primary sequence (22, 157, 179, 295). Fusions of this

signal to foreign proteins have been shown to promote secretion, although there
seemed to be limitations related to the size and structure of the passenger (27,

89,102,145,237)

Type II Secretion Pathway: Main Terminal Branch (MTB) of the General
Secretion Pathway (GSP)

Type II secretion pathway (TZSS) is encoded by at least 12 genes to secrete
different extracellular enzymes and toxins from a wide variety of Gram-negative
bacteria (Table 1-1, next page) (104, 252, 258, 271, 278, 279). Since the
sequence of pa! gene cluster, which is responsible for the secretion of
pullulanase from KIebsie/Ia oxytoca, has been reported more than a decade ago,
sets of homologous genes were subsequently discovered in several species of
Gram-negative bacteria (78, 279). Some of well-studied TZSSs are from K.
oxytoca: pul (76, 78), Pseudomonas species: xcp (4, 18, 72-74, 103, 104, 182,
192, 321), Erwinia species: out (127, 265), Xanthomonas campestn's: xps (87,
152), Aeromonas species: exe (150, 161, 168) and Vibrio cholerae: eps (215,
280, 284). Functional TZSSs were also examined in enterohemorrhagic E. coli
(EHEC) 0157: etp (190, 289), enterotoxigenic E. coli (ETEC): gsp (147, 316),
Burkholden'a species: gsp (80, 99) and Legionella pneumophila: Isp (122, 203).
A cluster of genes homologous to the type II secretion genes seems to be
widespread among Gram-negative bacteria according to DNA sequence analysis
from microbial genome data (279). Most of these organisms are animal and

plant pathogens and the secretion is thought to play an important role in

 

 

 

 

 

 

 

 

 

 

 

 

 

BACTERIUM PROTEIN GENES

CT eps B C D E F G H J K L M

Lipase vcp D
Vibrio Protease
cholerae Chitlnase

Neuraminldase
Aeromonas Aerolysln exe B C D E F G H J K L M
hydrophila tap D
Klebsiella Pullulanase puI B C D E F G H J K L M O
oxytoca
Erwinia Cellulase out B C D E F G H J K L M O
chrysanthemi Pectinase
Erwinia Cellulase out B C D E F G H J K L M O
carotovora Pectinase

ExotoxlnA xcp P Q R S T U W X Y Z A
Pseudomonas Protease
aeruginosa Elastase

Lipase

Type IV PIII pi! Q B C A D
Xanthomonas Cellulase xps D E F G J K L O
campestris Pectinase
Aeromonas Acetyl exe C D E F G H J K L
salmoniclda transferase
Escherichia Chltlnase gsp C D E F G H J K L 0
call

C

SUBCELLULAR LOCATION OF IM * * * * * * * * *
GENE PRODUCTS OM * a I. .

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 1-1. Similarities between proteins essential for extracellular protein
secretion and subcellular locations of each gene product. Homologous genes
appear in the same column except for puIA of K. oxytoca, which is a structural
gene for pullulanase and is not homologous to epsA, exeA and gspA, which are
components of the GSP. Two different names, piID and xcpA, are used to
designate the same gene encoding a prepilin peptidase for the type IV pili (Tfp)
and the type II secretion, respectively, in P. aeruginosa. V. cholerae vch is
also called piID. Many more unlinked genes are required in addition to the genes
listed in the table for the Tfp biogenesis of P. aeruginosa. Abbreviations for
subcellular locations are cytoplasm (C), inner membrane (IM), and outer
membrane (OM). For orientation and spatial organization of each gene, refer to
the reference (279).

 

pathogenesis since mutants, which are unable to secrete proteins, showed
reduced pathogenicity or became totally avirulent (202, 240, 279). Although
nomenclature of genes encoding the T2885 are different depending on the
organism, individual designations for homologues carry the same letter code
except in Pseudomonas species (Table 1-1) (258, 279).

Unlike type I, type III, or type IV secretion pathways, which can transport
proteins directly from the cytoplasm to the extracellular environment in a single
step, complete secretion of type II substrates occurs in two distinct steps (260).
The first step involves Sec-dependent translocation or GEP and it has been
extensively studied in E. coli (68, 84, 93, 227, 252, 259). Six sec genes, secA,
secB, secD, secE, secF and secY, have been shown to be required for
processing of the signal peptide in precursor form of pullulanase and secretion in
pul system of Klebsiella reconstituted in E. coli K12 (259). Sec homologues are
expected to be present in all Gram-negative bacteria with GSP. Indeed, recently
completed V. cholerae genome sequence does show homologues of Sec
proteins (129). Therefore, substrate proteins of type II secretion pathway which
contain N-terminal signal peptides are likely to be processed by signal peptidase
when translocated across the IM via the Sec machinery (128, 248, 259).
Following the removal of their signal peptides and release into the periplasm,
they fold and assemble into translocation competent conformations to enter the
T288 and cross the OM (29, 33, 124, 128, 139, 255, 311, 329). When
substrates of GSP are introduced into an E. coli strain, whose gsp genes are

known not to be expressed under the normal laboratory growth conditions, or into

host organisms with mutation(s) in TZSS genes, they are found exclusively in the
periplasm (108, 109, 210). Therefore, the type II secretion pathway actually
refers to the second step or translocation of the periplasmic intermediates across
the OM and is also referred to MTB of the GSP (252).

Recently, a novel type of TZSS involving twin-arginine translocation (Tat)
pathway, rather than GEP, to translocate its substrates across the IM and to feed
substrates into the secreton has been described for P. aeruginosa (328). Tat
system operates in parallel to the Sec system to export periplasmic proteins and
has remarkable ability to transport folded enzymes (21). Thus, P. aeruginosa
xcp secreton recognizes elastase and exotoxin A, bearing Sec-dependent signal
peptide, and phospholipase C, bearing a twin-arginine signal peptide, in the
periplasm and release them across the OM via the same T283 (193, 211, 212,
216, 328). Although TZSS genes are well conserved among members of
proteobacteria, the transported proteins are diverse and most of them seem to be
lacking any similarity in amino acid sequences and structures (279).
Characterization of individual components and up to date information on TZSS
will be further described in detail using eps system of V. cholerae as an example

on page 21.

Type III Secretion Pathway

Type III secretion systems (TTSSs) are found in many plant and animal
pathogens and they play a central role in the pathogenicity (9, 37, 43, 50, 60,
113-115, 156, 194, 325). Genes encoding the 'I‘I'SS and genes encoding the

proteins released by the TTSS are generally located on virulence plasmids and
pathogenicity islands (PAls) (8, 59, 121, 136, 219, 344). More than 20 gene
products are required for successful secretion of unfolded or partially folded
proteins and at least nine are highly conserved between different bacterial
species and probably constitute the core components of the secretion apparatus
(126, 176, 198). Eight of these conserved proteins are homologous to
components of the flagellar basal body or IM transport channel, which secretes
structural components of the flagellum (2, 7, 20, 44, 125, 214, 247). However,
the sequence and host cell activities of the secreted proteins differ in the
individual pathogens (53, 115). Some of these proteins are effectors or virulence
factors that are delivered by extracellular bacteria into the cytosol of the target
cell while the others are translocators or translocons (discussed below),
structural components that help the effectors to cross the membrane of the
eukaryotic host cell (57, 315). The secretion is triggered by close physical
contact between the bacterium and the host cell which initiates injection of
effector molecules directly into the cytoplasm of eukaryotic host cells. Thus, it is
designated contact-dependent secretion pathway (79, 194). In addition to cell
contact, other factors such as environmental cues also play important roles in
regulation of complex TTSS (110, 172, 218, 225). The effectors secreted by
TTSS are very diverse and often resemble eukaryotic proteins, enabling them to
interfere with signaling pathways and suppress host defenses (58, 61, 301, 304).
Some of the effector proteins such as SopE from Salmonella are encoded by

bacteriophage (31, 330). Secretion of some, if not all, effector proteins require

binding of distinct cytosolic chaperones, which stabilize their substrates and
prevent premature interactions with other partners (20, 23, 158, 235, 236, 303,
340). Chaperones are also thought to present a secretion-competent
conformation of the effector to the TTSS and loss of chaperone reduces
secretion of their specific substrate proteins (20, 158, 303). One of a number of
effectors from Y. pseudotuberculosis, YopE, has been suggested to possess
several distinct domains required for secretion from the bacterial cell, release
from the bacterial cell surface into the culture supernatant, formation of a stable
complex with its chaperon, YerA, and traversing the eukaryotic membranes (288)

In the exemplary type III secretion of Yop proteins in Yersinia spp., the
secretion signal is thought to be located either in the N-terminal 15-20 amino acid
residues of Yops or in the mRNA rather than in the protein itself (6, 13, 14, 43,
52, 60, 198, 206, 207, 263, 288, 298). Supramolecular structures of TTSS in
animal pathogens Salmonella typhimurium, Shigella flexnen‘, Yersinia
enterocolitica and enteropathogenic E. coli (EPEC) have been shown to
resemble a needle. The complex is composed of a cylindrical base that
resembles flagellar basal bodies and anchors the whole structure to the IM and
OM, and a needle-like structure which extends from the bacterial cell surfaces
(28, 144, 173, 187, 188, 293, 314). One of the proteins comprising the needle
complex belongs to the family of secretins, a group of OM proteins involved in
the transport of various macromolecules and filamentous phages across the OM
(116, 187). YscC of Yersinia has been shown to form ring-shaped multimeric

structure In the OM as do the other secretins involved in Type II secretion, Type

10

IV pili (Tfp) biogenesis and filamentous phage extrusion (181). In EPEC, a
sheath-like structure, EspA filament, is linked to the needle and may allow
passage of effector proteins through the eukaryotic cell membrane, forming a
physical bridge between the bacteria and the host cell (175, 245, 293).

Extracellular surface appendages named Hrp pili have also been
observed in plant pathogenic bacteria such as Enlvinr'a amylovora, Pseudomonas
syn'ngae and Ralstonia solanacearum, and they are thought to penetrate the
plant cell wall (155, 176, 269, 326, 341). In P. syringae pv. tomato, hrpA gene,
which encodes the major subunit of the Hrp pilus, is required for type III secretion
in vitro and the pili were shown to serve as conduits to deliver proteins outside
the cell (162, 163).

In several bacteria, transmembrane protein complexes termed translocons
have been identified and they are predicted to be channels that are inserted into
the host cell membrane since some of them have been shown to form pores (37,
38, 315). Effector proteins presumably pass through the eukaryotic cell
membrane via such translocons. Therefore, bacteria defective in translocons are
not able to inject effector proteins into the host cell cytoplasm and they are just
secreted into the surrounding medium in vitro (37). Translocation via the type III
pathway is Sec-independent and transit of effectors from the cytoplasm of the
bacterium to that of the host cell is thought to occur in one step through the TTSS

apparatus and the translocon.

11

Type IV Secretion Pathway

Type IV macromolecular transfer systems share a common ancestry with
the conjugation machines of Gram-negative bacteria (36, 39, 46, 47, 64, 343).
Type IV secretion systems (TFSSs) are able to transport proteins or nucleic acids
and their substrates not only across the bacterial envelope, but also the plasma
membrane or vacuolar vesicles of host cells. The family includes the E. coli
conjugative pilus or transfer (Tra) systems of the conjugative plasmids pK101
(IncN), F (lncF) and RP4 (lncP); the Agrobacterium tumefaciens T-DNA transfer
system required for exporting T-DNA to susceptible plant cells; the B. pertussis
ptl system for secretion of pertussis toxin (PT); the cag system of Helicobacter
pylori and Rickettsia, Legionella (dot/icm) and Bruce/Ia (VirB) systems for
delivery of effector molecules to the eukaryotic cell cytoplasm or to the vacuolar
membrane (19, 30, 36,46, 64, 343).

TFSS of A. tumefaciens, which delivers not only T-DNA, but also effector
proteins to plant cells, has been studied in most detail and serves as a model
system. The genes of the 12 TFSS components from A. tumefaciens, virB1 to
virB11 and virD4, are encoded in two operons on the Ti (tumor-inducing) plasmid
and other type IV systems are composed of a complete or incomplete sets of
genes homologous to VirB and virD4 (46, 64). The corresponding genes are
often collinearly arranged in the respective operons. Genetic organization,
function and localization of each member in several TFSSs are well described in
several references (47, 63, 189). VirB proteins can be categorized into three

functional groups: (i) proteins localized exocellulary forming the T-pilus or other

12

adhesive structures (VirB1, VirB2 and VirB5); (ii) putative channel components
spanning the two membranes (VirB3, VirB6, VirB7, VirB8, VirB9 and VirB10); and
(iii) cytoplasmic ATPases, including VirB4, VirB11 and VirD4 (47, 63). All of
these proteins are probably assembled into a supramolecular structure and
recent studies of protein-protein interaction involving yeast two-hybrid assay,
peptide linkage mapping, protein fusions and detergent extraction will help to
define the structure along with studies of protein localization (70, 81, 183, 338).
VirB7, VirB8, VirB9, and VirBlO, which form a structural and functional core,
interact with each other and form a high molecular weight complex containing
VirB7-10 that may be extracted with a detergent (183). Major T-pilus component
VirB2 is associated with VirB5 and VirB7 (183). Several hypothetical models of
Agrobacterium TFSS assembly were derived from the above results (64, 70, 183,
338). Further identification of protein-protein interactions will bring a more
detailed structure of the type IV secretion machinery. The delivery routes and
cellular consequences of various known effector proteins from A. tumefaciens
and other Gram-negative bacteria have been reviewed recently (46, 47).

Despite the overall similarity in subunit composition between the T-DNA
transfer and ptI secretion systems of A. tumefaciens and B. pertussis,
respectively, the secretion route of PT from B. pertussis is thought to be quite
different from the A. tumefaciens system. Substrates of A. tumefaciens TFSS,
for example VirD2 and VirE2, lack signal sequences and it has been thought that
the virB system and other conjugal DNA transfer systems transport their

substrates across both bacterial membranes simultaneously in one step.

13

However, the secretion of PT from B. pertussis seems to be a two-step process
as in the general secretion pathway (36). Each of the individual subunits of PT
(S1-SS), which is a member of the AB5 toxin family, is synthesized with their own
signal sequence and they are likely to cross the inner membrane in a Sec-
dependent manner. Holotoxin assembly in the periplasm is essential for effective
secretion of PT across the outer membrane by the Ptl system (95, 306).
Interestingly, unlike those of other A85 toxins, five B subunits of PT are not
identical and PT B oligomer, which consists of one copy each of subunits 82, S3,
and S5 and two copies of subunit S4, cannot be secreted effectively in the
absence of S1, which corresponds to A component (95, 186). The PT, therefore,
is different from the B subunit pentamers of cholera toxin (CT) or the E. coli heat
labile enterotoxin (LT) , which can be secreted effectively by the type II secretion
in the absence of A subunit (141 ).

There are several similarities between 'I'I'SSs and TFSSs of various
pathogens: (i) TFSSs of some organisms are encoded in PAls as in TTSSs (45,
63, 135); (ii) both PAI-encoded secretion systems are contact-dependent and
directly deliver virulence factors or effectors to interfere with the functions of host
cells with the exception of the B. pertussis Ptl system, which exports PT to the
extracellular milieu (40); (iii) with the exception of the Ptl system, substrate
translocation by TFSS probably does not require periplasmic intermediates as in
TTSS (19). However, very recently Pantoja et al. (239)proposed a two-step
model for the Agrobacterium TFSS as in the Ptl system and it needs to be

clarified; (iv) surface appendages might be involved in translocating DNA and

14

effector proteins into eukaryotic host cells (16, 176). The Hrp pilus of TTSS in P.
syringae can function as a conduit for protein delivery (162, 163). The TFSS-
associated T-pilus has also been observed in A. tumefaciens and it has been
hypothesized to serve as a channel for T-DNA transfer, but this has never been
shown directly (111, 176, 189); (v) substrate-specific Chaperones and coupling
proteins are necessary for presentation of substrates to the mating channel (46);
(vi) the TFSS secretes proteins which form a membrane channel or pore in the
host cell membrane to aid transfer of DNA or effectors to eukaryotic cytoplasm,
just like translocons of TTSS (37). Agrobacterium VirE2 forms a membrane
channel that transfers the T-strand through the plant plasma membrane (86).
VirE2 is one of the most abundant Vir proteins and it performs an unusually large

number of functions as described by Ward and Zambryski (339).

Type V Secretion Pathway: Autotransporters

Descriptive designation for the type V secretion pathway is the
autotransporter secretion system and it has been described as type IV in some
past literatures, but recently the nomenclature for classification of secretion
systems has been re-established by a group of researchers (133). As the name
implies, the proteins secreted by this pathway mediate their own translocation
across the OM and no other element is necessary for it (130, 132, 134). The
best known member of this family is IgA1 proteases of Neisseria gonorrhoeae
and the number of proteins that are secreted by an analogous mechanism is

rapidly increasing (174). These proteins possess unusually long N-tenninal

15

signal sequence and are thought to traverse the IM through Sec machinery just
like the proteins secreted by the type II pathway. However, they differ in how
they pass through the OM. In autotransporters, N-terminal signal sequence is
followed by a passenger domain corresponding to the mature secreted protein,
which is translocated to the cell surface through an OM pore formed by a C-
terrninal B-domain (174, 294, 327). After exiting the pore, the IgA1 proteases are
cleaved by the catalytic activity of their serine protease active sites and release
itself from the B-barrel structure (174). Therefore, it is assumed that all the
requirements for secretion across the OM are contained within a single molecule
and the secretion is probably energy-independent. Apparently, it is the simplest
secretion mechanism for that reason and can be simply described as an all-in-
one system.

In addition to the autoproteolytic processing of passenger domains for
release into the external milieu, there can be two other outcomes depending on
the autotransporter protein protruding through the OM. First, the protein may
remain intact as a large polyprotein with a membrane-bound C-terminal domain
and an N-terminal domain extending into the external milieu (62, 300, 322).
Alternatively, the protein may be cleaved by an OM protease and the passenger
domain may remain in contact with the surface via a non-covalent interaction with
the B—domain (134, 305). Generally, B-domains of autotransporter proteins are
highly conserved, consistent with their highly conserved function, although the
passenger domains are widely divergent. There are several different types of

autotransporter proteins secreted from diverse Gram-negative bacteria and they

16

can be grouped together according to functions of their passenger domains.
Many of them are suspected to be involved in pathogenesis, but more studies
have to be carried to determine their exact roles in virulence (132).

Recently, a new pathway called two-partner secretion (TPS) pathway has
been described (160). As the name implies, TPS pathway involves a single
accessory protein specifically devoted to the secretion of the exoprotein across
the OM. The proteins being secreted, named TpsA family, are synthesized as
precursors with an N-terminal cleavable signal peptide and has N-proximal
module called the secretion domain, while the partner transporter proteins,
named TpsB family, are channel-fonning fi-barrel proteins (160). Two partners
are usually organized in an operon. The components of TPS are very similar to
classic autotransporters, but the passenger protein and the pore-forming ,6-
domain of the unlinked autotransporters are translated as two separate proteins
rather than a single protein with two separate domains. For that reason, the
term, “unlinked autotransporter’ has been proposed in place of TPS (130, 131,

159).

Mechanism of Type II Secretion in Vibrio cholerae
And Its Significance in Cholera Pathogenesis
Cholera is a life-threatening epidemic disease that occurs worldwide
(165). The main symptom of cholera is characterized by a severe watery
diarrhea caused by ingestion of contaminated food or water containing Vibrio

cholerae, which colonizes the small intestine after being ingested (96, 220). A

17

number of pathogenic factors produced by toxigenic V. cholerae are responsible
for cholera pathogenesis and some of them are known to be secreted out of the
cells. V. cholerae is a motile, Gram-negative curved rod-shaped bacterium
belonging to the family Vibrionaceae and they are often found in estuarine and
aquatic environments (266). V. cholerae genome contains two unique circular
chromosomes that have been sequenced (129, 323). Genome of CTX¢, 6.9-kb
single-stranded covalently closed DNA filamentous bacteriophage, harboring
ctxAB, which encodes cholera toxin (CT), is integrated into chromosome I or
larger chromosome by phage lysogenic conversion (129, 332, 333). CT is
responsible for profuse watery diarrhea and it contributes to the fecal-oral
transmission of V. cholerae (292, 330). The gene encoding a member of the
type IV pili (Tfp) family, toxin-coregulated pili (TCP), which are required for
intestinal colonization and also serve as receptors for CTX¢, is on V. cholerae
pathogenicity island (VPI) (166, 332). VPI is a genome of another filamentous
bacteriophage , VPICD and it is also on chromosome l (129, 167, 333). This is a
good example demonstrating a critical role of bacteriophages in the emergence
of pathogenic bacteria by horizontal gene transfer and also shows importance of
bacteriophage-bacteriophage interactions in the evolution of pathogenic bacteria
(31, 32, 221, 233, 330, 331). Along with two bacteriophage genomes encoding
very important virulence factors, a cluster of TZSS genes designated eps
(extracellular protein secretion), which is required for the secretion of CT and

CTXCD from V. cholerae, is also on the same chromosome (71, 129, 284, 333). In

18

addition to already mentioned genes, most genes known to be essential in
cholera pathogenicity are encode by chromosome I (129).

Vibrio cholerae mutant strain that did not secrete CT has been reported
more than 25 years ago (146), but actual genes that are responsible for the
secretion have been identified within last 10 years (224, 284, 285). In addition to
CT, which is a major virulence factor, TZSS of V. cholerae, identified by our
laboratory, is also required for secretion of several different enzymes such as
hemagglutinin/protease (HAP), endochitinase, lipase, and neuraminidase (55,
112, 234, 284, 285). Since they do not show any sequence similarity, it is still
unknown how these heterologous proteins are recognized by the same
machinery. Although the mode of action for CT is well studied, it is not well
known how other potential virulence factors play roles in cholera pathogenesis
(292).

CT and E. coli heat-labile enterotoxin (LT) share similar structural and
functional properties due to ~80% protein sequence identity (292, 299). They are
structurally AB5 toxins composed of a single catalytically active A subunit and a
pentamer of B subunits responsible for binding to receptors, and functionally
belong to ADP-ribosylating exotoxins (94, 186, 238, 299). Biogenesis of CT and
LT from V. cholerae is well characterized (138). The genes which encode the
toxin subunits are organized into polycistronic operons. As depicted in Fig. 1-1
on page 39, individual A and B subunits of CT are synthesized In precursor
polypeptides with N-terminal signal peptides in the cytoplasm before translocated

across the IM via the Sec system (199). The signal sequences are proteolytically

l9

removed by signal peptidase during the translocation of the polypeptides across
the IM to yield mature protein in the periplasm (138). The mature A subunit (27.2
kDa) and five identical B subunits (11.6 kDa each) fold and assemble into the
AB5 holotoxin complex before being recognized by Eps secreton or type II
secretion apparatus via specific recognition of 35, which carries the putative
secretion signal (139, 141, 243, 349). The B subunit pentamer can be secreted
in the absence of A subunit, but A subunit alone will remain in the periplasm in
the absence of B subunit (141). Therefore, A subunit is secreted only when it
forms a multimer with the B subunit and the signal for OM translocation is
probably contained in the tertiary conformation of the 85 subunit (140, 141).
However, the exact signal responsible for type II secretion is not known yet.
The A35 complex is thought to be targeted somehow to the putative type II
secretion pore, EpsD, in the OM to reach the extracellular environment. Other
Eps proteins are expected to interact or communicate with each other and with
EpsD to form an active secretion apparatus spanning the cell envelope or to
bring the substrate proteins to the gate. Conformational changes caused by
these interactions will probably regulate the secretion process. Since the
multimeric exoproteins such as CT, with molecular weight of 84,000, are very
large and the resident proteins of periplasm have to be distinguished from the
substrate of the T288, the pore is expected to be gated, rather than being open
at all times.

CT binds to the GM1 ganglioside on intestinal epithelial cells via B subunit

pentamer and the A subunit ADP-ribosylates the stimulatory G protein of the

20

adenlylate cyclase complex. The resulting elevation of cyclic AMP causes
excessive secretion of salts and water leading to severe diarrhea followed by

dehydration (1 65).

Components of Type II Secretion System

In V. cholerae, eps genes, C through N, which seem to be organized into
an operon and a separate prepillin peptidase gene designated vch/piID (gspO
homologue) are required for type II secretion (112, 215, 284). Two more linked
genes, epsA and epsB, which are separated from the epsC-N cluster, were
identified by sequence similarity to exeA and exeB, which have been shown to
play an important role in type II secretion of aerolysin in A. hydrophila (15, 161,
278). However, epsA and epsB genes do not seem to be essential for secretion
(M. Sandkvist, personal communication). Although TZSS is required for
translocation of proteins across the OM, most components of the apparatus are
IM-associated proteins (Table 1-1). The only OM protein component in V.
cholerae is EpsD. More than half of the T288 components (EpsD, EpsE, EpsF,
EpsG, EpsH, Epsl, Esz, EpsK and chD) are homologous to proteins required
for the biogenesis of type IV pili (Tfp) or fimbriae (69, 191, 284). The proteins
involved in Tfp assembly and TZSS also resemble proteins involved in
filamentous phage assembly and natural competence or uptake of DNA (85, 273,
274)

Our laboratory has been characterizing individual components involved in

type II secretion of V. cholerae and detecting interactions between its

21

components for several years. In this section, some of the well-characterized
T288 components will be discussed with the results of experiments done by us
with V. cholerae system and also other researchers’ reports using other Gram-
negative bacteria as their TZSS model organisms. In the following section,
assembly of the type II secretion apparatus will be discussed combining all the
available data regarding interactions among type II secretion components from
different organisms. Eps designation will be used where the information is
derived directly from the study of V. cholerae while other designations or just one
letter code representing homologues will be used if the information is from

studies of other organisms.

Eps D protein

EpsD is an integral OM protein and association of this protein with the OM
has been demonstrated by sucrose density gradient separation (77, 148, 153).
This is the largest protein with 674 amino acid residues and predicted subunit
molecular weight of ~ 73,000 Da among the Eps components (284). It is initially
produced with a cleavable signal sequence before being processed to become a
mature protein (77, 153). Protein D belongs to a family of OM proteins termed
secretins, all of which form large complexes resistant to dissociation in sodium
dodecyl sulfate (SDS) at 100° (25, 116). They are all involved in transport
processes such as, type II and Ill secretions (YscC from Y. pestis, 48% similarity
and 22% identity with OutD from E. carotovora), Tfp biogenesis (PiIQ from P.

aeruginosa, 26% identity with EpsD), filamentous phage extrusion (pr from

22

M13, 54% similarity and 32% identity with OutD from E. carotovora), and DNA
uptake (ComG1 from Bacillus subtilis, 61% similarity and 38% identity with OutD
from E. carotovora) (83, 116, 170, 181, 265, 272). One of the secretins, XpsD in
X. campestn's is the only known lipoprotein among D homologues in the T288
and at least part of it is exposed to the cell surface as suggested by results of
trypsin digestion (153). Interestingly, its homologues PuID and chQ in
Klebsiella and Erwinia, respectively, require a small peripherally associated OM
lipoprotein PulS and OutS, respectively, for insertion into OM. They also stabilize
protein D, although the majority of other type II systems, including that of V.
cholerae, seem to lack this protein (66, 76, 123, 296). Similarly, a lipoprotein in
Neisseria gonorrhoeae, PiIP, is essential for Tfp biogenesis and is required for
stability of PilQ homomultimer, which is a member of the secretin family (83).
Similar OM Iipoproteins are also required for the proper localization of secretins
in TTSS (67).

The C-terminal half, the 8 domain, is highly conserved among the
secretins and is thought to be embedded in the OM (119). However, the N-
terrninus, which is variable and conserved only among related secretion
pathways, is predicted to face the periplasm, where it might interact with other
components of the secretion apparatus and/or with the secretable proteins (116).
Although the 8 domain is important for multimer formation and/or stability, both
the N and 8 domains are required for full multimerization (119). Some secretins
were shown to form large, stable oligomers of ~12 subunits in the OM (42, 123,

170). PulD of K. oxytoca and chQ of P. aeruginosa for TZSS, YscC of Y.

23

enterocolitr'ca and InvG of S. typhimurium for TTSS, Pile of P. aeruginosa and
N. meningitidis for Tfp biogenesis, and pIV required for the assembly and export
of filamentous phage f1, have been shown to form ring-like structures by electron
microscopic (EM) analyses (24, 34, 51, 65, 181, 205, 228). The cavity in these
structures probably forms the channel that accommodates the transport of folded
proteins, phages or pili (25). The sizes of these ring-shaped structures vary,
ranging from 14 to 20 nm for outer diameters and 5 to 10 nm for inner diameters,
but they are large enough to accommodate their own exoproteins that will go
through the channels (25). Fusion of proteoliposomes containing the purified D
complex with a planar lipid bilayer results in appearance of small, voltage-
activated, ion-conducting channels (228). Thus the channel is expected to be
gated for opening and closing.

EpsD has been shown to be required for secretion of both extracellular
proteins and filamentous phage CTX<l> from V. cholerae (71). The genome of
CTX<i> lacks a homologue of gene IV encoding the OM pore or secretin in the
related filamentous phage of E. coli, f1. CTXCD uses the host secretin, EpsD to
exit from its host although other Eps components are not necessary for this
process. Thus, EpsD plays a role both in pathogenicity and in horizontal transfer
of a key virulence gene (71). Interestingly, certain strains of A. hydrophila have
two different protein D homologues, ExeD, which is involved in the T288, and
SspD, which is independent of Exe T2SS. SspD (S-protein secretion D) appears
to be specific for secretion of S-protein or two-dimensional paracrystalline layer

and does not appear to be involved in T288 (320). In contrast, P. aeruginosa

24

also has two homologues of protein D, chQ involved in ch T288, and thA,
which is not required for efficient secretion in the wild type, but is solely
responsible for the residual export in Axch strain (216). Unlike SspD from A.
hydrophila, no other function has been discovered for thA except for its

involvement in the type II secretion (216).

PIIDNch (0) Protein

0 protein was initially Identified as a prepilin leader peptidase involved in
Tfp biogenesis (209). In P. aeruginosa, the gene (pilD/xcpA) encoding the
peptidase is located adjacent to piIA, the pilin structural gene, along with other
genes required for pilin assembly (229). The substrate of the enzyme is PiIA,
which Is synthesized as a prepilin, containing leader sequence that is notably
shorter than the typical signal sequences found in many substrate proteins of
T288 (229). The same gene turned out to be also required for secretion of
extracellular proteins by the T288, which contains five consecutive prepilin-like
componets (GspG-K, discussed below) (17, 240, 270, 311). Biochemistry of
prepilin peptidase has been extensively studied by Lory’s group (232, 308, 309,
312, 313). This integral IM protein is a single bifunctional enzyme, which
catalyzes cleavage of a short basic leader sequence in both type IV prepilin and
prepilin-like components of T288 and N-methylates their exposed phenylalanine
at the amino termini to generate mature proteins (17, 26, 191, 230, 231, 257,
264, 312, 313). Comparison of cleavages by signal peptidase for Sec-dependent

signal peptide and by prepilin peptidase for type IV prepilin signal peptide shows

25

a few differences. Signal peptidase has its catalytic site in the periplasmic face
of the IM and cleave after the hydrophobic region while prepilin peptidase has its
catalytic site in the cytoplasmic face of the IM and cleave in front of the
hydrophobic region (101). Interestingly, a homologue of O, ComC, is also
present in DNA transformation system ComG of Gram-positive Bacillus subtilis
(5, 49, 226). Though the region containing leader sequences are conserved
among different organisms, the enzymatic activities seem to be quite specific to
its native substrates, with some exceptions. For example, neither chA/PiID
from P. aeruginosa nor ComC from B. subtilis is able to process prePulG of K.
oxytoca in vivo (257). Without the processing, PulG cannot be transformed to
mature protein, which is required for proper assembly into a subcellular structure
in the T288 (92). Thus, genes encoding prepilin peptidase for P. aeruginosa and
B. subtilis cannot correct secretion defect of an E. coli strain carrying all pul
genes except for pulO, while puIO gene complements this mutant in trans and its
product processes PulG protein (257). In contrast to these data, xcpA/pilD
mutant could be complemented by pulO (52% identity) and by xpsO (50%
identity) from X. campestn's, restoring the secretion of elastase in P. aeruginosa
(18, 154, 257). Furthermore, PuIO, chA and ComC can process type IV
prepilin, PiIE, from Neisseria gonorrhoeae, while EpsG and PulG from V.
cholerae and K. oxytoca, respectively, can be processed by PiID from N.
gonorrhoeae in E. coli (91, 92, 284). Further studies are required to determine
the efficiency of processing in heterologous combinations of enzyme and

substrate. Several bacteria with the T288 also have the Tfp and the prepilin

26

peptidase, encoded by a gene near other genes required for Tfp biogenesis, is
often shared between the two systems as in Pseudomonas species and A.
hydrophila (244). However, if the O homologue gene is essential solely for
TZSS, it is usually right next to the last gene of the T288 operon, next to M or N
gene as in K. oxytoca and Erwinia species, which seem to lack the Tfp (92, 204,
279). Lindeberg and Collmer suggested that outO of E. chrysanthemi is
transcribed independently from the transcription units consisting of outC through
outM (204).

In V. cholerae, there are two separate 0 homologues specific for Tfp
or/and T288. First, TcpJ is required to yield the mature, export-competent form
of the toxin-coregulated pilus (TCP), which is essential for colonization of the
intestine by V. cholerae, but it cannot process the prepilin-like components
involved in T288 (169, 284). That led to the discovery of another prepilin
peptidase chD/PIID that is absolutely necessary for both biogenesis of another
Tfp and T283 (112, 215). Processing of two prepilin-like components in TZSS,
EpsG and Epsl, by PIID has been demonstrated (215, 284).

DNA composition analysis of the secretion genes of V. cholerae suggests
that the eps genes are relatively old and that the vch gene has been acquired
recently by horizontal gene transfer. This suggests possibility of loss of epsO
gene, which was originally at the distal end of the eps cluster, due to the

acquisition of vch resulting in redundancy.

27

EpsG, EpsH, Epsl, Esz and EpsK Proteins (Pseudopilins)

As mentioned above, epsG-K encode products having hydrophobic N-
terminal a—helical regions that are highly homologous to leader sequences of the
type IV pilin subunit of Gram-negative bacteria (10, 17, 18, 26, 143, 204, 217,
231, 256, 342). Similar prepilin-like proteins are also found in the DNA uptake
system, ComG, of B. subtilis (217, 265). All these proteins are associated with
lM, with the extreme N-terminus in the cytoplasm and the 16—18 conserved
hydrophobic residues spanning the membrane (253, 264, 267, 310). However,
majority of these proteins are targeted to the periplasm and some might even
extend into the OM as shown with EpsG (148, 256, 264). In P. aeruginosa,
relative amounts of chT (G), chU (H), chV (I), and chW (J) in membrane
fractions have been estimated to be 16:1:1:4 (230, 264). Because of the
similarity in sequence with type IV pilin subunit, these prepilin-like components or
psuedopilins of T2SS have been thought to form a trans-periplasm pilus-like
structure or pseudopilus, resembling a Tfp, to facilitate extracellular secretion
(252). However, little data supported presence of this hypothetical structure.
EpsG and some homologues of pseudopilins have been shown to be associated
with both membranes by density gradient separation (18, 148, 230). Direct
evidence for relocalization or assembly of multiprotein complex from processed
pseudopilins has not been shown until PulG, which Is the most abundant
pseudopilin, has been demonstrated to assemble into pilus-like bundles by
immunogoId-labelling and EM when the 15 genes encoding the pullulanse

secreton of K. oxytoca were expressed on a high copy number plasmid in E. coli

28

(253, 261, 287). Most components of TZSS, including all components with
homologues in Tfp biogenesis, were necessary for PulG to form pili (287). G
protein is probably the major component of pseudopilus (287). High level
expression of the puIG gene interfered with pullulanse secretion, possibly due to
the unnatural stoichiometry of the secretion machinery components preventing
assembly of the proper secretion machinery (254). These results strongly
suggest that pseudopilus or individual pseudopilins interact with other secreton
components. Crosslinking analysis of P. aeruginosa revealed that major
component of pseudopilus, chT (G) forms dimers with chU (H), chV (l),
chW (J), and also with PiIA, major subunit of Tfp (213). The authors suggested
that these dimers could be the components of the secreton or intermediates
involved in assembly of the secreton. One unexpected result in this report is that
PilA is required for efficient secretion since pilA mutants, which are unable to
express this protein, could not secrete extracellular proteins as efficiently as the
wild type strain does. Similarly, a type IV fimbrial subunit gene, fimA of an
anaerobic sheep pathogen, Dichelobacter nodosus, has been shown to be
essential for pilus subunit production, protease secretion, and also natural
competence (171 ). This shows close relationships among similar macromolecule
transfer systems involving Tfp-like components and suggests possible roles of

the pseduodpilus in the type II secretion process.

29

EpsE Protein

EpsE is another component of TZSS with its homologues in various
macromolecular transport systems required for Tfp biogenesis (PilB from P.
aeruginosa, 44% identity), TFSS (VirBI1 from A. tumefaciens, 14% identity with
PuIE from K. oxytoca, 50% similarity and 24% identity with OutE from E.
carotovora ssp.), and DNA transport in conjugation (TrbB from RP4 plasmid) and
natural transformation (ComG1 from B. subtilis, 42% identity with PulE) (246,
249, 284). In addition, XpsE from X. campestn's shows significant overall
homology to the ABC exporter components, PrtD (47% similarity, 19% identity)
and HIyB (45% similarity, 19% identity) from E. chrysanthemi and E. coli,
respectively (87). These homologues are generally hydrophilic and lack obvious
transmembrane domains and N-terminal export signals (184, 249). These
proteins are characterized by presence of nucleotide-binding motifs called
Walker A and B boxes, and for that reason they are putative NTPases, probably
providing energy for the assembly of the secreton, gating of the OM channel, or
for the secretion process itself (249). In addition to Walker boxes, they also
possess two conserved regions designated as the Asp and His boxes (184, 246,
250, 268).

Although ATP binding and autophosphorylation or autokinase activity have
been shown for purified EpsE protein, actual ATPase activity has not been
reported for any T288 (250, 281). Very recently, Sandkvist’s lab was able to
demonstrate ATPase activity by purified EpsE proteins (M. Sandkvist, personal

communication). ATP hydrolysis by some homologues in other systems has also

30

been demonstrated. According to the phylogenetic analysis, TZSS E protein
homologues are most closely related to putative NTPase proteins of TFSS
although clear distinctions can be made between the two families, which are
generally called PulE-VirB11 family (246). Two purified proteins of TFSS in A.
tumefaciens, VirB4 and VirB11», have been reported to bind and hydrolyze ATP in
addition to autophosphorylation activity for VirB11 (48, 297). According to Planet
et al., VirB11 is evolutionarily closely related to EpsE homologues in TZSS, both
possessing all four conserved domains mentioned above (246). Another purified
protein, Ter, encoded from conjugative transfer region of a lncW plasmid could
hydrolyze ATP and intact Walker A box was essential for the function (268).
Walker A box has also been shown to be essential for the function of EpsE and
other TZSS homologues in secretion (250, 281 ). Furthermore, the mutated Ter
whose lysine residue in Walker A box has been replaced with glutamine showed
dominant negative effect when introduced into a donor strain containing the wild
type plasmid, decreasing conjugation frequency 1,000-fold (268). Similar
phenomena of transdominance have been observed for EpsE and PuIE (250,
281). These results indicate that those proteins with mutation in the Walker A
boxes compete with the wild type proteins to interact with either itself or other
components of secretion or conjugative machinery, suggesting that these
proteins function as multimers (184, 281). Furthermore, it was shown that N-
terminal region of E gene is required and is sufficient for transdominant effect
(250, 281). Sandkvist et al. (281) showed that the N-terminal half of EpsE

contains the domain that associates with EpsL. In the same line with that, they

31

also showed that the N-terminus of EpsE determines species specificity by
demonstrating effective complementation of epsE mutant by a chimeric protein
composed of the N-terminal half of EpsE and the C-terminal half of Eer from A.
hydrophila, but not by the whole Eer or a chimera with N-tenninal half of Eer.
Similar results have been shown for complementation of P. aeruginosa chR (E)
mutants by the gene fusion composed of two different domains from two
different chR's of P. aeruginosa and P. putida (74). These results suggested
that species specificity or the reason Eer cannot substitute for EpsE in V.
cholerae is most likely due to its inability to interact with EpsL (281). EpsE is
soluble and found in the cytoplasm when produced alone without other Eps
components in E. coli but a fraction of EpsE is membrane associated in the
presence of IM protein EpsL. Thus, EpsL is responsible for the association of
EpsE with the IM in the native WT V. cholerae strain (249, 250, 281). A
filamentous phage-encoded pl, which is not a part of the virus particle, might be
equivalent to fused E and L components because it contains an essential
nucleotide-binding motif in the cytoplasmic domain and it spans the IM. Upon
binding of packaging signal in phage DNA, cytoplasmic domain of pi causes a
conformational change in the periplasmic domains of pi and interacts with a
secretin homologue pr, which is not part of the virion, triggering pIV channel
opening to allow phage particle to assemble and exit (273). Requirement of ATP
hydrolysis for filamentous phage assembly has been demonstrated (100).
Several reports suggest that E protein might be a homodimer although purified

EpsE was shown to be a monomer (250, 280).

32

Interestingly, EM studies determined that above mentioned TI'WD and two
other related secretion NTPases (TrbB and HP0525) encoded by the conjugative
transfer region of the broad host range plasmid RP4 and by the cag PAI (TFSS)
of H. pylori, respectively, share similar hexameric ring structures (184). The
hexameric ring with diameter of 12 nm seemed to be composed from a trimer of
dimers and a central channel of about 3 nm in diameter was exhibited (184, 185).
The authors suggested that these hexameric ring-structured proteins might aid or
catalyze a repetitive step and successive rounds of NTP hydrolysis might support
translocation of cognate substrat(s) across the IM (184). Crystal structure of
binary complex of HP0525 bound to ADP suggested a model that this hexameric
ATPase functions as an IM pore, the closure and opening of which is regulated
by ATP binding and ADP release (348). However, utility of forming rings in the
mechanisms of these AAA (ATPases associated with different cellular activities)
ATPase is still poorly understood (241, 324).

Another indirect indication of EpsE’s function as an ATPase providing
energy comes from the study of its homologue PilT, from N. gonon'hoeae Tfp
system (324). PilT protein, which is dispensable for Tfp biosynthesis, but is
essential for both twitching motility and for the DNA uptake step of genetic
transformation, has been demonstrated to be required for pilus retraction (222).
Therefore, piIT mutations lead to defects in competence for natural
transformation and in twitching motility despite the expression of structurally and

morphologically normal Tfp (345). This observation supports a model in which

33

ATPase activity of PiIT is responsible for cell movement and macromolecular
transport (164).

Other Less Characterized Proteins (EpsC, EpsF, EpsL, EpsM, EpsN, and
EpsA-EpsB)

None of these proteins are well-characterized structurally or functionally,
but interactions between T288 components seem to involve some of these
proteins. According to the density gradient separation of V. cholerae membranes
performed by Hough, the majority of EpsC fractionated with IM proteins, but
significant amount of this protein also seemed to be associated with OM,
suggesting direct or indirect association of this protein with the OM component(s)
(148). Relatively little information is available about EpsC, which is the main
subject of this thesis, and more discussion about EpsC and its homologues will
be covered in the next chapter.

EpsF protein is another component of T288, which has similar
components in Tfp biogenesis (PiIC from P. aeruginosa, 29% identity) and other
related systems (284). Similarly to homology between XpsE and ABC exporter
components, it is interesting to see XpsF of X. campestris share a significant
degree of homology with PrtE (46% similarity, 18% identity) and HIyD (42%
similarity, 18% identity), MFP components of the type I secretion system from E.
chrysanthemi and E. coli, respectively (87). This protein is also similar to ComG2
of B. subtilis, which is involved in DNA uptake (87). F protein has large
hydrophobic domains and it may be a polytopic IM protein spanning the IM three

times, with a large N-terminal tail and a large central loop being exposed in the

34

cytoplasm (87, 249, 265). Indeed, OutF from E. carotovora has been shown to
have a small periplasmic loop and two larger cytoplasmic domains connected by
three transmembrane regions by OutF-BIaM topological analysis and OutF may
be involved in interactions on both sides of the IM (319). Thus, this might act as
a platform for the assembly of a putative pseudopilus or secreton (265). EpsF
with (His)3-tags has been purified from E. coli by Hough (148) and 0.5% SDS
was used to solubilize the EpsF protein in the insoluble fraction of the cell, while
it was not extractable in 1% Triton X-100 with or without 10mM EDTA .

EpsL homologues share a single hydrophobic region located in the C-
tenninal half of each protein. The distribution of positively charged residues in
the proximity of this hydrophobic region suggests that the larger N-terminal
domain is cytoplasmic and the rest of the polypeptide is periplasmic (149, 262,
265). Cell fractionation studies showed that these L homologue proteins are
mainly associated with IM (148, 262, 319). Contradictory to this prediction, while
most of EpsL was fractionated with the IM, some of them were also fractionated
with the OM fractions (148). EpsL forms dimers and stabilizes EpsE protein by
attracting it to the IM for interaction (281, 282).

EpsM homologues display a single hydrophobic segment, preceded by
two or more positively charge residues, which is located in the N-terminal regions
of these proteins. This region might function as a signal sequence or a
membrane anchor (262, 265). An interactive relationship between the L and the
M proteins were suggested by observation of mutual stabilization or proteolytic

degradation of one protein in the absence of the other protein (195, 223, 282).

35

Physical interaction between two proteins has been strengthened further by co-
immunoprecipitation experiments (251, 282). We also observed co-elution of
EpsL with (His)6-tagged EpsM from the metal affinity chromatography column
(unpublished results). Sandkvist et al. (283) demonstrated that the first half of
the C-terminal half of EpsL, which is predicted to be transmembrane sequence,
is likely to be involved in its complex formation with EpsM and suggested that
these two proteins probably interact through their transmembrane regions. EpsM
has also been shown to form dimers as EpsL does (282).

EpsN homologues are not found in every organism with T288 (see Table
1-1). These proteins contain an N-terminal hydrophobic region and are probably
anchored in the IM, while majority of the proteins are exposed to the periplasm
(196, 262, 264, 265). Significant studies about this protein have been carried out
only in X. campestris. The gene encoding the XpsN protein has been identified
by the location of the gene, which is right downstream of XpsM, and the protein
has been shown to have a molecular weight similar to other N proteins of the
T288 (196). In accordance with the topology prediction, major part of the XpsN
seemed to face the periplasm, and the possibility of interaction between XpsN
and OM protein XpsD has been suggested by Lee et al. (196). They showed co-
immunoprecipitation of XpsN with XpsD and also co-elution of XpsN with (His);-
tagged XpsD from the metal affinity chromatography column, suggesting XpsN
protein indeed forms a stable complex with XpsD protein. The authors also
found that C-terminal region of XpsD protein is involved in complex formation

with XpsN. In addition to the association with XpsD, IM protein XpsN seems to

36

be associated with XpsL and XpsM, and XpsL-XpsM complex formation is
enhanced and stabilized by XpsN (195). Inhibition of secretion was observed by
overproduction of wild type XpsL and XpsM together, but not separately, in the
wild type strain of X. campestris (195). These data suggest the presence of
trimeric complex composed of XpsL, XpsM and XpsN. Therefore, XpsN protein
is a candidate for a bridge component connecting the IM XpsL-XpsM complex of
the secreton to the OM exit pore and its role may be similar to TonB that serves
as the energy transducer for the uptake of iron-siderophore complexes and
vitamin B12 in E. coli as the authors suggested (195). TonB opens gated pores
for the inward translocation of ligands across the OM. Similarly to XpsN, TonB is
an IM protein with its N-terminus in the cytoplasm, signal sequence anchored in
the IM, and most of the protein extending into the periplasm. TonB is thought to
shuttle between the IM and the OM to contact both IM and OM components
triggering conformational changes (41, 137, 200). If it is true that N protein has a
similar role as TonB, it must be a very important essential component of T288,
but PulN of K. oxytoca is dispensable for secretion and organisms such as E.
chrysanthemi and P. aeruginosa do not even have N homologue (Table 1) (251 ).
Thus, it was suggested that C homologues, whose molecular size and
membrane topology are similar to those of the N protein, might play a similar role
(195). Interestingly, C homologue has not been identified in X. campestris and it
makes the hypothesis stronger.

Although some T2885 possess protein B, only V. cholerae and A.

hydrophila possess both A and B proteins among well-characterized organisms

37

(161). ExeA from A. hydrophila is a hydrophilic protein with a consensus ATP
binding site (161 ). ExeB bears sequence as well as topological similarity to TonB
mentioned earlier (151). ExeA and ExeB forms a stable complex and ExeAB
complex is required to assemble and/or stabilize the ExeD secretin in the CM
(15). In E. chrysanthemi, fractionation experiments indicated that OutB could be
associated with the OM through its C-terminal part and an interaction between
OutB and OutD has been suggested by several experiments (54). By analogy to
TonB, ExeAB may act to transduce metabolic energy to the opening of a
secretion pore in the OM (151). In contrast to ExeAB and OutB, PulB in K.
oxytoca is not required for secretion (251, 252). EpsA and EpsB of V. cholerae

have not been studied yet.

Assembly and Polar Localization of Eps Complex In V. cholerae cells

Since multiple proteins are required for T288, it is reasonable to anticipate
that these proteins would form a multimer complex acting as a secretion
apparatus. Since T288 is responsible for the transport of proteins from the
periplasm across the OM to the extracellular milieu, it has been thought that the
secretion machinery will span the periplasm, thus envisioning a channel-like
structure connecting the IM and the OM (Fig. 1-1). Researchers are now trying
to figure out interactions between proteins involved in the T288 to dissect the
structure of this yet hypothetical apparatus and quite a number of protein-protein

interactions have been detected.

38

However, the location of the secretion apparatus assembly in the cell has
never been questioned before. Recently, a very elegant study, demonstrating
the location of secretion apparatus on single poles of V. cholerae cells was
carried out by Scott et al. (291). This study employed real-time monitoring of
green fluorescent protein (GFP) fused to EpsM to locate the Eps complex at the
old pole after cell division. They also visualized secretion of the protease from
the site of the polar Eps apparatus, confirming the coincidence of the locations
for Eps complex assembly and the secretion. Interestingly, detection of GFP

reporter fused to the EpsL, which is known to interact with EpsM, required EpsM

 

 

 

 

 

 

    
 

 

Pro-B
PIO'A Cytoplasm
Sec
IIIIIIIIIII' IIIIIIiIIII
“It!!! Kurt!!! C M
holotox'n
gj‘gfi 133—9- Periplasm
“Assembly
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
O M
FIG. 1-1 Assembly and secretion of cholera toxin shown with a

schematic hypothetical model of Eps type II secretion multiprotein complex
In V. cholerae, suggesting protein-protein interactions between the components.

39

expression for polar localization, while EpsM could localize to the pole in the
absence of other Eps components in E. coli (291). These results suggest that
EpsM carries information for polar localization. However, they pose other
Interesting questions about the subcellular structure of bacterial cell. For
example, what directs EpsM protein to its polar location? Polar secretion would
be more efficient in delivering virulence factors such as CT and enzymes directly
to the specific target rather than wasting proteins and energy by secreting all
over the surface of the cells. TCP is known to be required for colonization and
CT expression, thus a possible scenario would be that V. cholerae cells first
attach to its target in the small intestine, then produce and secrete CT and other
virulence factors in a directed manner. Still there are many questions to be
answered to figure out how the cells deliver virulence factors to the specific target
and this finding will provide important insights for pathogenesis of V. cholerae for
future research. Localization of EpsC protein in the cell is also under
investigation by Sandkvist’s group and the result would provide more information

valuable for understanding the role of the EpsC in the Eps complex.

40

10.

11.

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70

CHAPTER 2

COMPLEMENTATION OF THE epsC MUTANT

INDICATES INTERACTIONS BETWEEN EPSC AND EPSD

71

Abstract

The human intestinal pathogen Vibrio cholerae, which is responsible for
the severe diarrheal disease cholera, secretes cholera toxin and a number of
degradative enzymes via type II or general secretion pathway, which is common
In Gram-negative bacteria. Secretion of these extracellular proteins plays an
important role in pathogenesis. The secretion process requires products from a
cluster of eps genes, C-N, in addition to prepilin peptidase, which is required for
the processing of some Eps proteins. Requirement of these Eps proteins for the
secretion has been proven by inability of several eps mutants to secrete and
restoration of secretion when complemented by copies of these genes in trans.
One of the mutants, with Tn5 insertion in epsC, could not be complemented by
copies of epsC in trans. Here we show that this mutant can be complemented
very efficiently by providing epsCD in cis, but not by epsC and epsD genes
provided in trans to each other. We also found that the amount of multimeric
EpsD proteins is severely reduced in the epsC mutant and the restoration of the
secretion through the complementation by epsCD is accompanied by
reappearance of EpsD multimers. Overexpression of epsD in the mutant can
also restore the EpsD multimers, but not the secretion. These results indicate
that EpsC is required for the stability of EpsD, not for the formation of multimeric
EpsD complex. The failure of complementation by epsC and epsD in trans
suggests that coexpression of these two genes is important for stoichiometry,

likely required for the assembly of the secretion apparatus.

72

Introduction

Vibrio cholerae secretes a number of proteins including cholera toxin (CT)
and degradative enzymes via the type II secretion system (T288) involving eps
products. Our group has shown the requirement of these genes for secretion by
demonstrating inability of secretion in V. cholerae strains mutated in epsC, epsD,
epsE, epsF, epsG, epsL and epsM (36). Secretion was restored when a clone
of the corresponding gene was provided in trans to each mutant with the
exception of the epsC mutant, which could not be complemented with a plasmid
containing epsC gene. However, when a cosmid containing all eps genes was
introduced to the epsC mutant, the secretion defect could be relieved.

EpsC is approximately 33.5 kDa protein with 305 amino acid residues
(36). According to the sequence analysis of EpsC and its homologues from other
species, N-terminal 29 residues are cytoplasmic, residues 30~50 are potentially
transmembrane domain, and the rest are probably in the periplasm. (4, 12, 14,
29, 38). These domains have been dissected into smaller domains in some
homologues of EpsC ' and significance of each segment has been studied
(discussed below). Cytoplasmic N-terminus of EpsC homologues seems to be
not essential for function and specificity as it can be deleted or exchanged
without a loss of function (4, 6, 29). The transmembrane domain is necessary for
function of the C proteins and inner membrane (lM)-anchoring has been
considered important to promote interactions with other secretion components (4,
14). For chP (C), an EpsC homologue in Pseudomonas aeruginosa, the

membrane emerging periplasmic region, 35 residues at the C-end of the

73

transmembrane domain, was not exchangeable with the corresponding domain
of OutC from Erwinia chrysanthemi and this region has been suggested to be
involved in the specificity of the chP (C) protein (14).

Analysis of all sequences from EpsC homologues identified presence of
PDZ domains (named after the three eukaryotic proteins, Post-synaptic density
protein, Disc large and Zo-1 proteins) in these proteins, except for P. aeruginosa
and P. alcaligenes , which contain coiled-coil domains in place of PDZ domains
for chP (C) (14, 25, 26). They are present in the C-terminal half region of the
periplasmic domain. PDZ domains were first discovered in eukaryotic proteins
and many proteins containing PDZ domains are associated with the cytoplasmic
membrane, in which the PDZ domains mediate the assembly of multiprotein
complexes that may initiate signal transduction (26, 28). Both PDZ domains and
coiled-coils are thought to be involved in protein-protein interactions and PDZ
domains within GspC proteins were suggested to be involved in recognition of
specific signals in the secreted proteins and/or interaction between the
components of secreton (28).

Topology studies of OutC, PulD, and chP (C) from Erwinia carotovora,
Klebsiella oxytoca, and P. aeruginosa, respectively, by fusion with topology
probes, bIaM and alkaline phosphatase (PhoA), revealed that C proteins are
really IM proteins with majority of the protein targeted to the periplasm, in
agreement with the predictions resulting from the sequence analysis (5, 12, 38).
When PuIC-PhoA fusion protein was subjected to isopycnic sucrose density

centrifugation of membrane vesicles, small amounts were also detected in the

74

OM fractions while majority was detected in the IM fractions (12). EpsC
associated with OM has also been observed when IM and OM proteins of V.
cholerae were separated by sucrose density gradient, raising the possibility of
interactions between EpsC and OM protein(s), possibly EpsD (17). Furthermore,
Indications of interactions between EpsC and OM protein EpsD have been
demonstrated by co-immunoprecipitation with or without crosslinking and also by
instability of EpsD protein when expressed alone in E. coli, in the absence of
EpsC (17). Cis expression of epsC and epsD in the same plasmid increased the
amount of EpsD detected dramatically when the whole cell samples of E. coli
expressing epsCD were subjected to SDS-PAGE and subsequently detected
with polyclonal anti-EpsD antibodies (17). A number of researchers studying
T288 with other Gram-negative bacteria also suggested possibility of the protein-
protein interaction between EpsC and EpsD homologues based on several
observations: (i) in P. aeruginosa, only xcpP (C) and xch (D) genes are
organized into a single operon being transcribed in the opposite direction
compared to the orientation of other Type II secretion genes. This raises the
possibility of coordinated action of the corresponding proteins (1, 4); (ii) chP
(C) was unstable in an xch (D) mutant (4); (iii) in E. chrysanthemi, OutC and
OutD have been proposed as gatekeepers of species-specific secretion (see
Discussion) (18).

In P. aeruginosa, decrease in the stability of chP (C) has been observed
in the absence of chY (L) or chZ (M) and this suggested interactions between

chP (C) and the already described and well-established chYZ (LM) complex

75

(14, 20). Similar results were also observed for the V. cholerae Eps system
homologues (35) (M. Sandkvist, personal communication). chZ (M)-dependent
stability of chY (L) has also been shown to be modulated by chP (C) and its N-
terminal transmembrane domain seemed to play an important role in the stability
of the chY-chZ complex (14, 32). These results suggest three partner
subcomplex composed of chP (C), chY (L), and chZ (M) along with the
results by Possot et al. (30), who demonstrated co-immunoprecipitation of PuIC
with PulE, PuIL, and PulM. Presence of another multimeric complex composed
of E, L, M and F proteins has also been suggested (31, 33). Thus, it is very likely
that EpsC homologues interact directly or indirectly with all these IM proteins to
form a multiprotein complex.

In this report, we characterize the epsC gene and its product. By
constructing a new cosmid harboring all eps genes and with the epsC gene
replaced by CmR cassette, we wanted to study the effect of the epsC mutation on
stability of other Eps proteins in E. coli in parallel with the study of the V. cholerae
chromosomal epsC mutant mentioned earlier. We also introduced several
different plasmids containing different lengths or combinations of eps genes to
the epsC mutant to determine the minimal requirement for complementation to
restore the secretion. The results indicate that EpsC is very important for stability
and/or function of EpsD and the requirement of epsCD in cis for
complementation of the epsC mutant suggest protein-protein interaction between

EpsC and EpsD.

76

Materials and Methods

Bacterial strains, plasmids and culture conditions. Bacterial strains
and plasmids used are listed in Table 2-1. E. coli strains were grown in Luria-
Bertani (LB) broth and V. cholerae strains were grown in LB, M9, or syncase
medium supplemented with 100 pg/ml thymine at 30°C, 37°C or 43°C. Antibiotics
were used at the following concentrations: ampicillin (Ap), 100 pg/ml; kanamycin
(Km), 100 pg/ml (50 pg/ml for V. cholerae KmR insertion mutants);
Chloramphenicol (Cm), 25 pg/ml; tetracycline (Tc), 10pg/ml (1ug/ml for V.
cholerae); polymyxin (Pm), 100pg/ml; streptomycin (Sm), 100pg/ml. Plasmids

were introduced to the cells by conjugation or electrotransformation.

Construction of epsC-6X histldine fusion. To construct a clone of epsC
fused to 6X histldine tags at the C-terminus, epsC gene was polymerase chain
reaction (PCR)—amplified with Pfu DNA polymerase (Stratagene, La Jolla, CA)
using primers EpsC1 (5’-ATGGAAT'I'TAA-ACAACTTCC-3’) and EpsCZ (5’-
CGCGGATCCAAATTGAATA- TATACATCAT-3’, BamHI site in bold) with
genomic DNA of V. cholerae TRH7000 as a template. The following touchdown
temperature cycle was used for amplification: 94°C, 2 min; 8 cycles of 94°C, 15
sec, 58°C (-1°C/cycle), 15 sec and 72°C, 45 sec; 17 cycles of 94°C, 15 sec, 50°C,
15 sec, and 72°C, 45 sec; 72°C, 7 min; and 4°C. The amplified DNA fragment
was digested with BamHI (New England Biolabs, Beverly, MA) and inserted
between the blunt-ended Sphl and the Bglll site of the vector plasmid pQE70

(Qiagen, Chatsworth, CA)).

77

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

 

 

Strain or plasmid Relevant characteristics Source or reference
Strains
V. cholerae
TRH7000 ElTor thy Hg' A(cbrA-cer) (16)
ve12 (PUG) TRH7000 epsC::KmR (24)
E. coll
MC1061 F' araD139 Nara-leu)7697 A(Iac)X74 rpsL (7)
hstZ mcrA mchi
TG1 supE thi-1 A(Iac-proAB) Mmch-hstMw Amersham
(r..-m.,-) [F’ traD36 proAB laci‘ZAMiSj
XL1-Blue MRF’ A(mcrA)1 83MmchB-hstMR-mrn1 73 Stratagene

endAi supE44 thi-1 recA1 gyrA96 relA1 lac
[F' proAB racr'znms rmo (Tc‘)

Plasmids

E26 Cosmid pLARF5::epsC to epsN (36)

pAR3 Arablnose-Induclble expression vector (27)

pKD3 Template plasmid for CmR (10)

pKD46 Red recombinase expression vector (10)

pMMBZO7I208 Broad-host-range cloning vectors; CmR (22)

pMMB§03 Broad-host-range cloning vector; SmR (21)

pMMBG11 epscmm In pQE70 This study

pMMB741 pMMBSO3zzepsC (17)

pMMB771 pMMBZO7zzepsD T his study

pMMB781 pMMBZOB::epsC This study

pMMB79O pAR3 omR replaced by Km" This study

pMMBT94 pMMB790::epsD This study

pMMB799 pMMBZO7zzepsCD with ~1.2 kb upstream This study
sequence

pMMBB31 526 epanCmR This study

pMMBB35 pMM8208zzepsCD’ This study

pQE70 Cloznlng vector Inserting C-termlnal 6X His, Qlagen
AP

pWD615 eth (E. coli heat-labile enterotoxin B (9)

subunit gene), TcR

78

Purification of EpsC protein and generation of antibodies. E. coli TG1
carrying plasmid pMMB611 [epsCmisp] was grown with shaking in 2 liters of LB
medium containing 100 ug/ml ampicillin at 37°C. At ODssonm of 0.3, isopropyl-B—
D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.05 mM
and growth was continued overnight. Cells were harvested by centrifugation,
suspended in Buffer I (50 mM NaPO. buffer pH 8.0, 300 mM NaCl), and lysed by
sonication in the presence of 1.0 mg/ml lysozyme. DNase I was added to a final
concentration of 10 U/ml in the presence of 10 mM MgCl2 and incubated at room
temperature for 10 min. Cell membranes were separated by centrifugation for 30
min at 70,000 x g and extracted with Buffer ll (50 mM NaPO4 buffer pH 8.0, 300
mM NaCl, 5% Glycerol, 0.5% Triton X-100, 20 mM imidazole). Insoluble material
was removed by centrifugation for 30 min at 70,000 x g and the supernatant was
applied to a 25.0-ml metal chelate column of POROS 20MC (PerSeptive
Biosystems, University Park, MA) charged with Ni2+ and equilibrated with Buffer
ll. After washing the column with Buffer ll until the ODmnm = 0.05, adsorbed
proteins were eluted with a linear gradient of 20 - 300 mM imidazole in Buffer l.
Protein EpsC eluted at approximately 130mM imidazole and was visualized by
separating the proteins on a sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) followed by Coomassie Blue staining. Using the
purified EpsCmisys protein, EpsC-specific antibodies were raised in rabbits

according to Harlow and Lane (15).

79

Quantitative determination of LT B-subunit pentamer secretion. The
gene eth, encoding the B subunit of LT, was introduced into V. cholerae on
plasmid pW0615 (8). LT B-subunit pentamers present in the growth medium
and sonicated cells were determined by GM1 enzyme-linked immunosorbent

assay (ELISA) (37) as described previously (23).

Construction of In-frame Insertion mutants of E26 cosmid. To
inactivate epsC gene in cosmid E26, containing epsC through epsN, procedures
described by Datsenko and Wanner were followed (10). PCR products were
generated by using a pair of primers, pKD3-C1 (GCGTAAAAAGTACAGAA-
AGGAATAACGTGGAAA‘I'I'ngtgtaggctggagctgcttc) and pKD3-CZ (TCGT'I'AC-
TCGCCTTAGCGTTAAAATTGAATATATACcatatgaatatcctccttag), which include
homology extension sequences in the epsC gene (capital letters) and 20-nt
priming sequences (small letters) for pKD3 as template. The linear PCR
products containing CmR gene with homology extension were gel-purified,
digested with Dpnl before electrotransforrnation into E. coli M01060 harboring

recombinase expression plasmid pKD46 and E26.

Extraction of proteins from V. cholerae and E. coli. E. coli or V.
cholerae cells grown in liquid medium were harvested by centrifuging in a
Beckman JA 20 rotor at 6,000 rpm for 10 min at 4°C. The cell pellet was
resuspended in B-PER bacterial protein extraction reagent (Pierce, Rockville, IL)

and then the suspension was centrifuged in a Beckman JA 20 rotor at 14,000

80

rpm for 15 min at 4°C to collect the soluble proteins from the supernatant. Both
IM and OM proteins were soluble in the B-PER bacterial protein extraction

reagent.

Gel electrophoresis and immunoblotting. B-PER extracts or whole
cells in sample buffer containing 2% sodium dodecyl sulfate (SDS) and 1% ,8-
mercaptoethanol were heated at 100°C for 5 min and proteins were separated by
10% or 12% SDS-PAGE and transferred onto BA-S 83 nitrocellulose (Schleicher
and Schuell, Keene, NH) by discontinuous semi-dry electroblotting using the
TransBlot SD apparatus (Bio-Rad, Hercules, CA). Gels were transferred for 1 hr
at ~2 mA/cmz. Membranes were blocked with blocking buffer, 5% skim milk in
1X PBS-T (PBS + 0.5% Tween 20) for 30 min at room temperature or overnight
at 4°C. Blots were incubated with rabbit-raised polyclonal primary antibodies
specific for the Eps protein at appropriate dilutions for 1 hr at room temperature.
PBS-washed blots were incubated with secondary antibodies, peroxidase-
labeled goat anti-rabbit immunoglobuoin G (lgG) (Pierce), at 120,000 dilution in
blocking buffer for 1 hr. After washing, peroxidase activity on the blot was
visualized with SuperSIngal WestPico chemiluminescent substrate (Pierce)

followed by exposure to Kodak Biomax MR film.

81

Results

Purification of histldine-tagged EpsC protein and detection of EpsC
by anti-EpsC antibodies. Upon induction with IPTG the cells of E. coli TG1
carrying plasmid pMMB611 produced a protein of the apparent molecular weight
(MW) of 33.5 kDa. When Triton X-100 extracts from the membranes of these
cells were chromatographed on the Ni2*-NTA Sepharose column, this protein
was eluted at approximately 130mM imidazole. The purified EpsCmisy, migrated
on the SDS-PAGE as a single, 33.5 kDa band that could be visualized with
Coomassie Blue staining (Fig. 2-1 A, lane 2). This MW agreed well with that
predicted for EpsC based on the nucleotide sequence of its gene (36). The
epsC(His)6 gene could not complement an epsC mutant of V. cholerae for the
secretion of toxin, similarly as the previously reported for the wild type epsC gene
(36). Interestingly, when epsC gene was overexpressed in E. coli, several
protein bands were detected by the immunoblot with anti-EpsC antibodies (Fig.
2-1 B, lane 3). This was in contrast to the results obtained in wild type V.
cholerae where only a single EpsC band was observed upon reaction with the
same antiserum (Fig. 2-1 B, lane 1). This indicates that in the absence of other

Eps proteins EpsC is unstable and presumably undergoes proteolytic cleavage.
EpsC stabilizes EpsD when expressed in trans In E. coli. In the

previous work (17), when epsD gene was expressed in E. coli under

bacteriophage T7 CD10 promoter control, the protein was hardly detectable by

82

A. Coomassie Blue Stain B. Western Blot

   

kDa
43.2 —
28.3 — _ Epsc
18.3 —
1 2 1 2 3
MW EpsC Genotype: WT epsC” epsC'

(Hls)6 _ (Him
Strain: V cholerae E. coll

FIG. 2-1 Purified EpsC and detection of EpsC by anti-EpsC antibodies.
(A) SDS-PAGE of purified EpsC(His)6 after Coomassie Blue staining. Lanes: 1,
MW=mo|ecu|ar weight standard: 2, purified EpsC(His)6. (B) Western blot analysis
of EpsC and EpsC(His)6 with anti-EpsC(His)6 antibodies. Lanes: 1, a single band
for EpsC in the whole cell sample of wild type V. cholerae; 2, absence of EpsC in
the V. cholerae epsC mutant; 3, multiple EpSC(His)6 bands, presumably degraded
products of EpsCmisx; from E. coli expressing epsC(H,s)6 from pMMBG11. (Lanes
in B, 1=VB1, 2=VB12, 3=CB2077)

Western blot with anti-EpsD antibodies. However, coexpression of epsCD in cis
from the same promoter increased the amount of EpsD protein considerably (17).
Hough (17) interpreted these results as protection of EpsD protein by EpsC from
proteolysis. However, it is also possible that epsD expression requires epsC
expression. To distinguish these possibilities, I placed epsD under the are
promoter (pMMB794) in the presence (pMMB741) or absence (pMMBSOB) of the
epsC, which was expressed from the tac promoter in E. coli. Whole cell samples
in the SDS sample buffer were subjected to separation by SDS-PAGE followed

by Western blot with anti-EpsD antibodies. As shown in lanes 1 and 2 of Fig.

2-2, the era promoter was very tightly regulated, and in the absence of

83

kDa

Stacking gel EpsD multimer
2°3_ EpsD dimer?
120—

EpsD monomer
52—

EpsC
34—

 

1_2 LA §_§ 7_8
epsC + - + - + - + - (IPTG so uM)
Arablnose (uM) - - 1o 10 20 20 40 4o (forepsD)

FIG. 2-2 Stabilization of EpsD by EpsC in trans. E. coli strains harboring
two plasmids, pMMB741 for epsC expression (lanes 1, 3, 5, and 7) or empty
vector pMMBSO3 (lanes 2, 4, 6, and 8), and pMMB794 for epsD expression.
EpsC was produced in a constant level by induction with 30pM IPTG, and EpsD
was produced by induction with increasing concentrations of arabinose. Pairwise
comparison of EpsD level with or without EpsC shows the effect of EpsC protein
on the stability of EpsD protein (compare lanes 3 with 4, 5 with 6, and 7 with 8).
As more arabinose was added to induce the epsD gene, more EpsD monomer,
probable dimer, and multimer forms are observed. (Lanes 1, 3, 5, and
7=CBZ413, lanes 2, 4, 6, and 8=CBZ450)

arabinose, EpsD was not produced. As the arabinose concentration increased
the amount of EpsD detected also increased (compare lanes 1, 3, 5 and 7, with
lanes 2, 4, 6 and 8, Fig. 2-2). Not only monomer, but also multimer forms of
EpsD were observed. These observations suggest that increased amount of
EpsD detected in the presence of EpsC is due to interaction at the protein level,

but not to the improved transcription of the epsD gene in the presence of the

epsC gene.

84

Blot 2

KDa
148 ——

—EpsD oligomer

98—
64—
50—

—-EpsD monomer

'—Epsc
36 -

 

+

1 2 3 4
Cosmid Genotype (eps) WT C' C' 0'
eps gene(s) on plasmid - - CD+ D+

0")“

FIG. 2-3 Stability of EpsD is affected by the epsC mutation in the
cosmid (epsC-epsM) when expressed in E. coli. Although expression of both
epsC and epsCD from pMMB781 and pMMB799, respectively, could raise the
EpsD protein amount, it could not be determined which plasmid was more
effective from this experiment since two different blots with the same samples
showed different results (lanes 3 and 5 for blots 1 and 2). (Lanes 1=CB2464,
2=CB2478, 3=CB2534, 4=CBZ533, 5:082532)

Effect of the epsC mutation in a cosmid harboring epsC-N on the
steady-state level of cosmid-expressed EpsD protein in E. coli. Since EpsD
has been shown to be unstable in the absence of EpsC supplied in trans, we
wanted to observe the effect of epsC mutation on EpsD in a cosmid system
containing all eps genes, C-N, to determine if the presence of other Eps proteins
make difference for EpsD in the absence of EpsC. Compared to the wild type
cosmid E26 (epsC-N) (lane 1), pMM8831 (E26 epsC::Cm) (lane 2) showed much
less EpsD and addition of the epsC-expressing plasmid pMMB781 to the mutant
(lane 5) increased the EpsD level without IPTG induction (Fig. 2-3). pMMB799,

a plasmid containing epsCD, was also introduced to the mutant to see if it would

make any difference compared to the introduction of epsC alone (lane 3). It also

85

increased the amount of detected EpsD compared to the EpsD level in the epsC
mutant (lane 3). However, it was hard to determine whether the plasmid
expressing the epsCD was more efficient than the plasmid expressing only epsC
because the results from two immunoblot experiments with the same samples
were different even though both plasmids were regulated without IPTG induction.
According to blot 1, epsCD was more effective than epsC alone to restore the
EpsD level, but result on the blot 2 was opposite (Fig. 2-3). This discrepancy
may be due to the inconsistent transfer of large multimeric proteins to the blot
since multimeric EpsD proteins could not be separated well by SDS-PAGE, but
stayed in the stacking gel, although the difference between the wild type and the
epsC mutant was always reproducible. Additional copies of epsD gene
expressed from pMMB771 in the epsC mutant cosmid-harboring E. coli did not

affect the amount of EpsD (lane 4, Fig. 2-3).

Effect of the epsC mutation on the steady-state level of EpsD protein
in V. cholerae. Since the stability of EpsD was affected by the epsC mutation of
E26 cosmid in E. coli (Figs. 2-2 and 2-3), we expected that the V. cholerae epsC
mutant (VB12) would also contain less EpsD compared to the wild type cells. To
verify this, proteins extracted from the previously constructed V. cholerae epsC
mutant and the wild type cells were subjected to Western blot analysis with anti-
EpsD antibodies (23, 36). In V. cholerae, EpsD protein was detected exclusively
as a multimeric form, which is probably closer to the native form of this protein

(Fig. 2-4). As expected, compared to the wild type (lane 1), the epsC mutant

86

(lane 2) contained an extremely low amount of EpsD (Fig. 2-4), often not
detectable when less amount of samples was loaded and/or with shorter film
exposure. Supply in trans of the epsC gene (pMMB781) under the control of the
tac promoter without IPTG induction to the mutant could increase the amount of
EpsD, but not to the wild type level (Fig. 2-4, lane 3). IPTG induction of the tee
promoter-controlled epsC gene did not further increase the level of EpsD,
although it produced more EpsC when analyzed by immunoblot with anti-EpsC
antibodies (data not shown). This is probably because the amount of EpsC
encoded by the chromosomal gene of the wild type V. cholerae and the EpsC
encoded by the plasmid without IPTG induction were similar (lanes 1 and 3 in the
panel for EpsC). These results confirmed that the lack of EpsC affected stability

of EpsD protein in V. cholerae.

EpsD multimer IFS q

EpsC

  

1 2 3
V. cholerae genotype (eps) WT C' 0'
eps gene on plasmid - - 0"

FIG 24 Effect of the epsC mutation on steady-state level of EpsD
protein in V. cholerae. B-PER extracts of V. cholerae strains were separated
by SDS-PAGE and subsequently immunoblotted with the anti-EpsD antibodies
after transfer to nitrocellulose membrane. The same blot was re-probed with
anti-EpsC antibodies to confirm the presence of EpsC. EpsD protein is almost
undetectable in the epsC mutant (lane 2), but it can be restored when
complemented with epsC gene (lane 3) although not quite up to the wild type
level (lane 1). (Lanes 1=VB1, 2=VB12, 3=VB$39)

87

Steady-state level of EpsD multimer is not affected by absence of
epsE, epsF, epsG, epsL, and epsM in V. cholerae genome. Since the epsC
mutant affected the formation of EpsD multimer and there was an indication of
interaction between EpsD and EpsL (17), the ability of multimeric EpsD formation
was examined in various available mutants to see whether absence of any other
Eps proteins can also affect the formation of EpsD multimer. All mutants tested,
except for two different epsC mutants, could form the EpsD multimer and no
difference was observed in levels of EpsD protein compared to the wild type
strain of V. cholerae (Fig. 2-5). These results do not exclude the possibility of
interactions between the tested Eps proteins and EpsD protein, but suggest the
absolute requirement of EpsC for the stability of EpsD, indicating a very close

relationship between the two proteins.

 

EpsD multimer

 

1 2 3 4 5 6 7 8 9 10
eps genotype M '1 M '2 E '1 G ' F '
WT 6’1 6'2 E '2 L '

FIG. 2-5 Effect of various eps mutants on the stability of EpsD. Two
different epsC mutants, generated by Tn5 insertion, are virtually missing EpsD
protein (lanes 3 and 5) while the wild type (lane 1) and all other mutants shown
seem to produce similar amounts of EpsD. (Lanes 1=VB1, 2=VBQ, 3=VB10,
4=VB11, 5=VB12, 6=VB35, 7=VB36, 8=VBB7, 9=VB106, 10=VB109)

88

Effective complementation of the epsC mutant by the epsCD
expressed In cis and restoration of EpsD multimer in and LT secretion from
V. cholerae cells. Previously, this epsC mutant created by insertion of Tn5, was
the only mutant that could not be complemented by its own gene while epsD,
epsE, epsF, epsG, epsL and epsM mutants were successfully complemented by
their own genes to restore the secretion of the plasmid-encoded E. coli LT B
subunit (2, 19, 36). The plasmid initially used for the complementation of the
epsC mutant possesses about 200 bp upstream and 700 bp downstream
sequences in addition to the epsC gene, thus including the sequence encoding
about one third of EpsD (36). The above results of EpsC stabilizing the EpsD
multimeric protein and the requirement of the outCD to restore secretion from the
outD mutant in E. chrysanthemi were quite encouraging to try complementing the
epsC mutant with epsCD (18). As shown in Fig. 2-6A (next page), while
pMMB781 expressing epsC alone could not restore the secretion from the epsC
mutant, pMMB799 expressing epsCD in cis could restore the secretion to the
wild type level (4th vs. 5th bars on the graph). However, supply of the plasmid
expressing the epsC only to the mutant consistently resulted in unignorable
increases of LT secretion, compared to the mutant itself in several trials (2"d vs.
4th bars in Fig. 2-6A, not all data shown). This probably correlates with results
demonstrating partial restoration of EpsD by copies of plasmid-encoded epsC in
both the chromosomal and the cosmid epsC mutants (Figs. 2-3 and 2-4). Since
the epsC mutant retained so little EpsD, a Western blot experiment with anti-

EpsD antibodies as probes was performed to see if the restoration of the

89

A. Restoration of secretion by epsCD

 

 

100

 

Secretlon('/o)
0388882388

 

 

 

WT epsC- epSC- epsC- epsC-
Genotype & (plasmid) Introduced

 

 

 

B. Restoration of EpsD by epsCD

kDa — EpsD multimer
43—
— EpsC
1 8-

 

Chromosomal epsC + - - - .
eps gene(s) on plasmid - - 0+ 0+ (30"

FIG. 2-6 Complementation of the epsC mutant by a plasmid expressing
epsCD. (A) Secretion level is restored to the wild type level only when epsCD
(pMMB799) is provided to the mutant. LT B subunit pentamers present in the
growth medium and sonicated cells were determined by the GM1 enzyme-linked
immunosorbent assay (ELISA). (B) Effective secretion is correlated with the
restoration of EpsD protein in the mutant when epsCD is provided. Western blot
was initially detected with anti-EpsD antibodies and then re-probed with anti-
EpsC antibodies. (Lanes 1=VB101, 2=VB345, 3=VB347, 4=VB348, 5=VB349)

90

secretion accompanies the restoration of EpsD multimer. As shown in Fig. 2-6B,
the amount of EpsD in the epsC mutant was restored to the level comparable to
the wild type level in the presence of the plasmid that could restore the secretion
(lane 5). Therefore, there is a correlation between the presences of EpsD
multimer and the ability of the cells to secrete. It has been shown that
overexpression of epsD gene can produce the multimeric form of EpsD as well
as the monomer in E. coli (data not shown). When pMMB771 expressing epsD
only was introduced into the epsC mutant, although the epsD gene was
expressed from the same vector plasmid used to express epsC and also epsCD
without IPTG induction, EpsD was not detected in the epsC mutant, thus failing
to restore the secretion (Fig. 2-6A and B, 3rd samples). That is not really
surprising considering that EpsD is unstable in the absence of EpsC. Therefore,
it can be concluded that the epsC mutant requires epsCD to restore the
formation of EpsD multimer and secretion, indicating that both EpsC and EpsD
contribute equally to the formation and/or the function of the secretion apparatus,

probably interacting each other.

Failure of the complementation for the epsC mutant by epsC and
epsD expressed in trans in V. cholerae. As mentioned earlier, the formation of
the multimeric EpsD is probably very important for assembly and/or integrity of
the secretion apparatus. It was found that pMMBB35 containing the epsC and
about a third of epsD in tandem could restore the EpsD multimer almost to the

wild type level in the epsC mutant, but this plasmid could not rescue the

91

EpsD Multimer

 

 

KDa
148—
98— EpsD Monomer
64—
Lane 1 2 3 4 5
EpsC
eps genome WT C ' C ' C ' C '
epsCD’ clone - - + - -
epsC clone - - - + +
epsD clone - - - +__+
(inducible by arabinose)
arabinose - - - - +
secretion +++ - +I++ + +
FIG. 2-7 V. cholerae epsC mutant cannot be complemented by the

clones of epsC and epsD supplied in trans. Supplying additional copies of
epsD in trans or even partial sequences of epsD at the 5’ region in cis, in addition
to the epsC gene, can help formation of EpsD multimers in the epsC mutant, but
they are not functional, thus failing to restore the secretion to the full extent
compared to the wild type and the epsC mutant supplied with epsCD in cis.
Secretion levels of these mutants, which can restore the EpsD almost to the wild
type level, are similar to the epsC mutant strain supplied with epsC alone (see
Fig. 2-6A). (Lanes 1=VB1, 2=VB12, 3=VB366, 4 and 5=VB367)

secretion defect of the mutant, although the strain secreted some amount of LT B
subunit similar to the level accomplished by complementation of the epsC mutant
by the epsC clone alone (Fig. 2-7, lane 3). This means in spite of the correlation
between the multimeric EpsD formation and restoration of the secretion,
presence of the multimeric EpsD does not always indicate the functionality of the
EpsD associated with other components of the T288 apparatus. According to
the results mentioned earlier, epsC alone could not complement the epsC mutant
completely, but could restore the secretion slightly. Also epsD alone could
restore the EpsD multimer if the expression was induced although it cannot
restore the secretion, indicating absolute requirement of epsC to complement the
epsC mutant. When epsCD was expressed from the same plasmid (pMMB799),
it could efficiently complement the epsC mutant. All these results lead to an
important question with a highly probable expectation. What if the epsC and the
epsD genes were expressed in trans from the separate plasmids in the epsC
mutant? Providing a functional clone of epsC and the epsD clone, which is able
to restore formation of EpsD, in combination seems very tempting for
complementation of the epsC mutant. So tac promoter-controlled epsC
(pMMB781) and araB promoter-regulated epsD (pMMB794) were provided
separately to the epsC mutant. The results were similar to that of the epsC
mutant, harboring the epsC clone (pMMB781) only, which could consistently
restore the secretion level slightly, about 10 to 20% more than the level of the
epsC mutant (4th sample, Fig. 2-6A). Induction of the epsD expression with

arabinose did not make any difference for secretion in the presence of epsC,

93

which was constitutively expressed from the tac promoter (lanes 4 and 5, the
panel for EpsC in Fig. 2-7) although it did increase the amount of EpsD multimer
to the level comparable to the wild type level (Fig. 2-7, lanes 4 and 5). These
results show that the addition of the epsD to the epsC mutant harboring a clone
of the epsC does not help functionally and the multimeric forms of EpsD detected
on the blot are not functionally viable forms. It would be interesting to investigate
the structure of nonfunctional EpsD multimers generated by these strains.
Alternatively, the EpsD might produce a native complex, but cannot be regulated
properly to open the channel due to the ineffective coordination of conformation
change caused directly or indirectly by the EpsC. The main reason for the failure
of complementation by in trans expression of epsC and epsD separately, while
the epsC mutant can be complemented effectively by coexpression of the epsCD
in cis, is probably due to the lack of the fine tuning between EpsC and EpsD that

can be achieved when coexpressed from the same promoter in cis.

Analysis of the epsD mutant and complementation of the epsD
mutant. Our efforts to construct an epsD mutant in El Tor strain of V. cholerae
TRH7000 were never successful, but there have been reports by other
researchers, involving the epsD mutants in other strains (2, 11). The reason for
the failure of the epsD mutant generation is probably due to impaired growth
caused by EpsD deficiency as indicated by other researchers (11). This
explanation is also supported by the nature of those epsD mutants acquired.

They are mutants generated by the KmR cassette insertion only 30 amino acids

94

away from the C-terminus of EpsD. Although these mutants are viable, they are
all severely impaired for the secretion as measured by CT secretion (2, 11). We
received one of the mutant strains, Bah—2 epsD:: Km along with the wild type
Bah-2 strain, from Sandkvist and used them to characterize the mutant (11).
First, we checked to see if they are still able to form the multimer in the absence
of the N-terminus. According to the Western blot analysis with anti-EpsD
antibodies, the bands appearing as multimeric form in the stacking gel were
apparently same for both the wild type and the epsD mutant (data not shown).
Although the actual EpsD protein cannot be examined thoroughly, it is very
probable that the EpsD multimer formed by the epsD mutant is defective in an
unknown manner since the secretion is known to be severely affected.
Alternatively, the non-encoded or KmR-inserted region might inhibit interaction of
EpsD with EpsC or other Eps proteins, preventing the opening of the channel.
Secondly, we checked to see if the mutation of the epsD affects other Eps
proteins. Again, Western blots with available anti-Eps protein antibodies were
performed to measure the levels of EpsC, EpsF, EpsG, EpsL, and EpsM proteins
in the mutant. No difference was detected between the wild type Bah-2 and the
Bah-2 epsD:: Km (data not shown). Thirdly, since Bah-2 strain is non-toxigenic,
the authors who used this strain could not measure the secretion in terms of
percentage for the toxin secreted into the medium, but could show reduced
amount of hemagglutin/protease secreted from the epsD mutant compared to the
wild type (11). Therefore, pWD615 encoding the LT B subunit was transformed

into Bah-2 strains to measure the amount of LT B pentamer secreted into the

95

 

 

Strain Genotype Plasmid IPTG Secretion (%)
VB372 WT - - 93%
V8373 epsD' - - 30%
VB375 epsD' epsD” - 30%
VB375 epsD ' epsD+ + 88%
VB376 epsD' epsC‘D+ - 54%

 

 

 

 

 

 

 

Table 2-2 Complementation of the Bah-2 epsD::Km mutant strain. The
epsD mutant’s secretion is restored when the supplied copies of epsD gene from
pMMB771 is induced with IPTG, but when it is not induced no change is
observed for the secretion compared to the mutant. Introduction of epsCD by
pMMB799 to the mutant can help restore the secretion to some extent, but not to
the wild type level. In separate experiments, it has been shown that the induction
of epsCD with IPTG can also complement this mutant as efficiently as the epsD
expressed by IPTG induction.

growth medium by GM1-ELISA and also introduced plasmids expressing
epsD (pMMB771) and epsCD (pMMB799) into the mutant strain to check
for complementation. The results are shown in Table 2-2. Compared to the wild
type (93%), the epsD mutant’s secretion was very much reduced (30%).
Introduction of the plasmid expressing epsD, the same plasmid used in the
complementation of the epsC mutant, into the epsD mutant did not restore the
secretion without IPTG induction, but IPTG induction of the same plasmid
led to the very effective complementation. These results indicate that the
amount of EpsD produced from the plasmid is probably not enough to
complement or even if the epsD is expressed, as shown before, it may be
unstable and disappears although there is a chromosomal copy of epsC

apparently producing the same amount of EpsC as the wild type. Unlike the

epsC mutant, the epsD mutant does not seem to require the coexpression of the

96

epsCD and the epsD alone is sufficient to complement the mutant as long as it
produces enough EpsD as also shown by other researchers (2, 11). Since the
uninduced epsD clone was not able to complement the epsD mutant, it was
interesting to see the results of complementation by a plasmid expressing
epsCD. It could complement the mutant, but not as effectively as induced epsD
alone (table 2-2). Two possible reasons for this phenomenon are: (i) expression
of epsCD is somewhat inhibitory to the epsD mutant because of additional copies
of epsC expressed, causing stoichiometry imbalance; or (ii) epsCD does not
produce enough EpsD necessary for the complementation. To verify this, epsCD
under the contol of tac promoter was induced with IPTG in the epsD mutant. The
data collected from several independent experiments suggest that induction of
the epsCD can complement the epsD mutant as effectively as the IPTG-induced
epsD under tac promoter (near 100% secretion). Therefore, in contrast to the
results of the epsC mutant complementation, the epsD mutant does not require
epsCD to restore the secretion and additional copies of epsC does not interfere

with the assembly of the secretion apparatus or with the secretion.

97

Discussion

Type II secretion system in V. cholerae requires a concerted action of at
least 12 genes. The products of each of these genes are located in the
cytoplasmic membrane except for the EpsD protein which is in the outer
membrane. It is reasonable to expect that at least some of the Eps proteins
interact with one another and probably form some kind of a secretion apparatus.
Indeed, the interactions of EpsE, EpsL and EpsM have been demonstrated in the
previous work (33-35). The question of how the proteins located in the
cytoplasmic membrane may cooperate with the protein EpsD, which is believed
to constitute the exit pore in the outer membrane, has been asked in this work. It
has been shown that EpsD is protected by the presence of EpsC both in E. coli,
in the absence of other Eps proteins and in V. cholerae. In the epsC mutant, the
concentration of EpsD protein is so low that it cannot be detected by immunoblot
anlaysis. However, in the presence of EpsC protein, a considerable increase of
EpsD protein is observed. The expression of epsC gene alone in the epsC
mutant cannot restore the secretion of toxin, but when both epsC and epsD
genes are expressed in cis, the secretion is restored. These experiments cannot
rule out the possibility that the epsC mutation tested is polar. However, no
evidence of polarity could be observed on the other eps genes located
downstream of epsD and these genes were not essential for the
complementation of the epsC mutation. We believe, thus, that the results
showing the protection of EpsD by EpsC and the results from complementation

of the epsC mutant suggest the interaction of the EpsC and EpsD proteins.

98

Previously, Lindeberg et al. (18) suggested possible interactions between
OutC and OutD, homologues of EpsC and EpsD, in Erwinia chrysanthemi based
on the indirect observation that the OutC homologues are among the least
similar of the Out proteins (50% amino acid identity) (14). These authors could
complement individual E. chrysanthemi out genes (homologues of the eps
genes) by homologous genes from Erwinia carotovora. However, the outC and
outD mutations could not be complemented by individual E. carotovora
homologues. In order to complement these mutations, appropriate wild type
outC and outD homologues had to be expressed together. Complementations
requiring expression of more than one genes in cis have also been reported for
the type IV secretion system of Agrobacterium tumefaciens (3, 13). These
results suggest that sometimes precise stoichiometry can be achieved only by
coexpressing genes involved in the assembly of multi-protein complexes such as
the type II and IV secretion systems. Therefore, organization of these genes as
an operon is thought to be efficient for fine tuning of the components involved in
the assembly of the secretion apparatus.

It is at present unclear why epsC and epsD genes have to be present in
cis configuration in order to complement the epsC mutant. It is possible that
these proteins start interaction during the translation of the epsD. Alternatively,
another gene may be present on the DNA fragment carrying epsC-epsD cluster
and separation of these two genes onto different vector plasmids disrupts this
gene. Further work on this problem is clearly required to decide between these

possibilities.

99

10.

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cholera toxin and the filamentous phage, CTXphi. Science 288:333—5.

d'Enfert, C., l. Reyss, C. Wandersman, and A. P. Pugsley. 1989.
Protein secretion by gram-negative bacteria. Characterization of two
membrane proteins required for pullulanase secretion by Escherichia coli
K-12. J Biol Chem 264:17462-8.

Fernandez, D., G. M. Spudlch, X. R. Zhou, and P. J. Christie. 1996.
The Agrobacterium tumefaciens VirB7 lipoprotein is required for
stabilization of VirB proteins during assembly of the T-complex transport
apparatus. J Bacteriol 178:3168-76.

Gerard-Vincent, M., V. Robert, G. Ball, S. Bleves, G. P. Michel, A.
Lazdunski, and A. Filloux. 2002. Identification of chP domains that
confer functionality and specificity to the Pseudomonas aeruginosa type II
secretion apparatus. Mol Microbiol 44:1651-65.

Harlow, E., and Lane, D. 1988. Antibodies, a laboratory manual. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Hirst, T. R., L. L. Randall, and S. J. Hardy. 1984. Cellular location of
heat-labile enterotoxin in Escherichia coli. J Bacteriol 157:637-42.

Hough, L. P. 1999. Ph.D. thesis, "Proteins of the general secretion
pathway in Vibrio cholerae: indications for a multiprotein complex".
Michigan State University, East Lansing, MI.

Lindeberg, M., G. P. Salmond, and A. Collmer. 1996. Complementation
of deletion mutations in a cloned functional cluster of Erwinia
chrysanthemi out genes with Erwinia carotovora out homologues reveals
OutC and OutD as candidate gatekeepers of species-specific secretion of
proteins via the type II pathway. Mol Microbiol 20:175-90.

Marsh, J. W., and R. K. Taylor. 1998. Identification of the Vibrio cholerae
type 4 prepilin peptidase required for cholera toxin secretion and pilus
formation. Mol Microbiol 29:1481-92.

Michel, G., S. Bleves, G. Ball, A. Lazdunski, and A. Filloux. 1998.
Mutual stabilization of the chZ and chY components of the secretory
apparatus in Pseudomonas aeruginosa. Microbiology 144:3379-86.

Michel, L. 0., M. Sandkvist, and M. Bagdasarian. 1995. Specificity of
the protein secretory apparatus: secretion of the heat- labile enterotoxin B
subunit pentamers by different species of gram- bacteria. Gene 152:41-5.

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22.

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Morales, V. M., A. Beckman, and M. Bagdasarian. 1991. A series of
wide-host-range low-copy-number vectors that allow direct screening for
recombinants. Gene 97:39-47.

Overbye, L. J., M. Sandkvist, and M. Bagdasarian. 1993. Genes
required for extracellular secretion of enterotoxin are clustered in Vibrio
cholerae. Gene 132:101-6.

Overbye, L. J., M. Sandkvist, and M. Bagdasarian. 1993. Genes
required for extracellular secretion of enterotoxin are clustered in Vibrio
cholerae. Gene 132:101-106.

Pallen, M. J., G. Dougan, and G. Frankel. 1997. Coiled-coil domains in
proteins secreted by type III secretion systems. Mol Microbiol 25:423-5.

Pallen, M. J., and C. P. Ponting. 1997. PDZ domains in bacterial
proteins. Mol Microbiol 26:411-3.

Perez-Perez, J., and J. Gutierrez. 1995. An arabinose-inducible
expression vector, pAR3, compatible with CoIE1- derived plasmids. Gene
158:141-2.

Ponting. C. P., C. Phillips, K. E. Davies, and D. J. Blake. 1997. PDZ
domains: targeting signalling molecules to sub-membranous sites.
Bioessays 19:469-79.

Possot, O. M., M. Gerard-Vincent, and A. P. Pugsley. 1999. Membrane
association and multimerization of secreton component pulC. J Bacteriol
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Possot, O. M., G. Vignon, N. Bomchil, F. Ebel, and A. P. Pugsley.
2000. Multiple interactions between pullulanase secreton components
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Bacteriol 182:2142-52.

Py, B., L. Loiseau, and F. Barras. 2001. An inner membrane platform in
the type II secretion machinery of Gram-negative bacteria. EMBO Rep
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Robert, V., F. Hayes, A. Lazdunski, and G. P. Michel. 2002.
Identification of ch2 domains required for assembly of the secreton of
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Sandkvist, M., M. Bagdasarian, and S. P. Howard. 2000.
Characterization of the multimeric Eps complex required for cholera toxin
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102

34.

35.

36.

37.

38.

Sandkvist, M., M. Bagdasarian, S. P. Howard, and V. J. DiRita. 1995.
Interaction between the autokinase EpsE and EpsL in the cytoplasmic
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J 14:1664-73.

Sandkvist, M., L. P. Hough, M. M. Bagdasarian, and M. Bagdasarian.
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Sandkvist, M., L. O. Mlchel, L. P. Hough, V. M. Morales, M.
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membrane topology of OutC and OutF. Microbiology 143:713-20.

103

CHAPTER 3

A NEW GENE REQUIRED FOR TYPE ll SECRETION

IN VIBRIO CHOLERAE

104

Abstract

Out and Pul type II secretion systems from Erwinia chrysanthemi and
Klebsiella oxytoca respectively, are the only systems that have been successfully
reconstituted in Escherichia coli, which does not secrete proteins normally.
Introduction of their corresponding substrate proteins make the E. coli strain with
reconstituted genes to secrete them to the extracellular milieu. No other type II
secretion system of many other Gram-negative bacteria has been reported to be
reconstituted in E. coli. Eps system of Vibrio cholerae could not enable E. coli to
secrete introduced proteins when reconstituted with prepilin peptidase gene,
vch. Therefore, depending on the organism, some extra genes may be
required for the efficient secretion in addition to the known type II secretion
genes. During the course of studying the complementation of the epsC mutant,
an unknown open reading frame (ORF) located upstream of the epsC gene was
found to be required, in addition to epsCD, in the supplied plasmid to
complement this mutant. The ORF is homologous to heat shock protein, hsp15
of E. coli according to the sequence and homologues are highly conserved in
prokaryotes, including other Gram-negative bacteria with the type II secretion
system. Recently found Hsp15 is a ribosome-associated heat shock protein with
a novel RNA-binding motif. It has been proposed that Hsp15 takes part in the
translational cycle although its function has not been studied in detail yet. Here,
we show that this Hsp15 homologue is necessary for the type II secretion
function of V. cholerae by employing various plasmids with insertion or deletion

mutations of this ORF in complementation experiments with the epsC mutant.

105

Introduction

With the increasing number of genome sequences available for many
bacteria, type II secretion genes have been found in many bacteria. Generally,
these genes are well conserved in sequence and organization, but there are
exceptions (18). For example, C and N homologues are missing in
Xanthomonas campestris and Erwinia chrysanthemi, respectively, and the gene
organizations or the orientations of some genes are different in Pseudomonas
aeruginosa and X. campestris compared to the type II secretion genes in other
organisms. Out and Pul systems of E. chrysanthemi and Klebsiella oxytoca,
respectively, are the only two type II secretion systems that have been
successfully reconstituted in E. coli to secrete their own proteins produced from
the supplied plasmids (6, 9). Therefore, in these organisms, the known type II
secretion genes are minimal requirement for secretion although auxiliary
association of other genes with the type II secretion genes cannot be ruled out
completely. However, the type II secretion system of Vibrio cholerae could not
enable E. coli to secrete introduced proteins when all eps genes and vch gene
were reconstituted (unpublished data). This raises a possibility that yet
unidentified gene might also be required for the type II secretion in V. cholerae. .

We showed efficient complementation of the epsC mutant by providing
epsCD in a plasmid (described in chapter 2). This plasmid, pMMB799, also
contains additional ~1.2 kb sequences upstream of epsC (Fig. 3-1). We
constructed a new plasmid to remove the upstream sequence and tested the

plasmid to complement the epsC mutant. Unexpectedly, this plasmid,

106

pMMB800, containing epsCD with only ~200 bp upstream sequence, but ~1 kb
sequence deleted, could not complement the epsC mutant. Analysis of the
upstream sequence revealed a full open reading frame (ORF) and another partial
open reading frame (ORF2), further upstream of epsC, which are transcribed in
opposite orientation to the eps transcription (Fig. 3-1).

Sequence analysis of this ORF identified a homologue of this protein in E.
coli. The homologue, recently found Hsp15, is very abundant heat shock protein
which binds nucleic acids, is ribosome-associated, and contains a novel RNA-
binding motif (11, 12, 20). Its synthesis is induced massively at the RNA level
upon temperature shift and its specific target for binding has been identified to be
free 508 ribosomal subunit only when it is not a part of the 708 ribosome. It has
been proposed that Hsp15 plays a role in recognition and repair of 508 particles,
dissociated from the ribosome by error, which is likely to be prevalent under heat
shock conditions (11). Thus, Hsp15 is functionally different from many other heat
shock proteins known to act as molecular chaperones on protein substrates (11,
12).

In this report, we show that putative Hsp15 ORF is required to
complement the Vibrio epsC mutant in addition to epsCD. Therefore, we suggest
that the gene encoding the putative Hsp15 is a new gene required for type II

secretion in V. cholerae.

107

 

Materials and methods
Bacterial strains and plasmids, and culture conditions. Bacterial

strains and plasmids used are listed in Table 3-1.

Table 3-1 Strains and plasmids used in this study.

 

 

Strain or plasmid Relevant characteristics Source or
reference
Strains
V. cholerae
TRH7000 ElTor thy Hg“ A(ctxA-cth) (10)
van (PUG) TRH7000 epsC::Krn“ (1 a)
E. coli
MC1061 F' araD139 Mara-lean”? Miac)x74 rpsL (2)
hst2 mcrA mchf
BL21(DE3) F‘ ompT 17 gene 1 under lacp control on A (21)
prophage
Plasmids
pCP20 FLP recombinase expression vector, Ap", (22mR (5)
Temperature sensitive
pKD4 Template plasmid for KmR (5)
pKD46 Red recombinase expression vector, ApR (5)
Temperature sensltlve
pMM8207I208 Broad-host-range cloning vector; Cm" (14)
pMM8781 pMM3208zzepsC Chapter 2
pMMB799 pMM8207::~1.2 kb upstream sequence and Chapter 2
epsCD
pMMBBOO pMM8207::~200 bp upstream sequence and This study
epsCD
pMMBB15 pT7-6::Hsp15 ORF In pMMB799 This study
pMMBB43 pMM8207::~400 bp upstream sequence and This study
epsCD
pMMBBM pMM8799 Hsp15 ORF::KmR This study
pMMBs45 pMMB799 AHsp15 ORF This study
pT7-6 T7 010 promoter, ApR (23)
pWD615 eth (E. coli heat-labile enterotoxin B subunlt (4)
gene), TcR

 

108

- . .\-.. .o..-.-...E..._.Iwml

 

Construction of ln-frame Insertion/deletion mutants of pMMBT99
plasmid. To insert a KmR cassette into and inactivate the ORF in plasmid
pMMB799, procedures by Datsenko and Wanner have been followed as
described in chapter 2 (5). KmR - containing PCR products were generated by
using a pair of primers, S-Prim1 (5’-CCAGCGCAGAAGAGGTGAGACTGGAT-
AAATGGCTGTGthgtaggctggagctgcttc—3’) and S-Prim2 (5’-TGAATTTGAGTAG-
ATCTCGGCGCTGTI'TCTTGTCatatgaatatcctccttag-3’), which include homology
extension sequences in the ORF (capital letters) and 20-nt priming sequences
(small letters) for pKD4 as template (Fig. 3-1). After getting transformants
containing a plasmid (pMMB844) whose putative Hsp15 ORF is replaced by KmR
cassette, they were transformed with pCP20, which acts on directly repeated
FRT (FLP recognition target) sites flanking the KmR gene, to eliminate the
resistance gene. Ampicillin-resistant transformants were selected at 30°C and
then colony-purified at 43°C to remove KmR gene and pCP20. The final
transformants with a new plasmid pMMBB45, which is derived from pMMB799 by
deletion of Hsp15 ORF, were checked for the loss of ampicillin and kanamycin

resistance.

Construction of pMMBB43. To construct a plasmid containing epsCD
with the C-terminal half of the Hsp15 ORF removed, corresponding region was
PCR amplified using primers EPSC upst-2 (5’-GCTCTAGAGCATGCTTG-
ACGCAATGTGA‘ITI'GGG-3’, Xbal site in bold) and EPSDZ (5’-CGCGGATCCT-

TGCTTGGGTTCCATCTG-3’, BamHI site in bold) with E26 cosmid DNA as

109

template (Fig. 3-1). Both pMMBZOB vector and the insert PCR product were

digested with BamHI and Xbal, and ligated with Iigase (lnvitrogen, Carlsbad, CA).

Expression of Hsp15 ORF under bacteriophage T7 $10 promoter
control. pMMB815, pT7-6 containing the Hsp15 ORF (Fig. 3-1), was introduced
into BL21(DE3) and the strain was grown in LB supplemented with ampicillin at
37°C. When the 00350 of the culture became ~0.5, 50pM IPTG was added to
induce the expression and the cells were harvested after 1.5 hours. The cells
resuspended in sample buffer, containing fi-mercaptoethanol, were subjected to
12% SDS-PAGE to separate proteins. The proteins on the gel were visualized

by Coomassie Blue staining.

Quantitative determination of LT B subunit pentamer secretion, extraction

of proteins, gel electrophoresis and immunoblotting were as described in chapter

2.

110

 

Results

Putative Hsp15 ORF (~380bp) located upstream of epsC gene, in
addition to the epsCD, is required for complementation of the V. cholerae
epsC mutant. In chapter 2, we showed effective complementation of the epsC
mutant by pMMB799 expressing epsCD. The plasmid also contains additional
~1.2 kb sequence, which is located upstream of the epsC gene (Fig. 3-1, next
page). When pMMB800, which contains only ~200 bp upstream sequence of the
epsC in addition to epsCD, was introduced into the epsC it could not restore the
secretion of LT. We found that the expression levels of both epsC and epsD
from pMMB800 is much higher than the expression levels from pMMB799
without IPTG induction (Fig. 3-2, page 113). There have been reports
demonstrating inhibition of secretion from the wild type strain by overexpressing
one of the components. For example, overproduction of PuIG blocks secretion
and drastically reduce the cellular levels of PuIC, PulE, PulL, and PuIM as well as
PulD in K. oxytoca (17). Therefore we suspected overproduction of EpsC and
EpsD might be the reason for the complementation failure. To verify our
hypothesis, pMMB799 and pMMB800 were transformed into the wild type V.
cholerae and the secretion of LT was measured. Neither of the plasmids
affected the secretion from the wild type V. cholerae (data not shown).
Therefore, it is not likely that overexpression of epsCD is responsible for the
failure of the complementation by pMMB800.

According to the analysis of the sequence upstream of epsC, there is a

~380 bp ORF that could encode a 128 amino acid residue protein with homology

111

 

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112

0"" ' —E sD multimer
KDa j. p

250— .
148— w
.9 i» .

98—

64 " -EpsD monomer

so — -
36 — ,

pMMB 799 800
ORF + -

FIG. 3-2 Expression of epsC and epsD from pMMBBOO lacking the ORF
is much stronger than that of pMMB799 with the ORF. B-PER bacterial
protein extraction reagent-extracted samples of E. coli harboring the plasmids
were separated on SDS-PAGE and subsequently immunoblotted with anti-EpsD
antibodies after proteins were transferred to the membrane. Both plasmids are
under the control of tac promoter and they were not induced with IPTG. Both
monomers and multimers of EpsD (in the stacking gel) are shown. EpsC is not
shown here. (Strains pMMB799=CBZ416, pMM8800=CB2417)

to a heat shock protein Hsp15 of E. coli, raising an alternative possibility. Is the
Hsp15 ORF necessary for the secretion? To test this, different plasmids derived
from pMMB799 and with alterations in this ORF were constructed and introduced
into the epsC mutant (Figs. 3-1 and 3-3A). The results of complementations in
terms of LT B subunit secretion percentage are shown in Fig. 3—3B (next page).
The percentage of the secreted LT B for the epsC mutant was only 18% while
the wild type V. cholerae secreted almost 100% of LT B to the extracellular milieu
(strains 1 and 2). As shown in chapter 2, expression of epsC alone by pMMB781

in this mutant could increase the secretion level only slightly (36%), whereas the

complementation by pMMB799, containing epsCD and ~1.2 kb upstream

113

 

Hsp15 ORF epsC epsD

 

 

 

 

 

 

 

 

 

 

  
    

 

secretion (%)

 

 

 

 

Strain 1 2 3 4 5 6 7 8

Genotype WT epsC” epsC' epsC“ epsC“ epsC“ epsC' epsC'
Hsp1 5 ORF - - - + . + . .
Plasmid (pMMB) - - 781 799 800 840 844 845

FIG. 3-3 A putative Hsp15 ORF is required in a plasmid expressing
epsCD to restore secretion of the epsC mutant. (A) Schematic representation
of various plasmids used to complement the V. cholerae epsC mutant. (B)
Secretion of LT B subunit from various strains of V. cholerae as measured by
GM1 ELISA (16, 22) and effect of the ORF in the plasmid on secretion in the
epsC mutant. (Strains 1=VB101, 2=VB345, 3=VB348, 4=VB349, 5=VB385,
6=VB396, 7=VB403, 8=VB404)

114

sequence, could restore the secretion to the level comparable to that found in
the wild type strain (strains 3 and 4). Plasmid pMMB800, containing only
~200 bp upstream sequence lost the ability to complement. The plasmid
pMMB840 contains a frameshift in the last 17 residues of the Hsp15 ORF and it
was constructed by digesting pMMB799 with Aatll, blunt ending and religating
the plasmid. The resulting plasmid results In an early termination codon only
after two amino acid residues from the Aatll cleavage site, thus losing about one
tenth of amino acid residues at the C-terminus of the protein encoded by the
ORF. Although we do not know whether this alteration would change the
biochemical function of this protein, it is possible that such protein would still be
active. According to the complementation data, pMMB840 could restore the
secretion from the epsC mutant as effectively as pMMB799 (Fig. 3-3B). This
suggests that the frameshift mutation near the C-terminus of the ORF does not
affect the protein function. The plasmids, pMMB844 (ORF is replaced by the
KmR gene) and pMMB845 (derived from pMMB844 and the ORF is completely
deleted), could not complement the epsC mutant or could not rescue its secretion
defect (Fig. 3-3B). In an independently performed experiment to complement the
epsC mutant by pMMB843 whose ORF is truncated in the middle of the protein
(Fig. 3-1), the secretion percentage was only 28%, thus failing to complement the
mutant. All these data strongly indicate that the putative Hsp15 ORF is required
in addition to epsCD to complement the epsC mutant. These results also
suggest importance of this ORF in the type II secretion and indirectly show its

requirement for the type II secretion in V. cholerae. Direct evidence of this ORF’s

115

requirement in the type II secretion awaits the mutagenesis of this ORF in the

genome of V. cholerae.

Fllm Exposure

  

; 'i‘ufiLm-I' ’77 '71.": ‘17!

 

 

 

EpsC - ' -— - '4': 1 hour
EpsC E -' ‘1 7: 5mlnute
1 2 3 4 5
Plasmid (pMMB) 799 800 843 844 845
ORF + - - - -

FIG. 34 Expression levels of epsC in various plasmids show that only
pMMBBOO overproduce EpsC, suggesting the inability of plasmids pMMB800,
pMMBB43, pMMB844 and pMMBB46 to complement the epsC mutant is not due
to the overproduction of EpsC and EpsD.

Effect of the Hsp15 ORF on expression of epsCD. To understand the
reasons why pMMB800 produces much higher levels of EpsC and EpsD these
levels were checked in several pMMB799 derivatives in E. coli. As shown in
Fig. 3-4, all plasmids except for pMMBBOO produced about the same level of
EpsC according to the intensity of the band on the blot. These included plasmids
pMMBB43, pMMBB44 and pMMB855 which do not contain the complete Hsp15
ORF (Fig. 3-1 and lanes 3, 4 and 5 in Fig. 3-4). These results suggest that the
overproduction of EpsC and EpsD from pMMB800 is not directly due to the
absence of Hsp15 ORF or of its product. If the ORF is the real gene, the
promoter of the ORF must be present upstream of the ORF. Thus, one common

point for pMMB799, pMMB843, pMMBB44 and pMM3855 is that they all contain

116

 

the intact putative promoter for the Hsp15 ORF while pMMBBOO is likely not to
contain the complete promoter (Fig. 3-1). Therefore, it seems the reason for the
difference in expression levels between pMMB800 and other plasmids is due to
the presence of a putative promoter for the Hsp15 ORF acting in opposite
direction of tac promoter in the vector which expresses epsCD genes (see Fig. 3-
1). It has been known that counteracting promoters can affect the expression of a
gene from the plasmid. This means the expression level of epsCD from
pMMB800 is not actually overexpression, but normal while all other plasmids,
including pMMB799, pMMB843, pMMB844, and pMMB845, express less than
the normal level because of the presence of the promoter for the putative Hsp15

ORF.

Putative Hsp15 ORF expression under the control of the bacteriophage T7
CD10 promoter. To test if the putative Hsp15 ORF of V. cholerae is expressible,
it was cloned into the pT7-6 expression vector and induced with IPTG. E. coli
cells harboring this plasmid were resuspended in sample buffer containing 5-
mercaptoethanol and the proteins were separated by SDS-PAGE. As shown in
Fig. 3-5 (next page), the Coomassie Blue-stained gel shows distinct bands for
the E. coli with the ORF-containing plasmid (lanes 1, 2, 5, 6, 7 and 8), while
negative control, which contains the empty pT-6 vector plasmid, does not show
this band. According to Korber et al., the molecular mass values of the Hsp15
homologues in Gram-negative organisms are in the range of 15.1-15.5 kDa (12).

The predicted MW of the putative Hsp15 ORF is 14.85 kDa according to the

117

sequence analysis. The band on the gel, according to the molecular weight
standard used, shows somewhat bigger molecular mass, but it is still pretty close
to the expected size. Oftentimes, protein migration rate is affected by several
factors such as folding or disulfide bonds and the actual molecular mass might

not be reflected correctly on the SDS-PAGE.

kDa ;‘
.31
43 3

-.- ... ......- 9— .. c— J

29 ......
It —ORF expressed

- m_,,4—~MMa-fi :41.

Lanez12345678

ORF In the vector: + + - - + + + «i-
Sample amount(ul): 5 5 20 20 10 10 20 20
Expressed ORF: + + - - + + + +

Fig. 3-5 Expression of the putative Hsp15 homologue ORF of V.
cholerae under the control of the bacteriophage T7 010 promoter.
Compared to the proteins expressed from pT7-6 in E. coli BL21(DE3) (CBZ457),
pT7-6::ORF (CB2456) expresses an additional distinct protein and the band
appears between 18.4 and 29 kDa standards.

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Discussion

In this report, we identified a new ORF that may be important for type II
secretion of V. cholerae. As shown in the genetic complementation analysis for
the epsC mutant, only the plasmids containing this Hsp15 ORF in addition to
epsCD could complement for secretion restoration when introduced into the
mutant. Since site-directed mutagenesis in V. cholerae is still technically not
possible, we still do not have a mutation affecting putative hsp15 gene in this
host. Thus, the crucial experiment showing whether the function of hsp15 is
essential for toxin secretion or whether the gene might have an auxiliary role in
this process awaits further research.

At present, the role of the putative Hsp15 ORF is unclear. The epsC
mutant should have intact wild type Hsp15 ORF in the genome. Expression of
epsC and epsD from plasmids may result in some quantitative imbalance in the
stoichiometry of the secretion apparatus and the amount of Hsp15 produced from
the chromosomal gene may not be enough to complement. Since EpsC and
EpsD interact and the absence of the ORF affects their function, it is likely that
this ORF is involved in the regulation or fine tuning of these two components so
they can play their role in the secretion apparatus properly in combination with
other proteins. The main reason for the epsC mutant failing to secrete is caused
not only by the absence of EpsC but also by insufficient level of EpsD.
Therefore, to complement this mutant, very precise and well-balanced

expression of epsCD is important and the ORF might be necessary for that. It is

119

possible that this Hsp15 might actually interact with EpsC-EpsD complex and its
expression in cis may be essential for its action.

Although Hsp15 of E. coli has been studied to some extent, its
homologues in other Gram-negative bacteria were identified by sequence
similarity and none of them has been characterized yet. Thus, they are still listed
as hypothetical proteins. One interesting point is that the location of the hsp15
gene is well conserved among a few bacteria with the type II secretion system.
As seen in the literature, type II secretion system is quite well conserved, but
there are some variations of gene organization (18). Among them, type II
secretion systems of V. cholerae, Aeromonas and Shewanella species are most
closely conserved in terms of gene organization and they all have the gene
homologous to hsp15 gene right upstream of type II secretion gene C.
Therefore, it is quite probable that the homologues of Hsp15 in these organisms
might play a role in the secretion. Although, Hsp15 is a heat shock protein, the
role of heat shock proteins might be essential not only after heat shock but also
under normal conditions (11).

The indication that Hsp15 homologue of V. cholerae is somehow
associated with EpsC-D complex and might play a role in the type II secretion is
further supported by presence of several D protein-associated proteins in other
organisms. In Eminia and K. oxytoca, S protein is required for outer membrane
insertion, assembly and stability of the D protein (7, 8, 15, 19). In A. hydrophila,
ExeAB complex is required for the localization and assembly of the ExeD

multimer (1). Similarly, in E. chrysanthemi, OutB interacts with OutD (3). In X.

120

campestris which lacks EpsC homologue, XpsN protein has been shown to be
associated with OM protein XpsD (13). This shows that D protein, which forms
OM pore, interact with several proteins, suggesting that in addition to the
interaction with the C protein, interactions with other proteins are also necessary
to control the gated channel. Since the multimeric D protein channel, through
which substrate proteins of the type II secretion system pass, is such an
important part of the secretion apparatus, it seems to involve several interactions
to communicate with other mostly IM proteins.

In conclusion, we have identified a new gene, whose product might be
important for secretion. It may be involved in the functions of the EpsC, EpsD or
of the EpsC-EpsD complex. Further study is required to understand the role of

this protein in the function of Eps apparatus.

121

10.

11.

References

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CHAPTER 4

CONCLUSION

125

 

For the past decade our lab has been trying to understand the mechanism
of type II protein secretion in Vibrio cholerae. Since the identification of a cluster
of eps genes, which are required for type II protein secretion, we have been
characterizing individual proteins and also identifying interactions between the
proteins making up the secretion system. The only outer membrane component
of V. cholerae, EpsD homologues have been characterized well structurally by
electron microscopy studies and a role for EpsD as the exit pore through which
the proteins are secreted, has been proposed (1, 4). However, the link between
the outer membrane pore and the cytoplasmic membrane has been missing. In
this thesis, I present three experimental results, two results directly supporting
the interaction between EpsD and the periplasm-spanning cytoplasmic
membrane protein EpsC:

(i) EpsD multimer is unstable in the absence of EpsC and vulnerable to
proteolytic degradation (Figs. 2-2, 2-3 and 2-4);

(ii) EpsD can be stabilized by providing EpsC in trans (Figs 2-2, 2-3 and 2-
4) but to form functionally active EpsD, EpsC has to be supplied in cis
with EpsD (Figs. 2-6 and 2-7). This was demonstrated by effective
complementation of the V. cholerae epsC mutant achieved only by
supplying a plasmid expressing epsCD and ~1.2 kb sequence located
upstream of the epsC gene (Fig. 2-6).

(iii) Not only the epsCD, but also an open reading frame (ORF) in the
upstream sequence seems to play an important role In type II secretion

of V. cholerae (Fig. 3-3). The ORF is a homologue of Hsp15 in

126

Escherichia coli, which is a novel ribosome-associated heat shock
protein and the homologues are also found in many other Gram-

negative bacteria (2, 3, 5).

The actual function of Hsp15 is unclear and its homologues have never
been studied yet in organisms other than E. coli. Therefore, it is hard to predict
the function of this putative Hsp15 ORF in V. cholerae from the known
information of Hsp15 in E. coli. Although a direct evidence for its requirement in
the secretion was not shown, its requirement for the complementation of the
epsC mutant along with epsCD suggest that this Hsp15 homologue might be
involved in interactions with EpsC and/or EpsD. The fact that at least two more
species, Aeromonas and Shewanella, have the same conserved genome
organization of this ORF with the type II secretion genes makes the involvement
of this ORF in the secretion process more likely. Further characterization of this

ORF will be worthwhile to help understand the type II secretion of V. cholerae.

127

References

Bitter, W., M. Koster, M. Latljnhouwers, H. de Cock, and J.
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which are involved in protein transport across the outer membrane of
Pseudomonas aeruginosa. Mol Microbiol 27:209-19.

Korber, P., J. M. Stahl, K. H. Nierhaus, and J. C. Bardwell. 2000.
Hsp15: a ribosome-associated heat shock protein. Embo J 19:741-8.

Korber, P., T. Zander, D. Herschlag, and J. C. Bardwell. 1999. A new
heat shock protein that binds nucleic acids. J Biol Chem 274:249-56.

Nouwen, N., N. Ranson, H. Saibll, B. Wolpensinger, A. Engel, A.
Ghazl, and A. P. Pugsley. 1999. Secretin PulD: Association with pilot
PulS, structure, and ion- conducting channel formation. Proc Natl Acad Sci
U S A 96:8173-8177.

Staker, B. L., P. Korber, J. C. Bardwell. and M. A. Saper. 2000.
Structure of Hsp15 reveals a novel RNA-binding motif. Embo J 19:749-57.

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