Denning the Role of CipA in the

Pathogenesis of Campylobactet jejuni Infection

Jennifer Lynett

A thesis submitted in confohty with the requirements for the degree of Masters of Science

Department of Laboratory Medicine and Pathobiology University of Toronto

Copyright by iennifer Lynett, 1999

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Campylobacter jejwii is recopized as a leading cause of foodborne bacterial uifection,

with symptoms ranging fiom a mild watery diarrhea to more sevexe infiammatory

colitis. At present, the proposed model of discase includes colonization of the intestinal

tract through adhesion and invasion of intestinal surfaces leading to mucosal

inflammation and damage to epitheliai ceii huiction. Revious work in our laboratory

identified the CipA protein (C(1111pyIobucter bvasion Fhmotype) as a potential

vhlence factor. A cipA deletion mutant was constmcted by allelic replacement to

assess the effect of the disruption of the cipA dele. Using the gentamicin protection

assay, the CipA mutant showed a reduced abüity to invade HEp2 celis grown in tissue

culture (29.5% 2 5.3% relative to wild type, p < 0.05). However, there was no reduced

invasion when INT407 cells or nonpolarized Cam-2 ceiis were utilized. The mutant

and wild type C. jejuni strain exhibited similar rates of bacterial translocation across a

polarized epithelial cell monolayer (Caco-2 cells) without affecting transepithelial

resistance. Using polyclonal antibodies directcd against CipA, Western blot analysis

showed that CipA protein expression is not regulated in response to bacterial growth

phase or when C. jejuni is culturexi in MEM-15% FCS. A murine model of

Campylobacter infection was employed to assess the virulence of the CipA mutant C.

jejuni strain during infection in Mvo. Following orogastric inoculation of BALBlc

mice, the rate and duration of intestinal colonization can be monitond by the level of C.

jejuni fecal shedding. Unfortunately, a sustained colonization of mouse intestine could

not be established despite repeated challenges. Future studies employing a more

invasive strain of C. jejuni as the genetic background for a cipA mutation wili help to

further characterize the importance of CipA during the interaction between the

bacterium and the epithelial ceil suIface.

Table of Contents

Chapter 1 Microbiology and Clinical Relevance of Cumpylobacter jejuni 1-10

1.1 Introduction 1.2 Taxonomy 1.3 Celluiar, cultural and serological c h h s t i n f -4 Chnical features 1.5 Post-infectious sequelae 1.6 C. jejuni as a foodbome pathogen 1.7 Antimicrobial resistance

Chapter 2 Molecuiar Biology of C. jejmi and Mode1 S ystems of Infection 1 1- 19

2.1 Introduction 2.2 Molecular genetics of C. jejuni 2.3 In viiro models of C. jejmi infection 2.4 Zn vivo models of C. jejmi infection

Chapter 3 V i e n c e Mechanisms of C. jejuni

Introduction Motility F l a g e h as an adhesin Role of motility in invasion Adherence Pilus production in C. jejuni C jejmi adherence to fibronectin Toxin production Invasion of C. jejuni Intracelluiar survival of C. jejuni Regulation of virulence factors

Chapter 4 Project Rationaie

4.1 Introduction 4.2 Objectives

Chapter 5 Materials and Methods

Bacterid st rahs and culture conditions Mammalian ce11 culture Adherence and invasion assays B acteria1 transcytosis Construction of the recombinant p2E38-KD plasmid Natural transfoZrnafion of C. j e ju i Polymerase chah rraftion amplification Southem hybridization analysis of genomic DNA Expression of recombinant CipA CipA antisenim Electrophoretic separation of proteins and Western immunoblot analysis CipA protein expression in jejuni CipA expression following interaction with eukaryotic ceiis C. jejuni infection in mice

Chapter 6 Results

6 1 Construction of a kanarnycin-resistant cipA-deletion mutant of C. jejuni T G W 1 1

6.2 Generation of antiCipA polyclonal antibodies and detection of CipA within C. jejuni

6.3 CipA expression in C. jejuni TGH90 1 1 is not affected by phase of growth 6.4 CipA expression following interaction with HEp2 cell monolayers in tissue

culture 6.5 Comparing the levels of adhmnce and invasion of wild-type C. jejmi and the

cipA mutant strains to eukaryotic ceU monolayers in tissue culture 6.6 Wild type C. jejuni and saaui 901LK2 exhibit simila. rates of translocation across

a polarinxi Caco-2 monolayer without disrupting the transepithelia1 resistance of the monolayer

6.7 Efforts to establish transient colonization of BALBIc mice with wild-type and mutant strains of C. jejuni

Chapter 7 Discussion 102-113

7.1 Summary and interpretation of resuits 7.2 Future directions

Chapter 8 References

Figures

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 14.

Figure 15,

Figure 16.

A phylogenetic tree of rRNA homology group 1 within the rRNA superfarnily VI.

The physical map of C. jejuni TGH9ûll

Schematic diagram of the plasmids utilized to generate the c@A-deletion mutant suicide vector construct

PCR analysis of the ci'pA gene h m wild-type and cipA mutant strains

Restriction maps of the *A chromosomal loci within wild-type C. jejuni and the mutants 901LK1 and 901LK2

Southern blot anaiysis of chromosomal DNA from C. jejuni TGH9û11, 901LK1 and 90 lLK2

Southem biot analysis of chromosomai DNA to conf- the partial deletion of the cipA coding region h m 901LK2

Southem blot anaiysis of chromosomai DNA of the wild-type and cipA mutant strains using the kanarnycin resistance cassette as a DNA probe

Expression and purification of the CipA protein using the GST-Gene Fusion S ystern

Western immunoblot analysis of wholecell lysates h m the cipA mutant strains and the parental wild-type strain using anti-CipA antibody

CipA protein ievels throughout the growth curve of C. jejuni TGH9û11

CipA expression when C. jejuni is cultured in tissue culture medium or following contact witb HEp-2 celis

Levels of adherence and invasion of wild-type and 901LK2 to HEp-2 cell monolayers

Gentamicin protection assays using INT407 ceil monolayers

Adherence and invasion levels of wild-type and cipA mutant C. jejmi using nonpolarized Cam-2 cells

Rates of translocation of C. jejuni and 901 W across a polarized Caco-2 monolayer

Figure 17. Redicted coding regions adjacent to CipA using the Sanger Centre C. jejwi saain NCïC 11 168 genome database

Tables

Table 1. Bacterial strains and plasmids utilized in this study

Table 2. PCR primers u t i . in this study

List of Abbreviations

CDT

c m CHO

CT

EDTA

FCS

GBS

GST

IL-8

INT407

iPTG

LB

LPS

LT

MEM

MFS

MH

MOMP

ORF

PAGE

PCR

EUTARD

SDS

Stx

Cytolethal distending toxin

Cystic fibrosis aansmembrane conductance regulator

Chinese hamster ovary cells

ChoIera toxin

Eth ylenediaminetettaacetic acid

Fetal calf senun

Guillain-Bame syndrome

Glutathione S-transferase

Interleukin-8

Intestine 407 ceils

Isopropyl $-D-thiogalactoside

Luria-Bertani medium

Lipcpolysaccharide

Heat-labile toxin

Minimal essential medium

Miller Fisher syndrome

Mueller Hin ton

Major outer membrane protein

Open reading frame

Polyacrylamide gel electmphoresis

Polymerase chah reaction

Removable intestinal tie adult rabbit diarrhea

Sodium dodecyl sulphate

Shiga toxin

Acknowledgements

There have been so many people who have provided encouragement and support

ihroughout the past two years. 1 would like to thank my family; Mom, Dad and Sarah. who

always offered me i $ c e h m which to escape the big city. In addition. their love and

understanding have been invaluable gifts.

1 am gratefd to my supervisor, Dr. Chan who provided my first introduction into research

as an undergraduate. Later as my graâuate supe~sor , Dr. Chan taught me the importance of

patience in research. In addition Dr. Chan's scientific and technical knowledge have greatly

aided my research training. 1 wodd like to thank my CO-supervisor, Dr. Sherman who was

always willing to iisten to my ideas and whose enthusiasm and encouragment were always

uplifung. 1 also thank my cornmittee members. Drs. Richard EUen and Martin McGavin who

were extremely helpful by providing a different outlook, thus rerninding me of the importance of

collaborative research.

1 have been lucky to have met so many people fkom various labs who have helped me

throughout my MSc. Angela, whose expertise and work habits continuously amazed me. Eric,

Mark, Shahnaz and David who are the kind of labrnates that make the time fly by. In addition. I

would like to thank the members of the Sherman lab @oh, Nicola, Hilary, Gilbert and Andrew)

who were always available to offer advice. Finally 1 would like to thank my roommates; Kirk-

rational and organized but always fun to be with and Tina, whose caring and thoughtful nature 1

will always chensh. A speciai thanks to fnends; Mark, Shahnaz, Donna. Janine, Lisa and Devin

who reminded that there is life outside the lab.

Chapter 1

Microbiology and Chical Relevance

of Campylobacter jejuni

1.1 Introduction

Since its recognition as a human pathogen during the 1970s. Cmpylobacter jejuni

has emerged from obscurity to becorne recognized as a l d n g cause of bacterid ententis

worldwide (Alteknise et al. 1999; Skirrow, 1977). In addition, infection with certain

serotypes of C. jejuni is iinked to the onset of a polyneuropathy known as the Guillain-

Barre syndrome (Nachamkin et al. 1998). As a m e r complication, clinicai isolates of

C. jejwii are becoming increasingiy resistant to antimicrobial dmgs as a result of the use

of antibiotics in food production (Smith et al. 1999; Witte, 1998). Yet despite the

frequency of C. jejuni infection and the associateci public health concems, very Little is

understood about the pathogenic mechanisms of this organism. Recently, the Sanger

Centre announced the completion of the C. jejuni genome sequencing project.

Comparison of the C. jejuni genome with that of other related pathogens wiil surely

provide new insights into our understanding of vinilence strategies and aid in the

development of a vaccine against this important human pathogen (Pennisi, 1999).

1.2 Taxonomy

The taxonomy of the genus Cmpylobacter has undergone a number of revisions

over the past two decades. The genus has expanded h m four species in the original

classification (Veron and Chatelain, 1973) to 15 species and 6 subspecies which are

currently recognized within the genus CumpyZobucieer (Vandamme et ai. 1995; Alderton

et al. 1995). Furthexmore, several species have k e n moved between different genera,

including additions to the genus CampyZobactet of organisms previously known as

Wolinella and Bacteroides species, as well as the transfer of species h m the genus

Campylobacter to new 1 y formed genera, including Helicobacter and Arcobucter

(Vandamme et al. 1995; Vandamme et al. 1991b; Goodwin et ai. 1989).

Using DNA-rRNA hybridization and 16s rRNA sequence analysis, it has becorne

clear that the genus Cumpylobacter and related organisms are phylogenetically divergent

from the five previously recognized rRNA superfamilies within Gram-negative bactena

This led to formation of a s u t h rRNA superfamily within the class of Proteobocte~

(Vandamme et al. 1991b). The rRNA superfamiIy VI can k M e r dividcd into three

rRNA homology groups (Thompson et al. 1988). rRNA cluster 1 is shown in Figure 1

and contains the 15 species of Campylobacter as weil as one species h m the genus

Bacteroides. rRNA cluster II contains species from the newly defined genus Arcobacrer.

Due to the close genetic Linkage between the genera Cmnpylobacter and A r c o b ~ e r ,

Vandamme et al. (1991a) proposed that a new family, the CampyllooMeraceae, should

be used to encompass these taxa. The closest relatives of the family Cumpylobocteraceae

make up rRNA cluster III which contains a heterogeneous group of organisms with

members from three separate genera: Helicobacter, Wolinelln and Fïexispira.

1.3 Cellular, cultural and serologicai characteristics

C jejuni is a small(1.0-1.5p long, 0.2-0.5p wide), Gram-negative bacterium

which grows optimally under rnicroaerophilic (5% Oz, 10% CO2, 85% Nz) and

themophilic (37OC42'C) conditions. C. jejmi has a spiral morphology, a uni- or bi-

polar unsheathed flagellum and is characterized by a rapid darting motility. However,

Figure 1: A phylogenetic tree of rRNA homology group 1 within the rRNA supei'fdy VI.

Campylobacter rectus Campylobacter showM

[Bacteroides] gracilis

t Campylobaetet cun>us

Campylobacter concisus

Campylobacter sputoïum

- I r - Campylobacter mucosaZis

1 fl Cmpylobacter hyoîntestinalîs

Cumpylobacter helveticus Campylobacter upsalienris

Campylobacter hyoilei

CampyZobacter coli

Cmpylobacter jejuni

L Campylobacter lari - [Btzeteroides] ureolyticus

Adapted f r o z Van D a . et ai. (1995); On, (1996); Aiderton et al. (1995)

bacterial cultures in the later phases of growth are predominantiy composed of elongated

and coccoid foms of C. jejuni @-King et al. 1985; Thomas et al. 1999).

There are two serotyping schemes in use for C. jejuni: the Pemer serotyping

system detects differences in lipopolysaccharide and lipooligosacchari& antigens (Penner

et al. 1983) and the Lior method differentiates on the basis of heat-labile antigens (Lior et

ai. 1982). The Pemer system has been used extensively in epidemiological snidies

linking padCUIar serotypes with the development of cinmic neuromuscular sequelae

(Moran and Penner, 1999). A varîety of biochemical tests can be used for the

identification of C. jejuni. The organism is oxidase and catalase positive and wease

negative. C. jejuni is able to reduce nitrates and does not produce hydrogen sulfide

(Penner, 1988). Differentiation of C. jejuni h m the closely relateci C. coli often reIies

upon the hippurate hydrolase test (Penner, 1988).

1.4 Clinid Features

Following an incubation p e n d of up to seven d a y s , infection with C. jejuni leads

to the development of acute enteritis (Cover and Blaser, 1989). Symptoms range h m a

mild watery diarrhea to more severe inflammation affecting the smaii bowel and colon

(Allos and Blaser, 1995). In addition, patients may also experience abdominal pain,

fever, nausea and malaise. Stools are frequentiy bloody and contain mucus and

leukocytes (ie: a colitis). In severe cases, rectal biopsy specimens reved epithelial cell

injury, loss of mucus and crypt abscesses in epithelial glands (Blaser et al. 1980). The

infection is usually self-limiting; however, antirniaobial treatment ic recommended if

s ymptoms persist. Erythmmycin is most commonly recommended due to its high &gree

of efficacy and relatively low rates of toxicity (Alte- et al. 1999).

There are marked differences in the clinical feanires of C. jejuni infections in

developing and industrialized nations. Patients in developed nations usually present with

an inflammatory form of diarrheal illness. The two age p u p s at highest risk of infection

are young adults and infants flauxe, 1992). However, in the developing world, infection

with C. jejuni is ofhm asymptomatic or rtsults in W d watery di&= Disease is cisually

restricted to children under two yean of age. These contras& in disease presentatïoa and

age distribution are thought to reflezt ciifferences in host immunity rather than strain

variations (Cover and Blaser, 1989). This view is supported by studies showing that

travelers to developing nations develop the more severe infiammatory diarrhea upon

infection (Taylor, D.N., 1992). Therefore, in developing countries where C. jejuni is

endemic, increased exposure early in life likely translates into an acquired immunity

(Cover and Blaser, 1989).

1.5 Post-Infectious Sequelae

The Guillain-Barre syndrome (GBS) and the related Miller Fisher syndrome

(MFS) are autoimmune d i s o h of the peripheral nemous system which are frequently

preceàed by infection with certain serotypes of C. jejicni. GBS is the leading cause of

acute flaccid paralysis in the world and is charactenzod by areflexia, a ioss in muscle

strength and weabiess of the respiratory muscles (Lindsay, 1997). Patients with MFS

present with areflexia and opthalmoplegia (inability to move the eyes). The symptoms of

both disorders are self-limiting but patients often require weeks to months before a full or

partial recovery (Nachamkirn et al- 1998)-

Evidence of a iink between a preceding C. jejuni infection and the development of

GBS and MFS comes from both serologic and stool cuiture data. Numemus groups have

documented an increased prevalence of anti-C. jejwu' antibodies in the s e m of GBS

patients (Mishu et al. 1993; Ho et ai. 1995). Efforts to obtain positive stool cultures as

M e r confirmation often have been misuccessful. This is likely because neurologie

symptoms are not present until up to three weeks foilowing infection by which t h e the

patient is no longer excreting Campyiobacter organisms (Allos, 1997). Rees et al. (1995)

reporteci that of the 26% of GBS patients with evidence of a previous Ccunpylobacter

infection, only 8% were positive by stool cuiture while the remaining were positive by

serologic testing.

The autoimmune aspects of these diseases are thought to be mediated by

antiganglioside antibodies which are frequentiy elevated in GBS and MFS patients. GBS

patients have high titres of IgG anti-GMi mtibodies and IgG anti-GD*, antibodies while

MFS patients demonstrate elevated levels of IgG anti-GTi, and anti-GQlb antibodies

(Chiba et al. 1993; Ho et al. 1999; Neisser et ai.1997; Yuki et al. 1997). Molecular

mimicry between lipopolysaccharide (LPS) epitopes and structures present in human

gangliosides is thought to elicit the production of the antiganglioside antibodies. Using

the Penner O serotyping scheme to detect ciifferences in LPS antigens, a varïety of groups

have documented that specific serotypes of C. jejuni are more frwluently associated with

the development of GBS. C. jejuni serotype 0: 19 is predominately isolated h m GBS

patients while serotype 0:2 is most commonly isolated from patients with MFS (Yuli,

1997). Structural analysis of bacterial LPS and human gangliosides revealed that the core

oligosaccharide of 0 : 19 LPS shares homology with the terminai oligosaccharide of the

GMI and GDI, gangliosides (Aspinail et al. 1994). Anti-GQ~b antibodies fiam MFS

patients cross-react with LPS h m C. jejuni serotypes 0 : 2 and 0:23 (Neisser et al. 1997).

1.6 C. jejuni as a Foodborne Pathogen

Infection with C. jejuni is the leading cause of bacteriaï entemolitis in the United

States. There are an estimated 2 4 million cases per year which represents twice the

frequency of Salmonelln spp. infections and four times the prevalence of infection with

Escherichia coli 0157:H7 (Altelcnise et al. 1997). C. jejuni i s a zoonotic pathogen and is

part of the normal intestinal fiora of a wide range of wild and domestic birds and

mammals. Outbreaks in hurnans are rather infiequent and are u s d y associateci with the

consumption of contarninated raw milk or untreated water flauxe, 1992). The majority of

C jejuni infections are sporadic with the highest number of cases occurring during the

summer months (Altekruse et al. 1999). Risk factors for infection include handling of

raw poultry leading to noss-contamination of other foods and the consumptiou of

undercw ked chicken which has been ftcall y contarninated during processing (Tame,

1992; Altekruse et ai. 1999). Several studies have shown that between 5040% of

chicken carcasses are contaminated by the time of sale, thus illustrating the need for new

initiatives aimed at reducing contamination incurred during slaughter and prucessing

(Konkel et al. 1999a; Stern, 1992; Smith et al. 1999).

1.7 AntMcrobM Resistance

The widespread use of antibiotics in agriculture to increase production has led to a

strong selective pressure for microbial adaptation (Witte, 1998). This is a particula.

concem with foodbome pathogens, such as CI jejuni, in which there is an animal

resentoir h m which the bacteria is transmîtted to humans, While erythromycin

resistance in C. jejmi has remained relatively low, a ciramatic increa~e has been o b s e ~ e d

over the past decade in the level of resistance to fiuoroquinolones (Sanchez et al. 1994;

Gaudreau and Gilbert, 1998).

The rise in antimicrobid resistance in C. jejmi is linked to the use of

fluoroquinolones in feeds in the poultry industry (Gaunt et al. 19%). This has been best

documented in the Netherlands where fluoroquinolones were approved for veterinary use

in 1987. Prior to its approvai, there was no detectable level of ciprofloxacin resistance in

Campylobacter spp. isolates. Two years following the introduction of fluoroquinolones,

14% of Campylobacter spp. isolates h m poulûy products and 11% of human ciinical

isolates were ciprofioxacin resistant (Endtz et al. 1991). Similar increases have been

observed in Spain, where ciprofloxacin resistance in human clinical isolates increased

from 9% in 1990 to 51% in 1991 (Sanchez et al. 1994).

An increase in the leveI of fluoroquinolone resistance also has been observed in

isolates in the United States and Cm& In Canada, between 1995-1997,13% of c i i ~ c a l

isoIates were ciprofloxacin resistant whereas no resistant strains were detected between

1985-1986 (Gaudreau and Gilbert, 1998). In the United States, the number of

ciprofloxacin resistant human isolates rose fmm 1.3% in 1992 to 10.2% in 1998 (Smith et

al. 1999). This increase coincides with the iicensurc of fluoroquinolone for use in the

poulay industry in 1995. In addition, molecular subtyping analysis fouad an association

between quinolone-resistant C. jejuni fiom poultry pmducts and clinicai isolates in

humans (Smith et al. 1999)-

Chapter 2

Molecular Biology of C. jejuni and

Mode1 Systems of Infection

2.1 Introduction

Research aimed at defining the pathogenic mechanisms of C. jejuni has been met

wi th a nurnber of obstacles. Molecular genetic techniques and mutagenesis strategies for

use in C. jejuni are still largely in the developmental stages. Furthermore, the lack of

reproducible in vivo modeis of infection have hampercd the characterization of putative

C. jejuni virulence determinants (Taylor, D.E., 1992; Konkel and Cieplak, 19%).

However, advances have been made in the generation of C. jejuni site-specitk mutants

using either natural transformation or electroporation to induce the uptake of exogeneous

DNA (Wang and Taylor, 1990; MiUer, J.F., 1988; Wassenaar et ai. 1993a). In addition,

the use of cultured epithelial cell lines have provideci convenient in vitro systems for

studying bacteriai adherence, invasion and the host ceii factors involveci during infection.

2.2 Molecular Genetics of C. jejuni

Difficuities are often encountered when using molecular genetic techniques such

as cloning or expression of C. jejuni chromosomal genes. Possible explanations include

differences in G+C content between the C. jejuni and Escherichia coii genomes,

differences in codon usage or problems with promoter recognition flaylor, DE., 1992).

Wosten et al. (1998) found that promoters with high levels of activity in C. jejuni are

often non-îùnctional in E. coli. While the -10 region of the C. jejuni promoters closely

resemble the E. coli conserved sequences, the -35 region is strongly divergent. In

addition, a third consensus region was identified in the -16 region of the C. jejuni

promoters (Wosten et al. 1998).

S huttie Vectors:

One of the Iimiting factois in the molecular genetic analysis of C. jejmi was the

Iack of plasmids which replicated within both C. jejmi and E. coli (Guerry et al. 1994).

Furthemore, antibiotic resistance markers fiom E. coli do not fwiction in C. jejmi, yet

fortunately, resistauce genes h m Cumpylobucfer spp. do confier nsistance in E. coli

(Taylor, DE. 1992; Guerry et al. 1994). Numerous rwearch groups have sought to

construct chzmeric plasmï& capable of autonomous replication within both C jcjuni and

E. coli (Labigne-Roussel et al. 1987; Yao et al. 1993). 'Ihe first generation of shuttle

vectors contained both an origin of replication (oriR) and a kanamycin resistance cassette

from C. coli and an origin of transfa (oriT) Erom an Inc P plasmid (Labigne-Roussel et al.

1987). Subsequently, newer vectors have been describecl with expanded multiple cloning

sites and different antibiotic resistance markers (Yao et ai. 1993).

One of the potential uses of an E. d i - C jejuni shuttle vector is complementation

analysis of C. jejuni isogenic mutant strains. While some studies have reported success in

this area (Yao et al. 1997; Whitehouse et ai. 1998) others have been unable to

complement C. jejuni mutants in tram using the shuttle vector system (van Vliet et al.

1998; Konkel et al. 1999b). Shuttle vectors have also been used to analyze gene

expression and regdation by fusing a C. jejuni promoter to a promoterless reporter gene.

In C. jejzuzi, promoterless chloramphenicol acetyltransferase (eut) gene and &

gaiactosidase ( l a d ) and in C. coli a promoteriess bacterial luciferase (LM) ~~nt

within a shuttle vector have been used to quantitate the transcription of the target genes

under various conditions (Purdy and Park, 1993; Wooldcidge et al. 1994; Wostcn et al.

1998). Future studics focused on detmnining gene expression following contact with

host cells would provide a usehi strategy for the characterization of C. jejuni virulence

de terminants.

Allelic Exchange:

In the absence of an origin of replication recognized by Campyiobuczer spp.,

plasrnids c m be used as suicide vectors to generate site-specific C. jejuni chromosomal

mutants (TayIor, DE., 1992; Labigne-Roussel et ai. 1988). This strategy relies on the

homologous recombination between the non-replicatuig plasmid and the C. jejuni

chromosome at the gene of interest This method has ban proven effective and used

extensively to study putative C. jejmi vinilence factors (Konkel et al. 199%; Konkel et

al. 1997; Pei et al. 1998; Wassenaar et al. 1991).

Other genetic tools, such as transposon mutagenesis, which have been used to

generate mutants in other enteric pathogens have not been successful in C. jejuni

(Labigne-Roussel et al. 1988; Ketley, 1995). As an alternative, Yao et al. (1994) have

adapted a mutagenesis strategy originaily described for Huernophilus influenzue

(Sharetzsky et al. 1991). C. jejuni genornic DNA fragments are iigated to an antibiotic

resistance marker and then retumed to C. jejuni by natural transformation. The site-

specific chromosomal insertion of the exogenous DNA results in a bank of mutagenized

C. jejuni tmnsfomants. Subsequent screening of the library for clones exhibiting reduced

adherence and invasion has led to the identification of putative virulence factors. thaeby

demonstrating the useiùiness of this mutagenesis technique (Yao et al. 1994; Yao et al.

1997).

23 In vitro mdeIs of C. jejuni infection

Cultured eukaryotic ceil lines are often used to study bacteria-host cell

interactions. These celî culture mode1 systems offer a reductionist approach whereby a

uniform popuIation of cells is ùifected with a bactexial suspension under defined assay

conditions (ie: temperature, length of incubation, multiplicity of infection) (Elghghorst,

1994; Finlay and Falkow, 1997).

C. jejuni is able to adhere to a variety of eukaryotic cell lines in tissue culture

(Konkel et ai. 1992b). Experiments conducted with one isolate of C. jejuni showed that

while intemalization rates varied, similar levels of adherence were observed using celi

Iines derived from both human (Nï407, HEp-2, HeLa, 293) and non-human (Vero,

CHO-KI, MDCK) origins (Konkel et ai. 1992b). However, since this study focused on

one isolate, these results may not be representative of different strains. Retreatment of C.

jejuni with chioramphe~col does not afféct adherence suggesting that & novo bacterial

protein synthesis is not required and that the C. jejuni adhesins involveci in binding to

eukaxyotic receptors are likel y to be constitutively expresseci (Konkel et al. l992a).

Quantitation of microbiai invasion into host cells can be studied using the

gentamian protection assay (Elsinghorst, 1994). Infkcted cefl monolayers an treated

with an aminoglycoside antibiotic to kill extraceilular bacteria whereas intraceliuiar

bacteria remain protected since the antibiotic is impermeable to the eukaryotic cell

membrane (Elsinghorst, 1994). C. jejuni is capable of invading a wide variety of ceil

lines with maximal intemalization occuning in cells of human origin (Konkel et al.

1992b). Clinical isolates typically exhibit higher levels of invasion than nonclinical

strains and extensive passaging in vitro reduces invasion leveis (Konkel et al. 1990;

Konkel and Joens, 1989). Bacterial invasion is an energy-dependent process and relies

upon bacterial de novo protein synthesis. However, & novo protein synthesis h m the

eukaryotic cell is not required (Konkel et al, 1992b). Some studies employ mild

centrifugation of C. jejmi ont0 the celi monolayer to synchronize the infection and

facilitate contact between the bacteria and the monolayer (Wassenaar et al. 1991; Konkel

et al. 1992b).

Polarized epithelial celi lines scnicturally represent a doser approximation of the

in vivo setting at the intestinal surface. When grown on ponxis membrane fiïter supports,

the monolayers differentiate to develop apical microvilli, a defined brush border,

intercellular tight junctions and a measurable transepithelial mistance. Examples of

polarized celi lines include the Madin-Darby canine kidney (MDCK) celi as weli as

human derived intestinal ce11 fines such as Caco-2, HT-29 and T84 (Pucciarelli and

Finlay, 1994).

The Cam-2 ceil line, derived h m a human colonic carcinoma, has been

employed as a mode1 system in studies with C. jejuni. As shown by transmission electron

microscopy, C. jejuni is able to translocate across Caco-2 monolayem by both paracellular

and transcelldar routes (Konkei et al. 199%). Interestingly, one report showed that high

rates of translocation across polarized Cam-2 monolayers does not necessarily correlate

with a high level of tissue invasion using Caco-2 cells c u l W on nonpenneable tissue

culture wells. Therefore, it appears that C. jejwri translocation and invasion occur via

separate mechanisms (Harvey et al. 1999).

2.4 In vivo models of C. jejuni infection

The availability of suitable animal models in studies of bacterial pathogenesis

provides an efficient means of testing the contribution of putative vinilence factors during

coionization and infection. Understanding the pathogenic mechanisms of C. jejuni has

been difficult due to the lack of simple, reproducible in vivo models which mimic the

manifestations of human disease.

Oral inoculatim of C. jemi in immunocompetent mice and chicks does not result

in the production of diarrheal disease; rather, C. jejuni establishes a transient intestinal

colonization accompanied by fecal shedding of the organism (Blaser et al. 1983;

Wassenaar et al. 1993b). C. jejuni colonization of chickens is thought to be an important

factor for transmission of the bacterium to humans. Thetefore the chick mode1 of C.

jejwi cotonization has been utilized to define the bacterial factors required for the

persistent colonization of poultry (Konkel et al. 1998; van Vliet et al. 1998).

Colonization of the immunocompetent mouse intestine by C. jejuni involves microbial

proliferation within the intestinal mucus layer without adherence to the intestinal

epitheliwi (Lee et d. 1986). Studies in immunodeficient mice achieve higher levels of

bacterial shedding with a srnail proportion of the C. jejuni-infected mice developïng

diarrhea (Hodgson et ai. 1998).

Baqar et al. (1996) utilized an intranasal route of infection to evaluate the immune

responses to C. jejuni in immuncompetent mice. While the authors did not note the

occurrence of diarrhea, there was a high mortality rate associaîed with infection, and C.

jejuni could be recovered from the blood and various intemal organs of the infected mice.

Interestingly, the authors found that prior intranasal inoculation with a sublethal dose of

C jejuni was able to protect the mice h m a subsequent challenge. Therefore, this model

has k e n proposeci for the evaluation of candidate vaccine for C. jejuni (Baqar et al. 1996;

Pei et al. 1998).

Other animal models have been developed which approximate the clinical features

of C. jejuni infection in humans. Studies have b a n descxibed using gnotobiotic beagles,

colostrum-depnved piglets and infant monkeys (Prescott et al. 1980; Babakhani et ai.

1993; Russel1 et al. 1993). However, the two models most commonly used to reproduce

the disease pathology observed in humans are the ferret model of ententis and the

(removable intestinal tie adult rabbit diarrhea) RITARD model (Fox et al. 1987; CaldweU

et al. 1983).

Caldwell et al. (1983) describeci the RITARD mode1 for studies in C. jejuni.

Infection led to the onset of diarrhea in 64% of rabbits with a mortality rate of 53%.

Pathological findings indicated intestinal lesions with the presence of inflammatory cells

in both the lumen and the lamina propria. The methodology involves the introduction of

a temporary ligation of the terminal ileum followed by inoculation of the C. jejuni

bacterial suspension. The intestinal tie is maintaineci for a 4 hr period to prevent the

peristaltic clearing mechanism of the intestine thereby facilitating the establishwnt of

infection.

Oral or intravenous inoculation of ferrets with C. jejuni also leads to the

development of a self-limiteci diarrhea similar to that observed during infection in

humans. The stools are watery and often contain mucus and blood with symptoms

persisting for up to two weeks following infection (Fox et al. 1987). In addition, the

infected ferrets experience dehydration, weight loss and fiequently develop bacteraemia

(Doig et al. 1996)-

Chapter 3

Virulence Mechanisms of C. jejuni

3.1 Introduction

Recent advances in the study of mimbial pathogenesis have revealed novel

bacterial mechanisms aimed at directing the interaction between the pathogen and its

host. A significant discoveq was that several pathogenic bacteria encode clusters of

virulence determinants on extrachromosomal virulence plasmids or in segments of

chromosomal DNA hown as pathogenicity islands. In addition, several Oramnegative

pathogens u t i k the type IU secretion system to cicliver eff'tor molecules to the host

ce11 in order to influence or perturb signal transduction cascades (Hueck, 1998). Another

exciting area of research is unraveling how various environmental signals impact upon

the coordinate regulation of Wulence gene expression (Mekalanos, 1992).

In many respects, the vinilence strategies of C. jejuni remmain enigmatic. The role

of toxin production in the manifestation of disease remains elusive despite considerable

research (Wassenaar, 1997a). In addition, although invasion of C. jejmi into the

intestinal epithelium has been demonstrated, the involvement of the host ce11 cytoskeleton

during uptake is inconclusive (Konkel et ai. 1992b; Konkel et al. 1992~; Oelschlaeger et

al. 1993). While pathogenicity islands or a type III secretion apparatus have not been

identified in C. jejuni, recent evidence of bacterid secreted proteins which infiuence

bacterial in temalization will provide new avenues of investigation (Konkel et al. 1999b).

As an enteric pathogen, C. jejuni likely encounters a wide variety of environmental

conditions while establishing an infection. Currently, the presence of bile salts, iron-

depleted conditions, and temperature shifts have been used to assess differential gene

expression in C. jejuni. Further investigation into the regulation of Wulence

detenninants coupled with the available genome sequence data should provide clarity into

how C. jejuni is capable of inkting its human h o s ~

3.2 Motility

C jejuni possesses a single polar flageiium which is p m n t at one or both poles

of the bacterium. The motility of C. jejuni remains the best characterized virulence

detenninant of this organism. Studies in a number ofanimaI modeIs have shown that

non-motile C. jejuni mutants fail to colonize the intestinal tract of infant mice and chicks

and do not induce fluid secretion in rabbit ileal loops (Everest et al. 1993; Neweli et al.

1985; Wassenaar et al. 1993b). In addition, studies in human volunteers further support

the requirement for flagella in estabiishing an infection (Black et al. 1988).

Successful colonization by C. jejuni req- crossing the mucus layer overlying

the intestinal epithelium Studies in vitro demonstrate that C. jejuni is uniquely adapted

for hi& viscosity environments. C. jejuni display an enhanced motility in response to

increases in the viscosity of the sumunding medium. Mild i n ~ e a s e s in viscosity lead to

longer path lengths of smooth swimming with lower rates of tunbling, while a m e r

increase in viscosity induces a darting motility in C. jejuni (Shigematsu et al. 1998).

Interestingly, Szymanski et al. (1995) showed that increases in viscosity also Ieads to

increased levels of adherence and invasion by C. jejuni in Cam-2 celi monolayers.

Two flagellin genes m, floB) are encoded in a tandem arrangement within the

genome and are independently transcribed h m separate promoters (Gu- et al. 199 1,

Khawaja et ai. 1992). FiaA expression is regulated by a fl pmmoter which is typical of

other flageliar systems while theflaB gene is transcribed h m a d* promoter which is

regulated by environmentai factors including temperature, pH, phase of p w t h and the

presence of various cations and inorgMc salts (Alm et al. 1993). Although the two

flagellar genes are highly homologous ( 2 93% nucleotide sequence identity), the flagellar

filament is predominantly composed of the HaA subunit with o d y minimai amounts of

FlaB present throughout the filament (Guerry et al. 1991). Mutants containhg a

disruptedflmî gene (naK FlaBf) are oniy slightly motile and develop a short, truncated

f i ageilum. FIaA'maB- mutants possess a normaI Iength flagellum yet dispiay slightly

reduced motility (Guerry et al. 1991).

3 3 FlageHum as an Aàhesin

In addition to the motility imparted by the flageiium, roles in both bacterial

adherence and invasion also have been suggested Studies in vivo, using infant macaques,

found that the C. jejuni flageilum mediates adherence of the bacterium to the micmvilli of

the intestinal epithelium (Russell et al. 1993). Studïes by Neweil et al. (1985) employed

an infant mouse mode1 to show that a non-motile flagellaid mutant that is geneticdy

undefined colonizes as efficiently as wild-type C. jejuni. The mutant strain also exhibited

increased attachment to INT407 and HeLa ceils but not to HEp-2 cells in tissue culture.

In a centrifugation-assisted adherence assay, wild-type C. jejmi bound INT407 cell

monolayers using the tip of the flagellum as shown by scanning electron microscopy

(Konkel et al. 1992b). However, it is possible that the centrifbgation of the bacteria ont0

the ce11 rnonolayer may induce an artificial interaction between the bacterium and the host

celi surface. Nevertheless, collectively these findings suggest that a flagellar adhesin is

present in C. jejuni (Newell et al. 1985).

Studies utilizing C. jejuni strains specificaliy mutagcnized at either flagebn loci

have yielded contradictory results. Grant et al. (1993) did not observe a ciifference in the

Ievels of adherence to INT407 cell monolayen between wild-type. RaA- FiaB* and

FlaA'FlaB- C. jejuni. In addition. Wassenaar et al. (1991) found that the addition of

excess pwified flagella did not inhibit subsequent adherence of C. jejuni to INT407 ceil

monolayers. In contrast, Yao et ai. (1994) found that a FiaAaA FlaB+ mutant exhibited a

50-fold reduction in adherena to INT4û7 ceils dative to wild-type. Thus the possibility

that the FlaA subunit is capable of mediating adherence between the bacteriun and the

eukaryotic ce11 remains controversial.

3.4 Role of Motility in Invasion

More conclusive data demonstrate that motiiity is requinxi for maximal

intemalization of C. jejmi into epithelial cells. Studies by several groups have shown

that both non-motile, flageilated (FlaA- FlaB*) and aflagellated (FlaA- FlaB? mutants are

less invasive relative to wild-type C. jejuni (Wassenaar et al. 1991; Grant et al. 1993). In

addition, the mutant strains are unable to translocate across polarized Caco-2 ceil

monolayers (Grant et al. 1993). Taken together, these resdts indicate that the presence of

a flageliurn contaùring FlaA is essential for efficient invasion and translocation across

eukaryotic cell monolayers in tissue culture. Yao et al. (1994) characterized a C. jejuni

pflA mutant which is non-motile due to a paralyzed fiageilum but is HaA* FI@.

Interestingly, this mutant cannot inva& XNT'407 cells implying that active motility, rather

than simply the presence of a flagellum containing FlaA, is required for the process of

invasion.

35 Adherence

Microbial adhesion to the gastrointestinal mucosa is viewed as a critical step in

the development of diarrheal disease (Fïniay and Falkow, 1997). Attachment to the host

ceiI surface is mediated by the specific interaction between a bacterid adhesin and its

cognate eukaryotic receptor. Therefore, the presence and distribution of the host receptor

dictates the susceptibility to infcction and tissue tropism foc the pathogen (St. Geme,

1997). Studies by Fauchere et ai. (1986) suggest a connection between in vitro adherence

and disease presentation in humans. C. jejuni clinical isolates fimm patients with severe

disease syrnptoms are more adherent to HeLa celi monolayers than strains isolated either

from asymptomatic individuals or fiom patients with mild diarrhea. While the flagella

and various outer membrane proteins have been proposed as adherence factors, there is a

continuing search for C. jejuni adhesins and the corresponding eukaryotic receptors which

are required during infection.

PEB 1 as an Adhesin

PEBl was originally identified by Fauchere et ai. (1989) as a component of a C.

jejuni giycine-extract that is able to bùid HeLa ce11 monolayers. The pebla locus is

conserved in both C. jejuni and C. coli and shows significant homology to the binduig

component of bacterial ABC transport systems (Pei et al. 1993). Subsequent

characterization revealed that PEB 1 is locaiized to the bactena1 surface and is an

antigenic outer membrane protein recognized by convalescent smun obtained h m both

C. jejuni- and C. coli- infccted patients (Pei et al. 1991; KerveUa et al. 1993).

An isogenic PEB 1 mutant strain displays reduced adherence to HeLa ce11

monolayers (50-100 fold reduction relative to wild type) and a 15-fold reduction in

internalization using INT407 cells (Pei et al. 1998). In addition, adherence of C. jejuni to

HeLa ce11 monolayers is inhibited by the presence of anti-PEB 1 &bodies or purifieci

PEB 1 protein in a dose dependent mamer (Kervelia et al. 1993). Thus, the in vitro &ta

suggests that PEI3 1 plays a role in mediating the interaction between C. jejuni and

eukaryotic celfs. Studies using in vivo models of infection have shown that a PEBl

isogenic mutant is less efficient in colonking the gastrointestinal tract of BALBc mice

but it did not exhibit reduced infectivity using the chick mode1 of colonization (Pei et al.

1998; Meinersmann et al. 19%). It is likely that other bacterial adhesins, in addition to

PEB 1, are involved in adhesion to the intestinal mucosa since disruption of the pebla

gene did nat abolish adherence to HeLa ce11 monolayen (Pei et al. 1998).

Other Potential Adherence Factors

Preliminary studies have identified an open reading frame termed P95, as a

potential adhesin in C. jejmi (Kelle et ai. 1998). The gene was identified by Southem

hybridization of C. jejuni genomic DNA using an oligonucleotide probe encoding a

conserved motif present in adhesins from enterotoxigenic and uropathogenic E.coli

(Dadeuille-Michaud et al. 1986; Hoschutzky et al. 1989). Genomic DNA from C. jejuni

strains with high levels of adherence to eumot ic cells in vitro is more likely to

hybridize the oligonuclwtide probe than non-adheent strains. The corresponding DNA

fragment was cloned and shown to have homology to adhesins h m Haentophilus

influenzae involved in epithelial celi attachment (Kelle et al. 1998). Further

characterization of P9S and its protein product are needed to c0dk.n a mle in C. jejmi

adherence and in disease pathogenesis.

The 43kDa major outer membrane protein (MOMP) of C. jejuni has been

proposed as an adhesin capable of binding h t h to immobilized fibmnectin and to

INT407 cell membranes (Scbroder and Moser, 1997; Moser et al, 1997)- The binding

characteristics of the MOMP were assessed using an enzyme-lùiked immunosorbent

assay (ELISA) which involves coating the weIIs of a microtitcr plate with cither

fibronectin or a membrane fraction from an INT407 ce11 monolayer followed by the

addition of purified MOMP isolateci fkom native polyacrylamide gels. The presence of

excess MOMP partiaily inhibits (1520% reduction) subsequent binding of C. jejuni to

the INT407 membrane fiaction (Moser et ai. 1997). Concems mise with these results as

to whether binding of the MOMP to fibronectin and INT407 ceiis is a specific interaction

which can be competitively inhibited and whether the use of an INTW ce11 membrane

Eraction is t d y representative of the interaction between bacteria and a ceIl monolayer.

Further confimation of the MOMP as an adhesin requires the cornparison of the binding

abilities of an isogenic mutant and the wild-type to INT407 ce11 monolayen and

fibronec tin .

3.6 Pilus Production in C. jejuni

A variety of bacterial pathogens proàuce fine haïr-like appendages which extend

out from the micmbial surface. These pili, or fmbriae, have been implicated in

mediating such divene functions as adherence to host surfaces, twitching motility and

cell-ce11 interactions (St Geme, 1997). Doig et al. (1996) found that the presence of bile

salts induces the production of pili in C. jejuni. The peritrichous pili are 4-7 nm in

diameter, extend beyond lpm in length and fiequently form bundles Ieading to an

aggregative colony morphology. Since the piiin subunit has not been describecl, the C.

jejuni pilus cannot be classifiecl into one of the four known classes (Soto and Hultgren,

1999). However, the authors note that the bundle formation is a characteristic of the type

N pili expressed by several Gram negative pathogens @oig et al. 19%). Examples of

the type N pili include the bundle forming pilus of cnttropathogenic E.:colr' and the toxin

coregulated pilus of Vibrio cholerae, both of which are colonization factors subject to

environmental regulation and are requked for disease productionf (Bieber et al. 1998;

Hemngton et al. 1988).

Disruption of the C. jejuni pspA @ilus gynthesis ~rotease) gene ~wults in f d y

motile yet non-piliated phenotype in the presence of bile salts @oig et al. 19%). The

pspA gene shows significant sequence homology to proteases found in E. coli and,

therefore, could be involved in protein pcocessing events required for pilus production.

However, its precise role in pilus production has not ken identified (Doig et al. 1996).

Isogenic pspA mutants exhibit wild-type levels of adherence and invasion using INT407

ce11 monolayers. Studies using various animal models have show thaî pilus production

is not required for colonization in ferrets, mice or rabbits using the RITARD mode1 (Doig

et al. 1996). However, disease production in ferrets, including mucoid stools and

dehydration, was attenuated in the animals infected with the pspA mutant (Doig et al.

1996). Therefore, while C. jejuni pili do not appear to be involveci in epithelial cell

attachment, this does not preclude a separate role iq disease pathogencsis. For instance,

production of a pilus adhesin when the bactexïum encounters bile salts in the lumen of the

small bowel could aid in establishing later stages of infection. Characterkation of the

pilin subunit and its host rcceptor should aid in delineating whether the pilus is involved

in C. jejuni virulence.

3.7 C. jejuni Adherence to Fibmnecün

Binding to fibronectin is considerrd to be an important vinilence mechanism for a

variety of pathogens (Wcstcrlund and Korhonen, 1993). Following damape to the host

epithelium, the underlying extracellular matrix proteins may become exposed and serve

as sites for bacterial attachment (Westerlund and Korhonen, 1993). C. jejuni binding to

fibronectin, as well as other components of the extraceilular ma&, has been reported

previously (Kuusela et al. 1989). However, a bacterial factor responsible for fibronectin

binding has only recently been described. Konkel et al. (1997) identified the CadF

(Compylobacter gdhesion to Bbmnectin) protein as the only component within a C. jejuni

outer membrane protein extract which binds specificaiiy to radiolabellecl fibmnectin and

can be competitively infübited by the presence of excess unlabelleci fibronectin. The

corresponding open reading fiame (ORF) was isolated from a C. jejuni genomic

expression library. Isogenic CadF mutants are markedly reûuced in their ability to bind

fibronectin (Konkel et al. 1997). Further characterization of the interaction between the

CadF mutant and eukaryotic cells and the vinilence of the mutant within an in vivo mode1

system would contribute much information about the pathogenesis of C. jejuni.

3.8 Toxin Production

While the disease manifestations of C. jejuni infeçtion are consistent with the

possibility of enterotoxin production (in cases of watery diarrhea) or cytotoxin production

(in the dysentery-iike syndrome seen in inflamrnatory colitis) there has been very little

success in identifying the encoding genes. The~fore, while C. jejuni has been proposed to

elaborate numemus toxins only one, the cytolethd distending toxin (CDT), has been

confmed at the genetic level.

Enterotoxin

The secretory fonn of C. jejuni-induced diarrhea has long been considered to be

caused by an enterotoxin. In the past, much reseanih was focused on a proposed C. jejuni

enterotoxin which is both fùnctiondy and immunologic~y similar to the cholera toxin

(Cï) of Vibrio cholerae and the heat-labile toxin (LX') of Escherichia coli Yet despite

the considerable phenotypic evidence. there has been no confinnation of an enterotoxin at

the genetic level (Ruiz-Palacios et al. 1983; Klipstein and Engm 1985a; Olsvüc et al.

1984).

Both Cl' and LT are A-B subunit toxins capable of disrupting ion transport in the

intestinal epitheliurn and ultimately cause excess fluid secretion (Kaper et al. 1995;

Rabinowitz and Donnenberg, 19%). The B subunit fanns a pentamer and is responsible

for binding to the cell surface GMI ganglioside receptor. Following intemalization and

proteolytic cleavage of the enzymatic A subunit, the a subunit of the Gs protein is ADP

ribosylated causing activation of adenylate cyclase. This increased adenylate cyclase

activity leads to elevated levels of intraceliular cyck AMP (CAMP) and protein kinase A,

changes in ion flux due to the opening of the CE.TR chloride channel and, ultimately, to

the watery fonn of d i h e a (Kaper et al. 1995).

Enterotoxin activity in C. jejuni was demonstrated using a variety of in vitro

assays. C. jejuni culture supernatants cause elongation of Chinese hamster ovary (CHO)

cells with increased levels of intracellular CAMP and rounding of Y4 mouse adrenal

tumour ceUs (Ruiz-Paiacios et al. 1 983; Johnson and Lior, 1984). Studies using animal

models of enterotoxigenic activity have shown that concentrateci culture supernatants

cause fluid accumulation in the rat, but not the rabbit, ileal ligated Ioop (Ruiz-Palacios et

ai. 1983).

Evidence of a close immunological sirnilarity between the C. jejuni entmtoxin

and CT and LT was suggested since the heat-labile enterotoxin can be detected by

immunoassay using anti-LT sera (Klipstein and Engert, 198Sa). In addition, the cytotoxic

activity in CHO ceIls and the induced secretion in rat ileal loops is inhibited by the

presence of CT and LT immune sera (Ruiz-Palacios et al. 1983; KLipstein and Engert,

1984).

Daikoku et al. (1990) used a combination of ammonium sulphate precipitation,

gel filtration and affinity chromatography as a means to p w the enterotoxin h m

culture supernatants. Using an anti-Cï IgG affinity column led to the isolation of a single

68kDa band on SDS-PAGE while a ganglioside affinity column yielded two bands

(68kDa, 54kDa). Based on these observations, it was suggested that the C. jejuni

holotoxin was compriscd of multiple subunits. Unfortunately, the enterotoxigenic

activity of these purifieci proteins was not tested. Despite the in vitro and in vivo evidence

suggesting enterotoxin production in C. jejuni, attempts to identiry the correspondhg

open reading frames have been unsuccessful. Using Southern hybridization anaiysis,

DNA probes specific for the A and B subunits of CT and LT fail to hybridize genomic

DNA h m C. jejuni strains s h o w to possess enterotoxigenic activity (Olsvik et al. 1984).

In addition, attempts to amplify the toxin-coding region using the polymerase chah

reaction (PCR) with degenerate primers have not ied to the identification of an

enterotoxin gene (Konkel et al. 19924). Thercforc, whethcr C. jejmi disease

pathogenesis involves the elabration of an entemtoxin remains controvesid.

Cvtotoxin

Examples of cytotoxin production in other bacterial enteropathogens include the

S higa toxin (Stx) produced by Shigella dysenteriae type 1 and the related Shiga-like toxin

produced by enterohemorrhagic E. coli both of which lead to host ce11 death by blocking

protein synthesis (Finlay and Falkow, 1997). The Clostridium dificile toxins A and B

cause disxuption of the actin cytoskeleton (Finlay and Fallcow, 1997). A number of

studies suggest that C. jejruri culture supernatants also are cytopathic for a variety of

eukaryotic ce11 Iines (Wassenaar, 1997a). It appears that a 70kDa cytotoxin is active

against HeLa, CHO, HEp-2 and INT4û7 ceiis but is inactive against Vero ceils (Goossens

et al. 1985; Johnson and fior, 1986; Guerrant et al. 1987; McCardell et al. 1986). In

addition, the cytopathic effects cannot be inhibited by anti-Clostn'dium or anti-Sa

immune sera (Goosens et ai. 1985; Guerrant et al. 1987). There is also a reported

cytotoxin with activity toward Vem cells but this activity also cannot be inhibited by

immune sera against either S a or the C2ostridial toxins (Klipstein et al. 1985b; Johnson

and Lior, 1986). The genes encoding the cytotoxic activities have not been identified;

therefore, the precise role of cytotoxins during C. jejuni infection remains undefineci.

Cytolethal distending toxin

In 1996, Pickett et al. described the hrst cloning and characterization of a toxin

gene from C. jejuni. The cytolethai distending toxin (CDT), originally identified by

Johnson and Lior (1988), induces elmgation and a graduai distension of HeLa ceiis ovcr

a period of two to four days, after which the cells eventually die. Pickett et al. (19%)

used PCR with degenerate oligonucleotide primers based on CDT sequences fkom E. coli

snains to ampli@ the cdtB gene. The resulting amplicon was cloned, sequenced and used

to probe C. jejuni restriction DNA fragments. This led to the i&ntïfication of three

adjacent genes (CU, cdrB. cdC) which are sufficient for CDT expression. In a

subsequent report, the C. jejmi CDT was found to cause an amst in the G2 phase of the

ce11 cycle in bath HeLa and Caco-2 cells (Whitehouse et al. 1998). The arrest is iinked to

the accumulation of the inactive tyrosine phosphorylated form of the catalytic subunit of

the c yclin dependent kinase (CDC2) which is r e q u i . for entry into the M phase of the

ceIl cycle (Whïtehouse et al. 1998). The authon proposed that CDT activity against the

intestinal crypt ceils may prevent their maturation into villus epithelial cells thereby

damaging the intestinal epithelium and perhaps interferhg with the absorptive functions

of the intestine ieading to dianheal symptorns (Whïtehouse et al. 1998). Whether the

eventuai ceil death observed in vitro occurs via an apoptotic or necrotic mechanism has

not been determineci. However, this could have implications with regards to the role of

the CDT in disease pathogenesis. Interestingly, the CdtB protein from Actinobacillur

aciinomycetemcomitmi was recently shown to have p a t e r activity against T cells than

HeLa cells in inducing a G2 phase ams t (Shenker et al. 1999). The authors suggested

that CDT may serve, in fact, to impair the host immune response during infection thereby

aiding in microbial survival. Whether the C. jejmi CdîB protein. which exhibits 60%

homology to the A. acitnomycetemomitmrs CtdB protein, has a simiiar h c t i o n is

unknown.

3.9 Invasion of C. jejuni

The presence of b l d and 1eukocytes in the stools and evidence of mucosal

ulceration in rectal biopsy sarnples h m C. jejim-infécted patients are indicative of an

acute inflammatory coiitis which could result from bacterial invasion (Dm et al. 1980;

Blaser et al. 1980). hacellular organisms have been o b s e ~ e d by transmission electron

microscopy in biopsy samples from C. jejmi-infeaed individuals presenting with colitis

thereby lending support to the view that C. jejwi invasion is important in mediating

disease (Van S preeuwel et al. 1985). Furthemore, studies using animal models of

infection dso indicate an invasive mechanism for C. jejuni pathogenesis. Bacteriai

invasion is observed by transmission electmn microscopy in colonic epithelial ceils of C.

jejuni-infected infant monkeys and in the epithelial ceils of the large intestine in infecteci

newbom piglets (Russell et al. 1993; Babakhani et al. 1993).

Si mal transduction reswnses to invasion of C. ieiuni

While C. jejmi invasion into the ceils lining the intestine has ban proposeci as

part of the M e n c e strategy for this pathogen, very liale is known about the eukaryotic

ce11 signalling factors involved in mediating bacteriai uptake (Ketley, 1997; Konkel and

Cieplak, 1996). Other invasive pathogens such as Salmonella spp. and Shigella spp. are

able ta induce their uptake into epithelial cells foliowing polymerization of the host F-

actin cytoskeleton (Adam et al. 1995; Finlay et al. 199 1). Similar studies in C. jejuni

have been inconclusive in determining the involvement of host cytoskeleton

rearrangements in bacterial intenialization. Biochemical inhibitors, such as cytochalasin

D, which disrupts cellular mimfilaments, and colchicine, which inhibits microtubule

polymerization, Vary in their abilities to block invasion of C. jejmi depending on the

bacteriai strain and the tissue culture cell line employed (Konkel et al. 1992~;

Oelschlaeger et al. 1993; Russell et al. 1994). Receptor-mediated endocytosis has also

been proposed as a mechanism utilized by C. jejuni to invade host ceils. However, the

observation appears to be cell line specific since compounds which inhibit the formation

of clathrin-coated pits (g-strophantin and monodansylcadaverine) reduce the invasion of

C. jejuni into INT407 cells but not Caco-2 ceils (Oelschlaeger et al. 1993 ; Russell et al.

1994).

Recently, Wooldridge et al. (1996) proposed a novel mechanism for invasion of

C. jejuni involving the host ceil caveolae system. Pretreatment of Caco-2 cells with

filipin III inhibits the uptake of C. jejuni in a dose dependent manner. FiIipin III causes

the disruption of caveolae by sequestering cholesterol present in the plasma membrane

(S haul et al. 1998). The potential involvement of caveolae in C. jejuni entry is intriguing

since these small invaginations in the plasma membrane are highly enriched in a variety

of ce11 signalling molecules such as tyrosine kinases, G proteins, and lipid signalling

molecules (Shaul et al. 1998). Studies in othcr invasive enteropathogens have implicated

both tyrosine phosphorylation and activation of the lipid kinase phosphoinosi'tïde (PI) -3-

kinase as precursors to cytoskeletal rearrangement (Gossart, 1997; Ireton et al. 1996).

Future studies aimed at detemiinhg whether signal transduction responses originating

from caveolae are involved in C. jejuni uptake will greatly contribute to our

understanding of the host ce11 response to infection.

In response to the induced uptake of invasive pathogens into the intestinal

epithelium, the Uifected cell is capable of alertiag the host to the infection through

activation of host infiammatory and immune responses (Kagnoff and Eckmann. 1997).

Secretion of interleukin-8 (IL-8) by epithelial celis has been proposed as an initiating

factor in the inflammatory msponse to infection (Wïïson et al. 1998). IL-8 is a

proinfiammatory chemokine w hich acts as a c hemoattractant for p l ymorphonuclear cells

(PMN) (Eckmann et al. 1993). IL-8 release from various eukaryotic cell lines has been

associated with both bacterial invasion (Salmonella dublin, Yersinia enferocolirico,

Shigella dysentenae, Lisredu mmcytogenes) and in response to bacterial adherence

(enteroaggregative E. coli, Helicobocter pylon) (Eckmann et al. 1993; Crowe et al. 1995;

Sharma et al. 1995; Steiner et al. 1998).

Hickey et al. (1999) found that clinical isolates of C. jejmà induce the secretion of

interleukin-8 (IL-8) from INT4û7 cells. The cytokine release requires both & novo

bacterial protein synthesis and bactena-host ceU interactions. Clinicai isolates with

higher levels of invasion in vitro cause greater IL8 secretion than l a s invasive strains.

In addition, defned isogenic mutants which exhibit a rcduced adherence and invasion

also induce lower levels of IL8 secretion (Hickey et ai. 1999). The authors proposed

that IL-8 secretion is more Uely to depend upon C. jejuni invasion rather than adherence

since mutants defective in invasion but ody slightiy less adherent c a w lower levels of

IL-8 secretion (Hickey et al. 1999).

Bac tend factors involved in tissue invasion

Numerous studies have shown that de novo bacterid protein synthesis is necessary

for maximal invasion by C. jefini, since bacterial cultures treated with chloramphenicol

are markedly reduced in their ability to invade IN'I'407 ceil monolayers and translocate

across polarized Caco-2 ceU monolayers (Konkel et al. 1992e; Russell et al. 1993).

Furthermore, interaction between C. jejuni and eukaryotic ceils induces the expression of

novel proteins in C. jejuni which are required for maximal intenialization (Konkel and

Cieplak, 1992a; Panigrahi et ai. 1992). Using metaboiically labeiled C. jejuni, Konkel et

al. (1993) showed that following CO-cultivation with INT407 monolayers, both ce&

associated bacteria and organisms present in the overlying culture medium had newly

expresse4 or enhancexi expression of forneen different proteins compared to C. jejuni

grown in broth culture alone. Antiserum raised against C. jejuni which has been cultureci

in the presence of INT4û7 cells is able to inhibit C. jejuni invasion in a dose dependent

manner. The binding of C. jejuni to INT407 cells in the presence of the antisenim is not

affected. In contrast, immune semm raised agaînst C. jejuni p w n in cuinire alone

exhibits no effect on bacterial interactions with host cells (Konkel et al. 1993).

Recently, Konkel et al. (1999b) have shown that culturing C. jejuni in INT407-

conditioned medium not only induces the synthesis of novel bacterial proteins but also

leads to the secretion of eight C. jejzuzi proteins into the culture supernatant. Screening of

a C. jejuni genomic expression library with the immune serum raised against C. jejmi

cultured with INT407 ceils led to the identification of a novel open reading frame

required for bactenal invasion (Konkel et al. 1999b). The gene termeci ciaB

(Campylobacter @vasion Mtigen) encodes a protein of 610 amino acids and a culB nuli

mutant exhibits both reduced invasion into DIT407 cells and a secretion-defective

phenotype (Konkel et al. 1999b). Using immunofluorescence and confocai scannùig

microscopy, anti-CiaB senim showed intense staining of the cytoplasm of INT4û7 ceiis

infected with C. jejuni. This finding suggests that CiaB is WreIy to be a bactenal effectm

which is translocated into the host ceU cytoplasm (Konkel et al. 1999b). CiaB exhibits

amino acid sirnilarity (45.3%) to the P50 adhesin fiom Mycoplarnia homini's which is

localized to the bacterial surface and mediates adheence to HeLa ceU monolayers in vùro

(Henrich et al. 1993).

Xnterestingly, CiaB is also sirnilar in sequence to various proteins exporteci by the

type III secretion system, namely to the Shigella IpaB (40.6%), Salmonella SipB (45.0%)

and Yersinia YopB (45.4%) (Konkel et al. 1999b). The type III secretion system found in

several Gram-negative pathogens is used to deliver bacterial virulence factors into the

surrounding medium or directly into the eukaryotic cytosol (Hueck, 1998; Lee and

Schneewind, 1999). The type III secretion system is believed to have evolved h m the

flagellar apparatus and is usuaiiy encoded by a cluster of genes found on virulence

plasmids or within pathogenicity islands, indicative of acquisition following horizontal

transfer (Hueck, 1998; Young et al. 1999). Both IpaB fiam Shigella spp. and SipB fkom

Salmonella spp. are required for bacteriai invasion into epithelial cells and are capable of

inducing apoptosis in macrophages through the binding and activation Caspase-1 (Hash

et al. 1999; High et al. 1992; Hilbi et al. 1998; Hueck et al. 1995; Kaniga et al. 1995). In

contrast, the d e of the YopB protein in the pathogenesis of Yersinia spp. remains

controversial. Previously, evidence suggested that YopB functioned in concert with

YopD, to induce the formation of small pores within the eukaryotic plasma membrane

thereby facilitating the translocation of other effector Yops into the target cell (Cornelis et

al. 1998). However, a ment report suggests that YopB is se~eted into the surrounding

medium and is not essentid for the injection of effector Yops into the host cytosol (Lee

and Schneewina 1999). At prescrit, a type III secretion system has not been identifîed

within C. jejmi. Aithough the Cid3 protein is secret& following interaction with host

cells and appears to be translocated into the host cytosol, its precise role in bacterial

uptake needs to be defined

3.10 Intracellular Survivai of C. jejuni

Two studies have assessed the ability of C. jejuni to survive intracellularly within

epithelial cells by using the gentamicin protection assay. De Me10 et al. (1989) found that

C. jejuni is rapidly killed upon intemalization into HEp-2 ceii monolayers (within 36 hrs)

and similar results were obtained using DIT407 cell monolayers (Konkel et al. 199%).

However, these experiments were conducted using gentamicin throughout the entire assay

period. Konkel et al. (1992) pmposed that the viability of the intemalized organisms

may be affected due to a low level of gentarnicin penetration into the eukaryotic ceUs.

When gentamicin was removed h m the assay medium following 18 hrs of infection, the

number of intemalized bactexia inmaseci at 60 hrs of infection.

Tmsmission electron microscopy indicates that C. jejuni is present within

individual vacuoles of infecteci tissue culhm cells. Beyond 72 hrs, cytopathic effects and

a decline in the viability of the ce11 monolayer are evident (Konkel et al. 1992~). These

studies indicate that C. jejunii is capable of swiving within epitheliai ceUs and that C.

jejuni invasion is Wrely to be of biological significance during infection.

The ability of C. j e j d to resist killing following macrophage phagocytosis has

also been investigated. While an initial study reported that C. jejuni is able to survive

within human-derived macrophages for up to six days, subsequent studies could not

confim these results (Kïehibauch et al. 1985). Wassenaar et al- (1997b) noted that the

contrasting results may weli be due to ciifferences in experimental methodology. Firstly,

the human monocytes employed by Kiehlbauch et al. (1985) were not treated with

cytokines to induce theu differentiation into macrophages. Without cytokine treatment,

monocytes maintained in culture soon die via apoptosis (Mangan et al. 1991). Therefom,

the ability of C. jejuni to proliferate within these cells lïkely is not a reflection of bacterial

virulence. Secondly, surviving intracellular organisms were separated h m the adherent

and unphagocytosed bacteria by extensive washing of the monolayer. Therefore, it is

possible that bacteria recovered during the expriment were, in part, organisms which had

proliferated outside of the monocytes. A later study by Wassenaar et al. (1997b)

employed cytokine-stimulated human monocytes in a gentamicin protection assay. Under

these conditions, al l isolates of C. jejuni tested were rapidiy lcicilled upon phagocytic

uptake, with no suntiving organisms present following 48 hr of infection. These resuits

suggest that rapid killing of C. jejuni by macrophages, coupled with the bactericidal

activity of host serum components and polymorphonuclear leukocytes, is iikely to provide

an effective barrier to prevent C. jejmi nom reaching the bloodstreaxn or microbial

proliferation in the lamina propria of the gut.

3.1 1 Regulation of virulence factors

Colonization and infection of the gastrointestinal tract is likely to depend upon the

ability of enteropathogens to rapidly adapt to the environmental changes encountered

upon entering the hosto Certain environmental cues such as changes in ambient

temperature, pH and iron levels are involved in the regdation of bacterial vidence genes

thus ensuring that the appropriate virulence factors are expresseci at each stage of the

infection cycle (Guuiey, 1997). In C. jejuni, research has focused on two separate types

of global regdators; namely, the Fur response to intracellular iron levels and a mcently

described temperature-regulated two component regdatory system,

bon-remdation in C, ieiuni

Iron is a key p w t h requirement for most organisms due its widespread

involvement in various cellular functions. For example, imn is essential for the activity

of enzymes involved in oxygen metabolism and electron transport processes (Litwin and

Calderwood, 1993). Within the mammalian hosf the availability of free iron is extremely

Iimited due to the presence of high affinity femc iron binding proteins located both

intracelluiarly (heme, hemoglobin, ferritin) and extracellularly (lactoferrin, transfemn)

(Litwin and Caldefwood, 1993; Aisen and Leibman, 1972). In response, pathogenic

bacteria have evolved strategies to obtain enough iron h m the host for sunival. Some

bacterial species secrete siderophores to compete wïth host hn-binding proteins or

reductants to scavenge iron away fmm the iron-protein complexes of the host

(Wooldndge and Williams, 1993). Oüier species direcly utilize the iron complexed to

host iron-binding proteins rather than competing with the host for iron (Wooldridge and

Williams, 1993). While C. jejwii does not produce siderophores, the aganism may be

capable of obtaining iron by scavenging exogenous siderophores and by utilizing iron

bound to heme and hemoglobh wetley, 1997).

The transition from an iron-rich to an iron-limited environment semes as a cue to

the invading pathogen of its entry into the host (Mekalanos, 1992). Indeed, reduced iron

concentrations upregulate the transcription of genes involved in iron acquisition and also

the expression of certain vinilence determinants (Bjom et al. 1979; Litwi-n and

Calderwood, 1993; Calderwood and Mekalanos, 1987). Iron-responsive gene regdation

c m be mediated by thefit Cfemc wtake gegulator) locus. Fur is a DNA-bïnding protein

that represses transcription by binding to specific operator sites near the promoter region

of iron-regulated genes. The repressor activity of the Fur dimer requires the presence of

ferrous iron as a cofactor. Therefore, it is only under iron-depleted condition that Fur-

regulated genes are derepressed, to allow their expression.

A Fur homolog is present in C. jejuni (Chan et al. 1995; Wwldndge et al. 1994).

Iron-regulated proteins have been identified through the cornparison of protein profiles of

wild type and fur-mutant C. jejuni (van Vliet et al. 1998). This method hss identified

components of an enterochelin uptake system and a femc siderophore receptor.

Unfortunately, novel iron-regulated proteins with potential d e s in vinilence are still to

be identified. Screening methods used to da&, comparing Coomassie blue-stained one-

and two-dimensional polyacrylamide gels may not be sufficiently sensitive to recognize

minor differences in protein expression. Therefore, further study employing more

sensitive techniques, such as differentid display (Liang and Pardee, 1992). should aid in

the search for iron-regulated virulence determinants of C. jejmi.

Two comwnent regulatow svstem in C. ieiuni

Two-component regulatory systems are global regulators of gene transcription

commonly used by bacteria as a means to detect and to respond to environmental signals

(Miller J I . et al. 1989). This regdatory network consists of two proteins, a 'sensor'

histidine kinase and a response regulator. Upon detection of the appropziate signal, the

sensor protein is autophosphorylated at a histidine midue. The phosphate group is

subsequently transferred to an aspartate residpe wi- the msponse regdatoc

Phosphorylation of the response teguiator enables the transcriptional activation or

repression of the target genes (Miller, J.F. et al. 1989).

Regulation of bacterial vinilence factors by two-component systems has bten

demonstrated in severai organisms (Miiier J.F. et al. 1989; Arico et al. 1989; Miller, SJ.

et al. 1989). The PhoP/PhoQ regulon of Salmunellu, tesponds to the extracellular

concentration of M ~ ~ * and ca2+ to control the transcription of genes required for entry

into nonphagocytic celis, intraceiiuiar survival within macrophages, and resistance to host

antimicrobiai peptides (Miiler, SI. et al. 1989; Groisman 1998). Similarly, the

BvgWBvgA locus of Bordetella p e m s i s controfs the expression of adhesins and toxins

required for establishing an infection (Axico et al. 1989).

Since C. jejuni is likely to encounter a variety of ecological niches during both

transmission to and infection of the human host, the coordinate regdation of the

expression of vinilence deteminants would be an advantage for this pathogen. Using

degenerate primers, Bras et al. (1999) amplifiad and clonecl a sensor protein and response

regulator termed RacS (piuced gbility to olonize gensor) and RacR Creduced gbility to

olonize ~sponse), respectively. An isogenic mutant has reduced ability to grow at 42OC,

which is the optimal growth temperature for wïld-type C. jejuni. Since thïs temperature

coincides wi th the approximate core temperature of birds, Bras et al. (1999) proposed that

the two-component regulatory systcm is required for the colonization of chickens.

Indeed, the RacR/RacS mutant has a reduced ability to colonize chicks, suggesting that

enhanced growth at 42OC is a factor in the effective colonization of the main reservair for

C. jejuni (Bras et ai. 1999; KonkeI and Cieplak, 19%). Comparing the protein profiles of

the wild-type and the RacWRacS mutant grown at both 37OC and 42OC, showed that

RacR acts as a repressor and activator of several genes, includuig some genes which are

thermoregulated (Bras et al. 1999). One of the Rac-regulated genes identifieci was Dnal

which has previously been shown to be upregulated at 42OC and required for colonization

of the avian intestine (Konkel et al. 1998). Further characterization of other Rac-

regulated genes will likely identifv factors not only requùed for colonization of chickens

but may also reveal gens necessary for the successful transmission of C. jejuni from the

poultry reservoir to the hwnan host. Currently, two-component regulatory systems are

being examined as targets for therapeutic intervention since the disruption of a giobal

regulatory network could potentially render a pathogen avident (Bmtt et ai. 1998).

Chapter 4

Proj ect Rationale

4.1 Introduction

Previous work in the Iaboratory of Dr. V.L. Chan has focused on the genomic

mapping of C. jejuni TGH9011 using pulsed-field gel electrophoresis (PFGE). Using

restriction enzymes which cut infkequently in the C. jejuni genome a genetic map was

consmicted and genetic markers were locaiized on the rnap (Kim et al. 1992; Kim et al.

1993). Based upon the size (1.8 Mbp) and the G+C content (appximateIy 30%) of the

C. jejmi strain TGH9ûI 1 grnome, the Sol 1 restriction enzyme with a recognition site of

GTCGAC, would be predicted to cleave at approximately 110 sites throughout the

genome (McClelland et al. 1987). Interestingly, there are oniy 5 6 Sa11 restriction sites

identified within the genomes of the various C. jejuni strains examineci (Chang and

Taylor 1990; Kim et al. 1992; Nuijten et al. 1990). In TGH9011, three of the six sites are

found within the highly consenmi 23s rRNA genes (Kim et al. 1993). It was of interest

to identify the open reading frames harbouring the remaining Sul 1 sites, as they are also

Iikely to encode other conserved sequences.

Screening of a genomic library of C. jejuni TGH9ûlL identifieci the pE3-8 clone

which harboured a 7.5kb insert containing a previously uncharacterized Sa1 1 restriction

site. Partial sequencing of the insert indicated that the restriction enzyme recognition site

was contained within an open reading frame of 1392 nt.. which wouid later be designated

as cipA. Mapping studies concluded that the cipA gene was present on the Sal 1-F

fragment on the genomic map of C. jejuni TGH9û11 (Chan et al. 1998) (Figure 2).

Analysis of the nucleotide and prrdicted amino acid sequence of cipA using the

BLAST databank did not reveal simcant homology to other known sequences (h et

al. 1998). Examination of the prrdicted amino acid sequence did not indicate the

Figure 2: The physical map of C. jejuni T G H . 1 1

The restriction map was generated using the Sac II, Sul 1 and Sma 1 restriction enzymes

and PFGE separation of the DNA fragments. The cipA locus was mapped to the genome

by Southem blot hybridization of the Sui 1 restriction fragments using a c@A-specific

DNA probe. The c@A gene mapped to the junction of the Sal 1-F and Sul 1-D tÏagments.

Adapted hm: Chan et al. 1998

presence of a N-tenninal signal sequence nor were any conserved amino acid motifs

present using the pSort and pFam aigorithrns, Using Southern hybridization andysis, it

was found that the c@A gene was consemeci within the four strains of C. jejuni tested and

was also present within the type sÉrain of C. coli. However, hybridization to the c@A

probe was not observed in other related organisms such as C. lari, C. upsuiiensis, C.

sputonun subsp. bubulus and Arcokter nirmfigüis (Jœ et al. 1998).

The cipA gene within TGH9û11 was insmionaily disrupted uskg a kanamycin

cassette present on a suicide vector. The muuuit construct, designateci 9OlLKl. was

conf i ied by both PCR and Southern blot andysis (Joe et al. 1998). To investigate

potential functions of cipA, the bacteria-host cell interactions of the 9ûlLK1 strain were

investigated in vitro. Using the gentamicin protection assay, 901LK1 exhibited a reduced

adherence (42.5% & 10.5% relative to wild type) to an )[NT407 cell monolayer. In

addition, internalization of 90lLKl into the eukaryotic cells was also l a s efficient than

wild type (47.5% + 7.7% relative to wiid type) (Joe et al. 1998). Based upon these

results, the c@A gene thus named for Campyfobacter Invasion ghenotype.

4.2 Objectives

To further characterize the role of cipA gene in C. jejuni vinilence, the frrst

objective of this study was to constnict a second mutant strain by deletion-replacement

disruption of the cipA allele. This deletion mutant strain is more suitable for assessing the

mutant phenotype within the in Mvo setting since the absence of antibiotic selective

pressure within an animal mode1 could lead to the reversion the 901LK1 mutant back to

the parental genotype. The deletion mutant would also be utilized to study the effect of a

cipA disruption within in virro models of infection. Using the gentamich protection

assay, the levels of adherence to and invasion into host cells would be assesseci using

polarized and nonpolarized epithelial celi lines.

In order to characteriz. the regulation of the cipA locus at the protein level and to

determine the subcellular localization of the CipA protein within C. jejzuzi, polyclonal

antibodies were generated which recognized the CipA protein. Using the GST-fusion

system the CipA protein was expressed in E. cofi and the purifieci protein was then

utilized to immunize a New Zealand White rabbit, Immunoblot analysis performed by

Dr. A. Joe using subceliular fractions of C. jejuni revealed that the CipA protein was

localized to the cytopiasm of C. jejwri. In addition studies were undertaken to examine

the regulation of CipA expression during the growth curve of C. jejmi and following

interaction with host cells.

Chapter 5

Materials and Methods

Materials and Methods

5.1 Bacterial strains and culture conditions

The bacterial strains and plasmids used in this study are listed in Table 1. C.

jejwti T G W 1 1 (ATCC 4343 1) was routinely grown on Mueller-Hinton (MH) agar, at

37OC in an atmosphere of 5%Co2 -95% air. C. jejmi 81-176 was originally isolated h m

an outbreak of enteritis (Korlath et al.1985) and was kindly provided by Dr. M. Blaser

(Vanderbilt University, Nashville, TN). C. jejunà 81 -1 76 was grown on 5% bIood ag;a

plates in a microaerobic environment. E. coli JMlOl was used as a host for cloning

experiments and was grown on Luxia-Bertani (LB) agar at 37°C. When required, medium

was supplemented with ampicillin (lûûps/mL) or kanamych (1ûûCrglmL).

5.2 Mammalian ceii culture

Stock cultures of INT407 ceils (human embryonic intestine, ATCC CCL 6), HEp

2 (human laryngeal epidermoid carcinoma, ATCC CCL 23) and Caco-2 (human colonic

carcinoma ATCC HTB37) were obtaiaed h m the American Type Culture Collection.

INT 407 and HEp2 cells were p m as monolayers in 25-cm2 tissue culture flasks in

MEM supplemented with 15% Fetal calf senun @CS) (vlv). Cam-2 cells were culhued

using MEM with Earle's salts supplemented with 10% FCS, Lglutamine and non-

essential amino acids. Caco-2 ceUs w m p w n as monolayen in 25-cm2 tissue culhue

flasks or on membrane-filter units (Costar, pore size = 5.0 p. surface area = 0.33 cm2)

placed into the wells of a 24-well tissue culture plate. Each filter unit was seeded with

id Cacw2 cells and cultured using 1mL of medium on the basdateral side of the filter

Table 1: Bacterial saains and plasmids utilized in this study

Bacterial suain or pIacmid Relevant charactcnstics Source

Campylobacter jejuni TGH9011 (ATCC 4343 1) Campylobacter jejuni 90 1LK1

Campylobacrer jejuni 90 1LK.2

Compylobacter jejwi 8 1- 176 (ATCC 55026)

Eschenchia coli m(r10L

Plasmids

PGEX-2T

pBluescript II SK+

pKAN-S phB

Parental strain fm consauction of cipA mutants

c ip~XanR Uistraon mutant of TGH9û11

UtiIizcd as a positive c0~1trol for infection of BALWc mice

Utilized as a host for cloning experhents

wR. KanR. pUC19 vccmr contaïning a C. coli Iranarllycin mistaLlCC cassette

Cc jejwri TGH9û 1 1 chromosomal Library clone. pBluescript II SK+ vscm barbourhg a 7 5 kb insert of C. jejuni DNA which contains the cipA gcrie

Dcnvative of pE3-8 containing the cipA gene within a 2 kb insat of C jejuni DNA

Derivative of p2E3-8. contains the cipA:KanR insatioa constnrt

Mvative of p2E3-8, contains the cipA:KanR deleaion construct

Derivative of pGEXI2T. contains the translational

J L . Pennet

Joe a al. (1998)

This study

K o r M et aï. (1985)

Yanisch-Perron et ai. (L98s)

Joe a al. (1998)

Joe a al. (1998)

J a et al. (1998)

This shidy

This study fusion of GST witb cipA

unit and 0.2mL of medium on the apical side. The medium was changed every 48 hrs and

the polarized monolayers were used after 14-21 days of incubation at which time the

transepithelid resistance was above 200 0hms/cm2.

53 Adherence and invasion assays

Adherence and invasion assays were performed without centrifugation using

INT4û7 (Yao et al. 1994). HEp-2 (Konkei and Joens, 1989) and nonpolarid -0-2

ceiis (Grant et al. 1993). Bnefly, log cfu h m a mid-log phase bactezial culture (optical

density at 6OOnm = 0.5-0.6) was used to infect confluent celi monolayers grown in a 12-

weil tissue culture plate. The infected monolayers w m incubated for 3 houn at 3PC in

an atmosphere of 5%C02 -95% air. Medium containing non-adherent bacteria was then

removed h m each weil, the monolayers were washed three times with Dulbecco's

phosphate buffered saline (PBS) (1mL per wash) foliowed by the addition of frrsh MEM

with or without gentamicin at a concentration of SOpg/mL. Foilowing a M e r three

hou. incubation, the monolayers were washed five rimes with PBS (1mL per wash) with a

final wash using cation-free Dulbecco's PBS. The ceiis were removed h m the weils by

trypsinization (0.05% trypsin-0.53mM EDTA for 5 min at 3PC). The eukaryotic celis

were resuspended in steriie distilled water containing 0.1% bovine senun albumin or

0.1 % Triton X-10, followed by miId vortexing to lyse the ceiis. The released adhcrent

and intemaiïzed bactena were enumrated by plate counting. Adherent bacteria

represented the organisrns enumerated in the absence of antibiotic treatment and was

expressed as a perrentage of the initial inoculum. Invasion levels were expresscd as the

percentage of adherent bactena srrrviving gentamich treatment All assays were

conducted in duplicate and repeated independently three to s u rimes.

5.4 Bacterial transcytosis

Bacterial suspensions (OMmL containhg 10' cfu) w m added to the apical side

of the Caco-2 monolayer to which fksh medium (0.175mL) had been adnpd_ After each

1 .S hr of incubation, the filter units were transftrred to a fresh wefI in a 24-weff tissue

culture plate containing lmlr of prewarmed MEM. The number of translocated bacteria

in the basolateral medium was determined by plate count as described by Konkel et al.

(1992e). The transepithelia1 resistance of the Caco-2 monolayer was monitored using a

Milliceii-ERS apparatus (MiIlipore, Bedford, MA)

5 5 Constmction of the Recombinaat p2E38-KD Plasmid

An isogenic C. jejuni mutant containing a disrupted cipA gent was constructeci

using a gene replacement strategy (Labigne-Roussel et al. 1988). Plasmid p2E3-8

containing the cipA gene was digested with Stul to completion and partially digested with

HinclZ to remove a 561 bp Eragment intemal to the cipA coding region. The resulting

4.4kb band was purifieci from a 0.7% agarose gel using a gel extraction kit (Qiagen)

according to the manufacturer's protocol. A gel-purined 1.45kb Sd-digested ftagment

from plasmid pKanSphE containing a kanamycin resistance cassette was inserted into the

digested cipA gene to generate plasmid p2E38-KD. Briefly, the blunt mded 4.4kb cipA

fragment was dephosphorylated using alkaline phosphatase (Pharmacia) for 30 min at

37OC and then the enzyme was heat inactivated at 85OC for 15 min. The Sniol kanamycin

fragment was ligated to the digested and dephosphorylated cipA plasmid using DNA

ligase (Pharmacia) in the presence of 5% po1yerhyIeneglycol.

The recombinant plasmici, designated p2E38-KD, was transfomecl into E. coli

Ml01 using the rubidium chloride rnetliod (Sambrook et al. 1989). Plasmids h m the

obtained transfocmants were isolated using an alkaline lysis protocol (Sambrook et al.

1989) and digested with restriction enzymes to venfy that the proper p2E38-M) constmct

was generatexi

5.6 Naturd Transformation of C. je&ni

The disnipted cipA was retuxned to C. jejuni TGII9011 by naturai transformation

(Wang and TayIor 1990). Bnefly, an ovemight bacterial culnirie was diluted 1 in 100 in

MH broth and grown to mid-log phase in MEI-broth. The plasmid p2E38-KD (0.5 pg)

was then added, the culture incubated overnight, and aliquots were spread ont0 MH agar

plates supplemented with kanam ycin. Kanam ycin-resistant C. jejuni colonies were

isolated and the presence of a disrupted cipA gene was verified using the polymerase

chah reaction (PCR) and Southern hybridization analyses. Western irnmunoblot anaiysis

using anti-CipA polyclonal antibodies confumed that the mutant strains did not express

the CipA protein.

5.7 Polymerase chah reaction ampfficatfon

PCR was used to ampli@ a c@A fragment used as a probe in Southem

hybridization or to ampMy the c@A gene h m chromosomal DNA fimm wild type and

isogenic cipA mutant of C. jejuni. In addition, PCR also was used to amplify a fiagrnent

of the kanarnycin resistance cassette from plasmid pKanSphB to be used as a probe in

Southern hybridization. The oligonucleotide m e r s used in this study are listed in

Table 2.

PCR was canîed out in a final volume of 50pl with either 60ng chromosornai

DNA or 3 h g of plasmid DNA (p-2E38 or pKansphB), 2.m of Taq DNA polymerase

(Boehnnger Mannheim) in the presence of 200 p M of each dNTP, 1.5M M&12 and a

lpM concentration of each primer. FoiIowing an initial denaturation s e p of 940C for 3

min, amplification involved 25 cycles of 94OC for 30s. 4S°C for 30s and 72OC for 1 min.

There was a final extension at 72OC for 5 min.

5.8 Southern hybridization analysis of genornic DNA

Campylobacter genomic DNA (2.Spg) was digested with Xba 1 or double digested

with Cla I and Sa2 I (40 U per enzyme) for four hours at 37°C. Chromosornai DNA

fragments (1 pg) were resolved on 0.7% agarose gels and transferred ont0 a nylon

membrane using the sait transfer protocol (Sambrook et al. 1989). Southern hybridization

using the cipA probe was performed using the DIG (digoxigenin)-High Prime DNA

labeling and Cherniluminescent Deteetion kit (Boehringer Mannheim). A cipA-specific

DNA probe was generated using PCR and DE-tagged by random prime labeling.

Following a 4 hr prehybridization at 65'C, the heat denahued probe was added and

hybridization was perforwd ovemight at 65OC. Following washing, the hybridizing

bands were visualized using the DIG-CSPD cherniluminescent detection system

(Boehringer Mannheim). The biot was stripped using 0.2N NaOH and 0.1% SDS, as

recommended by the manufacturer.

Table 2: PCR primers utilized in this snidy

Primer name Primer sequence (5'-3') Location

AJPS ATG CGA A I T CTT ITA Complementq to the 5' end CTC TAT ATA AAA of cipA (nt. 1-27)

AJpr8 TCC AAG AAT CAA TAA Complementary to the 3' end TAT GAG GATG of cipA (1329-1353)

JLP~ GGC ATA GGC AGC GCG Complementary to the nt. CïT ATC AAT A 301-326 of the kanamycin

resistance cassette in plasrnid pKanSphB

J L P ~ GCï CGA CAT ACT GTT Complementary to the nt. CTLT CCC CGA TA 1295-1321 of the kanarnycin

resistance cassette in plasmid pKanSphB

Southern hybndization using a PCR amplified 1.02kb fkqpent derived from the

kanamycin resistance cassette (pKanSphB) was undertaken foilowing nick translation of

the probe with [a-32~] ATP (ICN Biochemicals) using the DNA polymemdDnase 1

enzyme (Gibco BRL). Briefly, 400ng of DNA (16 pi) was combined with Spi of solution

A l (0.2mM of aITP, d T P and dGTP), 5pi of [ a - 3 2 ~ ] ~ ~ ~ (lOmCi/ml), 5pl of DNA

polyrneraselDnase I (0.4 Ulpl). The reaction was incubated at 14OC for 1 hr and the

reaction was stopped foliowing the addition of S p i of 0.5M EDTA pH 8.0.

Unincorporated radionucleotide was removed h m the reaction using a G-50 spin column

(Pharmacia). The activity of the labeled probe was checked by adding 1 pl of the labeled

probe to lOmL of liquid scintillation solution (Beckman) and counted using a liquid

scintillation counter (Beckman LS 3801). The strïpped blot was prehybridized for 2hrs at

42OC in 50% deionized formamide, 1% ultrapure SDS and 10% dextran suifate in 1M

NaCl in the presence of denatrued salmon sperm DNA (lûûpg per mL of hybridization

fluid). Following prehybndîzation, the radioactive probe was heat denatured and added

to the hybndization fluid at a concentration of 5x16cprn per ml of hybndization fluid.

Hybridization proceeded for 20 hcs. at 42°C. The blot w u washed for 10 min at room

temperature using ZxSSC, foiiowed by 2 washes for 20 min st 42OC using 2xSSC. 1%

SDS and then 2 washes for 20 min at 42°C using 0.2 SSC, 1% SDS. The blot was then

wrapped in Saran wrap and exposed to X-ray nIm (Kodak XR5).

5.9 Expression of recombinant CipA

The translational in-frame fusion between glutahione S- transferase gene and

cïpA was constnicted by Dr. A. Joe. Briefly, a gel-purifiecl 1.4kb PCR fragment of the

cipA gene was ligated into the @EX-2T plasmid vector (Pharmacia) using compatible

Eco RI digested ends.

The resulting construct was designateà pXcpl2 and was transformed into E. coli

JMlOl. Expression and subsequent purification of the recombinant CipA was perfonned

according to the manufacturer's instmctions. Briefiy, an ovemight culture of E. coli

Ml01 harbouring the GST-cipA gene fusion on a plasmid was dïiuted 1 in 10 in LB

broth containing 100pg/mL of ampicillin and grown at 37°C to an ODm of

approximately 1 .O. Expression of the GST-CipA fusion protein was induced h m the rat

promoter using O.lmM isopropyl B-D-thiogalactoside @TG) for 4 hr at 3PC. The

culture was centrifuged at 7700xg for 10 min. at 4OC and the bacterial pellet resuspmded

in phosphate buffered saline (PBS). The celis were disrupted by sonication on ice with a

Branson Sonifier450 microtip (30% duty cycIe, output contrd 5) using 30 sec bursts for

a total of 10 min. The supematant fraction was gently mixed with 1% Triton X-100 for 30

min followed by cenûifugation at 12000xg for lOmin at 4OC to remove cellular debris.

The resulting clarified supernatant was incubated with 2mM ATP, lOmM MgS04, 50mM

Tris pH 7.4 for 10 min at 3T°C, and then mixed for 30 min at room temp with

glutathione-Sepharose 4B beads. The Sepharose 4B beads bound the fusion protein via

the GST moiety. Following 30 min of incubation, the beads wcre washed five times with

PBS and then incubated with bovine thrombin (Calbiochem) overnight at room

temperature with mild shaking to cleave the GST moiiety h m the fusion protein. The

next day, the beads with the attached GST moiety were pelleteci (500xg for 5 min at room

temp.) and washed 10 times with PBS. The recombinant CipA protein was collected

from the supernatant of each PBS wash. The CipA protein was then concentrated using a

Centricon-IO column (Amicon) and the protein concentration detemined using the

Bradford assay (BioRad Laboratones). The purity of the concentrated CipA protein

fraction was detennined by sodium-dodecyi-sdphate pdyacrylami& gel electrophortsis

(SDS-PAGE) analysis followed by staining with Coomassie R-250 blue (Laemmli 1970).

5.10 CipA antisenim

Polyclonai antiserum to CipA was genefifted by immunin'ng a New Zealand

White rabbit with 0.5mg of recombinant CipA protein combined with complete Freund's

adjuvant foilowed by a second injection three weeks later with 0.25mg of recombinant

CipA protein with incomplete Freund's adjuvant. Immune serum was collected 10 days

foüowing the second injection.

Affinity purification of the immune serum was performed by Dr. A. Joe using a

method originally described by Smith and Fisher (1984). Bnefly, the recombinant CipA

protein was immobiIized on a strip of nitrocellulose and incubated with the immune

senim in order to isolate antibodies specific for the CipA protein. In a second purification

strategy, the immune s e m was incubated with nitrocellulose disks which had been

coated with sonicated extracts h m the 901LK2 mutant strain (Iriarte et al. 1998). Thus,

antibodies cross-reacting to other C. jejuni proteins were absorbeci onto the nitroceilulose

disks and removed h m the immune serum. However, despite these purification

techniques the anti-CipA antibodies continued to cross-react non-specificdy with other

C. jejuni proteins.

5.11 Electrophoretic separation of proteins and Western immunoblot Pnalysis

Whole-cells lysates of C. jejzmi and protein samples were solubilized in sample

buffer (62SmM Tris-HCl @H6.8), 5% fbmeniaptoethanol, 1% SDS, 10% glycerol,

0.025% bromophenol blue) and heatcd in a boiiing waterbath for I0min. Roteins wert

separateci using SDS containing polyacrylamide gels as describecl by Laemmli (1970).

Proteins were visualized using Coomassie R-250 staining or were transferred to

nitrocellulose membrane (WV, 2 hrs) (Towbin et al. 1979). Western blot analysis was

performed using anti-CipA rabbit immune senun at a dilution of 1 in 250.

Immunoreactive proteins were detected using the ECL Western blotting system

(Amers ham).

5.12 CipA protein expression in C. jejuni

Using cultures of C. jejuni from early, mid and late log phase of growth, Western

blot analysis was used to investigate whether CipA expression is growth phase dependent.

An overnight culture of C. jejuni TGH9û11 was diluted 1 in 20 into 5mL aliquots of MH

broth and bacterial growth was monitored by optical density at %m. The bacterial

cultures at each phase of growth were normaiized by the optical density at 600nm (5mL

of bacterial culture at an 0.2) and îhe bacteria were hamesteci by centrifugation

(16000xg for 10 min). C. jejuni bactezial pellets were solubilized in sample buffer, the

proteins were resolved by SDS-PAGE using a 15% polyacrylamide gel and stsined with

Coornassie R-250 blue to ensure equal Ioading between ianes. A second gel was thm

transferred to nitrocellulose and CipA expression was assessed by Western blotting.

5.13 CipA expression foiiowing interaction with eukaryotic ceils

CipA expression was compared between C. jejmi cultures grown in the presence

and absence of eukaryotic cells. C. jejuni celis (10' cfu) w m incubated for 3 hours in

either 8m.L of MH broth, 8mL of MEM+ 15% FCS or with a HEp2 ce11 monolayer

containhg 8mL of MEM+15% FCS. Bacteria were coliected by centrifugation (16000xg

10 min), in addition the infectecl HEp2 monolayer was washed three times with PBS and

the washes were combined with the overIying medium before centrifugation. C. jejuni

bacterial pellets were solubiiized in sample buffer and the proteins were separated by

SDS-PAGE, transferred to nitrocellulose membrane and reacted with the anti-CipA

polyclonal antibodies.

5.14 C. jejuni infection in mice

Femaie BALB/c mice. 5 weeks old were purchased h m Jackson Laboratory (Bar

Harbor, Maine) and individually housed in microisolator cages. Following the protocols

described by Pei et al. (1998) and Yao et ai. (1997), mice were inoculateci with C. jejmi

at 6 and 9 weeks of age. Before inoculation, gastric acîdity was neurralized by orogastxic

administration of 0.2mL of 5% sodium bicarbonate followed by the bacterial culture 10

minutes later. C. jejuni TGH9011 and 8 1-176 p w n overnight in MH broth w m

harvested by centrifugation and resuspended in PBS to the desid ceii density. The

inocula comprised of 10' cfu in OlmL for the h t challenge at 6 weeks or 10" cfu in

0.4m.L for the second inoculwn at 9 weeks.

C. jejmi colonization was monitored by fecal excretion. Four to five fksh fecal

pellets were collected on alternate days and homogenized into 0.4mL PBS with mild

vortexing. The homogenate (0.2mL) was plated in duplicate onto modifieci

carnpylobacter blood-free selective plates of Modifieù Cefoperazone-Charcoal-

Desoxycholate-Amphotencin (CCDA)-Preston agar plates (Oxoid). PI- were

incubated micmaefobically for 56 hr a& 37°C. At 13 weeks, blood sampks were coilected

by cardiac puncture, ailowed to clot at m m temperature and the serum coilected by

centrifugation (3000xg for 10 min at m m temp) and then stored at -20°C.

Chapter 6

Results

6.1 Construction of a kanamycin-resistant c@A-àektion mutant of C. jejuni

TGH9011.

In addition to the cipA-insertion mutant (9û1LKI) which had been previously

constnicted (Joe et al. 1998), a cipA-deletion mutant was also developed for use in the

charactenzation of CipA. The mutant was generated by aUeic replacement using a

disrupted cipA gene present on a suicide vector (Figure 3). This mutant was constructed

since it would be more suitable for studying the effects of a c@A mutation within in vivo

rnodels of pathogenesis. Within an animal model, where there is no selection for

antibiotic resistance, concems aise with regard to the stability of a c i p ~ : ~ a n ~ consmict

present within the chromosome of an insertion mutant.

The cipA gene, cloned into the pBluescript vector (plamid p2E3-8) was paaially

deleted (nt. 33 1-892) using restriction enzyme digestion. The cipA gene was then

disrupted by the insertion of a kanamycin resistance cassette to generate plasmid p2E3-

8KD. This resulting plasmid, serving as a suicide vector, was introduced uito C. jejmi

TGH9û 1 1 by natural transformation (Wang and Taylor 1990). Transformants were

characterized by PCR and Southern blot hybridization to confinn the site-specific

insertion of the disrupted cipA gene into the chromosome. One transformant, designated

90 1LK2, was selected for m e r study.

PCR prime= Np5 and AJpr8 designeci to amplify the cipA gene wcrc utilized to

compare the respective sizes of the cipA allele h m mutant and parental strains of C.

jejuni. PCR analysis generated a 1.4 kb fiagment h m wild-type genomic DNA. while

Figure 3: Schematic diagram of the plasmids utilized to generate the cipA-deletion

mutant suicide vector construct.

Plasmid p2E3-8 was digested with the restriction enzymes Sm 1 and Hinc II to remove a

0.56 kb hgment intemal to the cipA gent. The kanamycin resistance cassette generaed

by Sma 1-digestion of plasmid pKanSphB, was ligated to the partially deleted cipA gene

to yield plasmid p2E3-8KD.

p2~3-8 vecto~ p~iuescript II SK+ insert: 2kb fiagment of C. jejuni

TGIW)11 genomic DNA

cassette from C. coli

amplicons of 2.2 kb and 2.7 kb were generated b m the genomic DNA of the cr'pA-

deletion mutant (901LKS) and the cipA-insertion mutant (WlLKl), respectively (Figure

4). The increased size of the cipA coding region within the chmmosomal DNA of the

mutant saains corresponds to the disuption of the gene by the lMkb kanamycin

resistance cassette. In addition, the absence of the wild type 1.4kb amplicon fkom the

mutant strains indicates the successfuI rephcement of the wifd type gene with the

c i p ~ : K a n ~ consûuct.

Southem blot hybndization using cipA-specific and JCanR-specific DNA probes

was also performed as further confirmation of the double crossover recombination event

between the C. jejwU genome and the plasGd p2E3-8M). The restriction maps of the

c@A region in TGH9û11,901LK1 and 90ILK2 are shown in Figure 5.

Genomic DNA from the Md-type and mutant strains was digestcd with Xba 1,

which does not cleave within the cipA gene, and hybridized with a cipA probe (Figure 6).

One hybridizing band of 6.2 kb was present within wild-type genomic DNA while

slightly larger DNA fragments of 7.6kb and 7.0kb hybndized the probe in the genomic

DNA from 901LK1 and 901LK2, respectively. This result suggests a single insertion of

the disrupted cipA gene into the chromosome.

Genomic DNA digested with the restriction enzymes, S d 1 and Ch 1, was dso

utilized in Southern blot analysis to confinn the deletion of the 0.56 kb c@A-fragment

from 901LK2. Since the hybridizing restriction hgments from the Sol 1 and Ch 1

genomic digest h m 901LK.2 (Figure 6 Iane 6) were nther fninf the Souhem blot using

S d 1. Cla 1-digested DNA was repeated and is shown in Figure 7. In this figure, the

Figure 4: PCR analysis of the c@A gene from wild type and cipA mutant strauis

Chromosornai DNA from C. jejmi TGH9û11 (lane 2), 901LK1 (lane 3) and 901LK.2

(Iane 4) was subjected to PCR using primes designed to ampiiry the c@A gene. The

PCR products were separated by gel electmphoresis using a 0.7% agarose gel. Molecular

sizes of the amplicons are indicated on the nght hand side of the gel. Laue 1 represents

the lambda Hird ID marker and lane 5 represents the 100 bp marker.

Figure 5: Restriction maps of the cipA chromosomal loci within wild-type C. jejuni and

the mutants 90ILKI and 901LK2.

The 901LK1 mutant was previously constructeci by the insertion of a ICanR cassette into

the unique Sm 1 site within cipA. The 901LK2 mutant was generated by the insertion of

the kanamycin cassette into a paiiially deleted cipA gene. The restriction enzyme Xba 1

cleaves at unhown sites outside of the sequenced cipA region. Restriction sites: C, Cla

1; S, Sul 1; St, Stu 1; x, Xba 1.

cipA DNA probe

K d DNA probe

Figure 6: Southern blot analysis of chromosomal DNA h m C. jejuni TGBûI 1,

901LK1 and 901LK.2.

Panel A: Chromosomal DNA kom wild-type C. jejuni (ianes 2.3). the cipA-insertion

mutant 901LK1 flanes 4,s) and the c@A-deletion mutant, 901LK2 (lanes 6,7) was

digested with ei ther Xba 1 (lanés 3 ,5 ,7 ) or double digested wiîb Sa1 1 and Cla 1 (ianes 2,

4,6). DNA fragments were separateci by gel electrophoresis on a 0.7 96 agarose gel.

Lane 1 represents the lambda Hind III marker and lane 8 represents the lOObp marker.

Panel B: The DNA restriction hgments were hybridized with a 1.3kb c@A-specific

probe generated by PCR b m plasmid p2E3-8 using primers N p 5 and AJpr8. Single

hybridizing bands are detected in the Xba 1-digested DNA from the parental strain (lane

3), 901LK1 (lane 5) and 901LK2 (iane 7). DNA digested with M I and Cla 1 yielded

three hybridizing bands in the wiid type (lane 2) and in 901LK1 (lane 4). Unfortunately,

there was only faint hybridization in lane 6, presumably due to a low level of

chromosomal DNA transferred to the nylon membrane. However, it appears that while

the 1.2 and 0.8 kb bands are present within 901LK2, the 0.55 kb band is absent h m this

mutant strain.

hybridizing bands of approx 0.8kb and 1.2kb representing the 5' and 3' ends of the cipA

gene respectively, were present in al l three strains. However, the 0.5 kb hybridiMg

fragment componding to n t 331-783 of cipA is clearly present ooly within the genomic

DNA from the wild type and the insertion mutant, 901LKl.

When the Southem blot h m Figure 6 was saipped and probed using the

kanamycin resistance cassette, the same 76kb and 7.0kb bands which hybridized the cipA

DNA probe were present within the Xba 1 digested DNA h m the two mutants strains but

not from the parental strain (Figure 8). Similarly, when genomic DNA digested with CCa

1 and Sa1 1 was hybridized with the ICanR-specific probe, a hybriduing band of 1.5kb was

present in the mutant strains but absent h m TGH9û11 (Figure 8). Collectively, these

results confirm that 901LK1 and 901LK2 do not contain the wild-type cipA allele and are

both while the parental strain is cipA + and -. In addition, the PCR analysis

and the Southem blot results in Figure 7 indicate that a deletion within the cipA gene has

k e n constmcted in the 901LK2 chromosome.

6.2 Generation of ad-CipA polyclond antibodies and detreaoa of CipA withia C.

jejuni,

The CipA protein was expressed in E. coli and subsequently affinity-purified

using the GST-Gene Fusion System (Pharmacia) (Figure 9). The CipA protein was

utilized as an antigen to generate polyclonal anti-CipA antibodies using a New Zealand

White rabbit Even though antibody purification techniques were employed, there

remaineci non-spocific binding of the antibodies to other C. jejrmi proteuis. However, the

Figure 7: Southern blot analysis of chromosornal DNA to confirm the partial deletion of

the c@A coding region h m WiLK2.

Panel A: Chromosomal DNA h m wild-type (lane 2), W U 1 (lane 3) and 901LK2

(lane 4) was digested with the restriction enzymes Sai 1 and Cla 1. The digested DNA

was subjected to gel electrophoresis (0.7% agarose gel). Lane 1 represents the lambda

Hind III marker and lane 5 represents the 100 bp marker.

Panel B : Southem blot hybridization was performed using the *A-specific DNA probe

as described in Figure 4. Three hybridizing bands were detcaed h m the chromosod

digests of the wild-type and 901LK2 strains (lane 2,3). The chromosornal DNA from the

cipA-deletion mutant, 90 1LK2, hybridizcd onIy with the t .2kb and 0.8kb bands. The

absence of the 0.55kb band from W 1 W denotes that the correct deletion of the cipA

coding region was generated

Figure 8: Southem blot analysis of chromosomal DNA of the wild type and cipA mutant

sttains using the kanamycin resîstance cassette as a DNA probe.

Panel A: Chromosornal DNA fiom wild-type C. jejmi Oanes 2.3). 901LK1 flanes 4-5)

and 901LK2 (lanes 6,7) were digested with either Xba 1 (lanes 3,5,7) or double digested

with Sa1 1 and Cla 1 restriction enzymes (lanes 2,4,6). The DNA fragments were

separated by gel electrophoresis on a 0.7 % agarose gel. Lano, 1 represents the lambda

Hind III marker and lane 8 represents the lOObp marker.

Panel B : The genomic DNA hgments from Panel A were subjected to Southem blot

hybndization using a 1.45kb kanamycin cassette DNA probe generated by PCR h m

plasmid pKanSphB using primefs JLpl and JLp2. Lanes 2 and 3 representhg DNA from

C. jejuni TGH9û11 did not hybridue the probe as expected Hybridizing bands were

detected in the chromosomal DNA fiom 901LX1 (lanes 4.5) and 901LKî (lanes 6,7).

Figure 9: Expression and pwification of the CipA protein using the GST-Gene Fusion

S ystem.

An N-terminal i n - h e fusion was genenited between the glutathione4-transferase gene

and the cipA coding region downstream of a tac promoter by Dr. A. Joe. The

recombinant plasmid harbouring the GST-cipA gene M o n was designated pXcpl2. The

expression of the GST-CipA protein within the E. coli host could be induccd in the

presence of IPTG. The fusion protein was subsequently purified using glutathione

coated beads to bind the GST moiety. The fusion protein was then subjected to cleavage

with the thrombin enzyme to release the CipA protein h m the glutathione beads. Lane S

represents the Biorad Broad-range protein marker, lane 1 represents a whole£eii lysate

from an uninduceci culture of E. coli JM101/ pXcpl2, lane 2 represents a wholeceil

lysate Çrom a culture of E. coti JMlOl/pXcplS following induction with IPTG, lane 3

represents the purified CipA protein following cleavage with the thrombin enzyme.

GST-CipA

CipA

antibodies could detect the native CipA protein within a whole-ceil lysate of C. jejuni

TGH9011. In addition, immunobIot analysis indicated that the mutant strains did not

express the 55kDa CipA protein (Figure 10).

6.3 CipA expression in C. jejuni TGH9011 ïs not affècted by pbase of growtb.

Studies in 0th- enteric pathogens have shown that the expression of virulence

determinants is O ften coordinately regdateci by envhnmental conditions (Mekalanos,

1992). Such regulatory mechanisms presumably aid in bacterial survival by preventing

the constitutive expression of virulence factors which would require the expenditure of

excess energy and could render the organism vuinerable to an immune-mediated attack.

Studies in C. jejuni have examined the effect of various environmental parameters on

gene expression including; depleted h n conditions, the presence of bile salts and shifts

in temperatures. Therefore, studies were undertaken to determine whether CipA

expression was regulated in response to growth phase.

Cell lysates fiom cultures at early, mid and late log phases of growth were utilized

to examine CipA expression throughout the C. jejuni growth curve (Figure 11A). The

ce11 lysate proteins were separated by SDS-PAGE and stained with Coomassie R-250

blue to show loading between lanes (Figure 11B). Western immunoblot analysis of the

ce11 lysates using anti-CipA antibodies did not reveal any differential expression of CipA

in response to the phase of growth (Figure 11C).

Figure 10: Western immunoblot analysis of whole-cell lysates h m the cipA mutant

strains and the parental wild type strztin using anti-CipA antibody.

The ce11 lysate proteins were separated by SDS-PAGE using 15% polyacrylamiâe gel.

The proteins were transferred to nitrocellulose and nacted with the anti-CipA antibody.

The CipA protein was detected within the protein profile of C. jejuni TGH9ûl1 (lane 3)

and was absent h m the protein profiles of the mutant strains (lanes 4,s).

Lanes: 1 BioRad Kaleidoscope protein marker, 2, purifieci CipA protein; 3, C. jejuni

TGH9011; 4,901LKl; 5,901LK2.

+ CipA

Figure 11: CipA protein levels throughout the gmwth cuve of C. jejuni TGH9û11.

Panel A: The TGWûl1 celi cultures were measured for the absorbante at 600nm at each

time point Serial dilutions were plated ont0 MH-agar to determine the ceU density of the

culture.

Panel B: TGH9ûl1 ce11 Iysates h m each time point were separated by SDS-PAGE

using a 12% polyacrylamide gel and stained with Coomassie blue R250 to illustrate equal

Ioading of protein from each celi lysate sample. Lane 1; BioRad Kaleidoscope protein

marker, lanes 2-7 represent the whole-ceil C. jejmi lysates (lane 2; T=6.5hrs, lane 3;

T=1 l.Shrs, lane 4; T=13 hrs, lane 5; T=16 hrs, lane 6; T=22 hrs, lane 7; T=24.5hrs), lane

8 is a whoie-ce11 lysate of 901LK1 as a negative control.

Panel C: Protein samples h m each the point were resolved by SDS-PAGE and

transferred to nitrocellulose. CipA expression during the different phases of growth was

determined by Western immunoblot analysis. Lane 1; purified CipA protein as a positive

control, lanes 2-7 are C. jejuni ceil lysates samples (lane 2; T=6.5hrs, lane 3; T=l l.Shrs,

lane 4; T=13 hrs, lane 5; T=16 hrs, lane 6; T=22 hrs, lane 7; T=24.5hrs), lane 8 is a

901LK1 cell lysate as a negative control. Densitometric analysis was performed using

ImageQuant 1.2 for Macintosh.

Cs jejuni Growth Curve

6.4 CipA expression foUowing Interaction with aEp-2 ceU monolayers in tissue

culture.

Previous studies in C. jejuni have shown that contact with eukaryotic cells induces

the expression of several bacterial proteins as shown by 2-D gel electrophoresis (Konkel

and Cieplak, 1992a; Konkel et al. 1993). The majority of these proteins remain

uncharacterized; however, a recent shidy has shown that at least one of the upregulated

bacteriai factors is secret& in the presence of eukaryotic c d s and is essential for

bacterial internalization into the host celi (Konkel e t al. 1999b). To examine whether

CipA expression was altered following incubation with host cells, C. jejuni was cultured

in either MH broth, MEM or -2- conditioned MEM. Whole-cell C. jejuni lysates

under each condition were resolved by SDS-PAGE and CipA protein expression was

determined by Western blot analysis. Densitometric analysis indicated that CipA protein

expression in C. jejuni was not affectecl by the presence of tissue culture medium or

fdlowing incubation with a HEp-2 cell monolayer (Figure 12).

6.5 Comparing the levels of adherence and invasion of wild type C. jejuni and the

c@A mutant strains to eukaryotic ceII monolayers in tissue culture.

Adherence foilowed by internalization into the intestinal epithelium have been

proposed as important pathogenic mechanisms of C. jejuni. Studies with C. jejuni, using

the gentamicin protection assay (Elsinghorst, 1994) indicate that adhesins are likely to be

constitutively expressed while the expression of invasins a ~ e likely induced by the

presence of eukaryotic cells (Konkel and Cieplak, 1993). Previous studies in out

Figure 12: CipA expression when C. jejuni is cul- in tissue culture medium or

following contact with HEp-2 ceiis.

C. jejuni TGH9û11 was grown in either MH broth (lanes 2,3) MEM supplemented with

15% FCS (lane 4) or incubated with a HEp2 ceU monolayer in a ~-25crn~ flask

containing MEM supplemented with 15%FCS (lane 5). Following a 3 hr incubation, the

bactena were collected by centrifugation and bacterial lysates were utilized to examine

CipA protein expression. Western immunoblot analysis was performed using anti-CipA

antibodies. Densitometnc analysis was performed using ImageQuant 1.2 for Macintosh.

Lane 1 ; purified CipA protein, lane 2; TGH9ûl1 grown in MH broth, lane 3; TGH9011

grown in MH broth, lane 4; TGH9011 grown in MEM+15% FCS, lane 5; TGH9û11

grown in mp-2-condi tioned MEM supplemented wi th 15% FCS.

CipA

laboratory suggested that a functional CipA protein is required for maximal adherence

and invasion of C. jejuni into INT407 celis. Using the 901LK1 mutant strain, the

disruption of cipA resulted in reduced adherence (42.5% 2 10.5% relative to wild type) to

the monolayer. Furthemore, fewer of the adherent bacteria were intemalized by the host

cells as compared to the parental strain (47.5% & 7.7% relative to wild type).

To further characterize the effect of a c@A mutation on bacterial-host ce11

interactions, studies were extended to include additional cultured epithelial cell lines and

the newly constructeci deletion mutant. Initialiy, the adherence and invasion assays

compared both 901LK1 and 901LK2 to the parentai TGH9û11 strain. However,

problems were encountered with the culturing of the 901LKl mutant, Ovemight cultures

of 901LK1 usually did not reach an optical density comparable to the wild-type or

901LK2 and therefore could not be included in the assay due to the low cell density and

variable growth phase of the culture. For this reason, after the completion of the series of

HEp-2 adherence and invasion assays, subsequent experiments focuseci on cornparhg the

wild-type with the 901LK2 deletion mutanL

HEP-2 cells, which originate fiorn a human laryngeal carcinoma have often been

employed as a mode1 system to shidy the adherence and invasive properties of pathogenic

E. coli (Donnenberg and Nataro, 1995). In addition, HEP-2 celi monolayers have also

been used in studies with C. jejuni comparing the adherence levels between clinical and

laboratory isolates (Konkel and Joens, 1989).

Disruption of the cipA locus in 901LK1 and 901LK2 had dissimilar effects on

bacterial intemakation (Fi- 13). While no difference was obsewed between the

wild-type and mutant levels of adherence, the deletion mutant (901LK2) exhibited a

reduction in the perçentage of adherent bacteria which were intemalized (29.5% + 5.3%

relative to wild type, ANOVA, p >O.OS). Invasion levels were not reduced in the 901LK1

strain. When different cell lines were employed, including INT407 ceus and

nonpolarized Caco-2 cells, the adherence and invasion phenotypes of 901LK2 were

indistinguishable h m wild type (Figures 14,lS).

6.6 Wild type C. jejuni and strain 901LK2 exhibit simiiar rates of translocation

across a polarizd Caca-2 monolsyer without dismpting the transepitheliai

resistance of the monaiuyer.

Polarized Caco-2 cell monolayers have bten widely used as a mode1 system to

study bacterial invasion and translocation into the intestinal epithelium. Differentiated

Caco-2 monolayers represent a closer approximation of the in vivo setring since they

develop defined apical and basdateral surfaces and express several markers of the small

intestine. Studies with C. jejmi have shown that this organism is capable of translocating

across Caco-2 monolayers, by both paracellular and transcellular routes, without affecting

the integrity of the monolayer (Konkel et al. 1992e).

To test whether the disruption of the cipA locus affected the rate of translocation across a

polarized monolayer, differentiated Cam-2 cells were infected with C. jejmi as descnbed

by KonkeI et al. (1992d). At 1.5 h . intervals, the filter unit containing the Caco-2 celï

monolayer was transferred to a new well and the numbcr of translocated organisms

Figure 13: Levels of adhereace and invasion of wild type and 901LK.2 to Ep-2 cell

monolayers

Panel A and B: The graphs represent the levels of bacteriai adherence (panel A) and

bacteria invasion (panel B) into HEp2 ceil monolayers. Bacterial adherence was

expressed as a percentage of the initial inoculum. Invasion levels of C. jejuni represented

the number of organisms which swived treatment with gentamich. Bactexial invasion is

expressed as the percentage of adherent bacteria intemaiized into the host cells.

Bars both graphs represent the mean t standard error, averaged over 4 - 6 independcnt

experiments. Statistical analysis was done using ANOVA, P > 0.05.

Panel A: Adherence to -2 cell monolayers

Adherence

Panel B: Bacterial invasion into HEP-2 cells

Invasion

Figure 14: Gentamich protection assays using INT4û7 celi monolayers

Panels A, B: The graphs represent the mean levels of adherence (panel A) and invasion

(panel B) of 90îLK2 ami C. jejmi TGH9û11 into IiVï40'7 ceH monolayers. The number

of adherent bacteria is expmsed as a percentage of the initiai inocuium while the level of

bacterial invasion is expressed the percentage of the aàherent bactena which survived in

the presence of gentamicin. Results represent the mean of three separate experiments 2

standard error. Statistical analysis was done using the unpaired, two-tailed StuQent's t-

test, p > 0.05.

Panel A: Adherence to INT407 ce11 monolayers

fi - Adherence

Panel B: Bacterial invasion into INT407 cells

Invasion

Figure 15: Adhe~nce and invasion levels of wild type and cipA mutant C. jejuni using

nonpolarized Caco-2 cells.

Pane1 A, B: Graphs represent the levels of bacterial adherence (expssed as a percentage

of the initial inoculum) and bactefial invasion (expressed as a percentage of adherent

bactena). The bars represent the mean t standard emr of three independent expenrnents.

S tatis tical anal ysis was perfomed using the unpairai, two-tailed S tudent's t-test, pAI.05.

Panel A: Adherence to unpolarized Caco-2 celi monolayers

A Adherenœ

Panel B: Bacterial invasion into unpolarized Caco-2 cells

Invasion

present in the basolateral medium was determinecl by plating serial dilutions (Figure 16).

The rates of translocation were indistinguishable between the wild-type and 901LK2.

After 1.5 hrs of infection approximately ld chi were recovered from the basolateral

medium, while at later time points the number of translocated organisms increased to

reach 10~cfu. The transepithelid resistance m) of the monolayer was masured

prior to the addition of the bactena and at time points following the completion of the

translocation assay. At no time was the integrity of the membrane affecteci by the

incubation with C. jejuni, even aftex 24 hrs of infection.

6.8 Efforts to estabiish transient colonization of BALB/c mice with wild type and

mutant strains of Ce jejuni.

Studies on the pathogenic mechanisms of C. jejuni have largely rrlied on in vilm

assays since in vivo models of campylobacteriosis have not been well characterized.

Immunocompetent mice have been utilized by some gmups as a m a n s to test the ability

of C. jejuni colonize the intestine (Pei et al. 1998; Yao et al. 1997). While the infected

mice do not deveiop disease, they become acymptomatïc caniers of C. jejuni, typically

shedding between IO*- 106 cWg of feces.

Foliowing the protocol descnbed by Pei et al. (1998) and Yao et al. (1997),

BALB/c mice were orally infécted with approximately 10' cfu of either C. jejuni

TGH9011 or 901 W. In addition, a group of mice was also infecteci with C. jejuni strain

81-176 which was utilized by Pei et al. (1998) and Yao et al. (1997) and thmby

Figure 16: Rates of translocation of C. j e j k and 9ûîLK2 across a polarized Caco-2

monolayer.

Caco-2 monolayers w m infecteci on the apical si& with 1o7cfu of either 9OlLKS (open

bars) or wild typt C. jejuni (batched bars). At 1.5 hr intervals, the number of organisms

present in the basolateral medium was detennined by plate count. Results are the mean

of three experiments & standard emr.

represented a positive contml. Fecal shedding of C. jejvni was monitored by plating a

homogenate of fresh fecal pellets ont0 campylobacter selective medi-

On &y 2 following the first oral inoculation (1.745 x 10' ch), C. jejmi was

recovered h m the fecai pellets of 2 out of 4 mice infected with TGIDûl 1.3 out of 4

mice infecteci with 901LK2 and 3 out of 4 mice infected with 81-176. However, by day 4

of infection none of the mice remained colonized The mice were infccted for a second

time with an increased inoculum (1.0-4.4 x 101° ch). On the following day, 4 out of 4

mice were colonized fimm the TGEW)11 and 8 1-176 groups and 3 out of 4 mice were

colonized with 901LK2. However again the colonization did not persist as none of the

mice remained colonized by &y 4. C. jejuni was identified by colony morphology on the

campyIobacter selective medium and was subcultured to MH agar with or without

kanamycin, as a M e r confirmation by colony morphology. A group of three sham

infected mice was included as a negative conrrol. At no tirne were the distinctive orange

colonies of C. jejuni 'rccovereci h m the fecd homogenate of the sham-infectcd mice.

Thus, contamination was unlikely to have occurred between the groups of mice.

Chapter 7

Discussion

Discussion

7.1 Interpretation of results

Construction of a c i ~ A deletion mutant

The focus of this study was to investigate the effect of a c@A mutation on the

virulence of C. jejuni using complementary m virro and Uc vivo experhental approaches.

Two separate mutant strains (901LK1,901LK2) were generated by allelic-replacement

mutagenesis. The 901LK1 s e was constructcd by the insertion of a KanRcassatt into

the middle of the ci'A coding region while the 901LK2 mutant contains a partial deletion

of the cipA gene followed by the integration of the cassette. The KanR+, c@A-

genotypes were confirmeci using PCR and Southem hybridization analyses. In addition,

the inability of the mutants to express the wild type CipA protein was confinned by

irnmunoblotting. The advantage of the deletion mutant (9ûiLK2) is the complete

abrogation of CipA protein expression even in the event of the excision of the kanamycin

resistance marker. In addition, the 901LK1 mutant, which retains the first 892nt of the

cipA coding region, could potentiaüy producce a truncated protein which might retain

some biological activity.

The effect of a ci3A mutation on adherence and invasion levels in vitro

Unexpectedly, the two isogenic mutants exhibited divergent phenotypes in vitra

using the gentamicin protection assay with HEp2 cell monolayers. While the two

mutant strains were not significantly altered in the levels of adherence as compared to

wild type, there was a difference when the mean adherence levels of the mutant straùis

were compand to one another (p = 0.02). In addition, the 901LK2 saain but not 901LK1

exhibited a significant reduction in the number of intemaiized bacteria.

Confounding results were also observed when the levels of bacterial adherence

and invasion for the 901LK2 mutant were assessed using different epithelial ceil lines in

tissue culture. While no differences in levels of adherence were observed between

901LK2 and the wild type strain regardless of the ceii line employed, a reduction in the

Ievels of invasion of 9OILK2 was obsemed onIy when HEp-2 cells were employd The

90 1LKî strain and the parental wild type exhibited indistinguishable levels of

internaiization into INT407 ceils and similar rates of translocation açross a polarized

Cam-2 ce11 monolayer. Therefore, it appears that the reduced levels of C. jejzuai

internalization as a result of a cipA dismption is a cefi-type specific phenomenon.

The construction of a cipA mutation using a more highly invasive sttain of C.

jejuni could also be employed to further substantiate the in vitro findings of this study.

While numerous reports have shown that C. jejuni is capable of adherence to and

invasion of a variety of eukaryotic cell Iines, levels of adherence and invasion Vary widely

between different C. jejuni strauis. Adherence levels cm range h m 0.02%-1.4% of the

initial inoculum, while the invasion levels can range h m 0.00003%-1.6% of the initial

inoculum depending on the cell üne and the bacterial strain employed (Konkel and Joens

1989; Konkel et al. 1999b; Doig et al. 1996; Yao et al. 1997). In the present study, both

adherence and invasion levels of TGH9011 were consistent between ce11 h e s but were

lower in compatison to other C. jejvni strains used for pathogenic studies. The levels of

adherence for TGH9011 ranged h m 0.08% to 0.15% of the initial inoculum while the

levels of invasion as expresseci as a pcrcentage of the initial ranged h m 0.01-

0.00042. Therefore, it would be of interest to introduce the cipA mutation into a parental

C. jejuni strain whîch exhibits higher levels of both adhmnce and invasion to tissue

culture ceUs.

Intestinal colo~zation in BALBfc mice

To obtain further evidence that the CipA protein is involved in C. jejuni vinilence, we

utilized an in vivo mode1 of infection. Oral challenge of BALB/c mice with C. jejuni (109

cfu) is reported to result in the colonization of mouse intestine, which can be monitored

by fecal shedding of the organism. Pei et al. (1998) found that 100% of BALBlc mice

infected with C. jejuni strain 81-176 weE colonized at day 9 following challenge and

three quarters of the mice remained colonized by &y 38. Yao et al. (1997) reported a

100% rate of coIonization up to three days following infection while 70% of the mice

continued to shed C. jejuni strain 81-176 at &y 9 p s t challenge.

In the present study, using an initial inaiulum of 4.5 x 109 ch, colonization of the

BALB/c mice with C. jejuni strain 81-176 was lower than reported (only 75% on &y 2

following the infection) and short lived (since none of the mice remained colonized by

day 4 following challenge). A second inoculum was administered to the mice (2.4 x 10''

cfu); however, none of the mice remained colonized by &y 4 ps t challenge.

The discrepancy in the efficiency of bacterial colonization may bt attributed to

differences in the culture techniques. In the prcsent study, bactexia used for infecting the

mice were grown ovemight in MH broth culture. Reports h m other groups utilized the

same C. jejuni strain but grown in a biphasic culture system (Pei et al. 1998; Yao et ai.

1997; Baqar et al. 1996). Colonization of moue intestine is thought to rely upon the

motility and chernotactic ability of C. jejuni since the bacteria colonize predominately

within the intestinal mucus layer without evidence of adherence to invasion into the

intestinal epithetium (Let et al. 1986). Increased levels and persistence of colonization

reported by Pei et al. (1998) and Yao et al. (1997) may have resulted h m culturing C.

jejuni wi thin the nutrient-rich biphasic system. Since C. jejmi strains T G W 1 1 and 8 1-

176 are commonly employed laboratory strains which have unàergone multiple passages

in vitro, it is possible that the efficiency of colonization in experimental animais,

including mice, might be increased if fiesh cIinical isolates were utilized,

Hodgson et al. (1998) f o n d that immunodeficient mice (CB-17SCID-Beige)

infected with clinical isolates of C. jejuni typically yield hifier levels of fecal shedding

(6.3~10~-i.0~10~ cWg of feces) than a laboratory-adapted C. jejmi strain (2 .3~10~ cfUg

of feces). Therefore. to assess the effect of a cipA mutation on the ability to colonize

mouse intestine, it may be usefd to clone and mutagenize the cipA gene h m a recent

clinical isolate.

Generation of anti-C~DA wlvclonal anti bodies

The CipA protcin was expressed in E coli using the GST-gene fusion system and

subsequently affinity-purifieci using Sepharose beads coated with glutathione. The

recombinant protein was then utilized as an antigen to immunize a New Zeaiand White

rabbit to generate polyvalent anti-CipA antibodies. Despite attempts to purify the rabbit

immune senim using affinity binding techniques (Smith and Fisher 1984; Iriarte et al.

1998), it recognized several other C. jejrmi proteins at an anb'body dilution required to

recognize the CipA protein within a C. jejuni whole-cell lysate. When utilized at a

dilution of 1 in 250 the antibodies could detect native CipA protein withui a whole-celi

lysate of C. jejuni. Western blotting indicated that CipA expression is not modulateci by

the bacteriai phase of growth nor was there was i n c d CipA protein expression

observed when C. jejuni was culturd in tissue culture medium (MI34 supplemented with

15% Fa).

The reasons for the additional immunoreactive proteins recognized by the anti-

CipA serum remain unclear. Although the purity of the recombinant CipA protein

preparation utilized for rabbit immunization was assessed by SDS-PAGE analysis, it is

possible that the protein preparation contained other proteins from the E. coli lysate used

to express the GST-CipA fusion protein. The presence of such contaminating proteins

could lead to the generation of additional antibodies which could cross-react with C.

jejuni proteins. Alternatively, the CipA protein may be antigenically similar to other C.

jejuni proteins thereby leading to the presence of other immunoreactive bands on the

Western blot. In this event, rabbit immunization using a peptide derived from the CipA

protein could be employed to d u c e the iikelihood of cross-reactivity with other C. jejMi

proteins. Another possibility is that CipA rnay be a minor protein within C. jejuni under

the growth conditions employed and at the relatively high concentration of antibody

required to detect the CipA protein, there axe nonspecific interactions (eg: hydrophobie)

between the antiCipA antibodies and other proteins on the nitrocellulose membrane.

Anaivsis of the ~redicted open readina frames adiacent to c i ~ A

Using the C. jejmi DNA sequence information available h m the Sanger Centre

website and the FASTA protein database, the predicted coding h e s adjacent to CipA

were determined with the aim of obtaining information about the passible function of

CipA (Figure II). CipA is immediately flanked by open reading fiames homologous to

the GCPE protein, which is a protein of unknown function in El coli, and the primosomal

protein N' which is involved in the assembIy of a replication-priming cornplex. Further

downstream are a number of open reading brames which encode for the components of a

potassium transport system and a sensor protein which responds to changes in turgor

pressure while a component of the flagellar basal body is located upstream of the c@A

gene. The CipA protein does not appear to resi& within a distinct cluster of proteins

with similar function which would aid in the characterization of the pratein.

Summarv

This study describes the initial chamcttnzation of the cipA locus within C. jejuni.

Disruption of the cipA allele resulted in a reduced internalization of the organism into

HEP-2 cells. A s idar phenotype was not observed when other celI lines were employed

It is well recognued that bacteriai vinilence involves a multitude of factors an4 as such,

the disruption of a single locus may not render the organism avident (Finlay and

Falkow, 1997). To c0nfi.m the biologicd rekvance of the in vitro findings of this study

and further investigate the d e of CipA in the bacteria-host cell interaction. it may be

necessary to utilize a more invasive s e of C. jejuni which could then be utilized for

both in vitro and in vivo analysis. In addition, the generation of anti-CipA antibodies

Figure 17: Redicted coding regions adjacent to CipA using the Sanger Centre

C. jejuni strain NCTC 11 168 genome database

NCTC 11168 Molecular Proposed function % id Predicted ORF weight Cj0678 1SkDa Potassium transporthg ATPase C chain 38% Cj0677 69kDa Sensor Protein KDPD 40% CjO68Oc 76kl)a Exinuclease ABC subunit B 64% Cj068 1 9kDa No homology Cj0682 9kDa No homology Cj0683 I7kDa No homology Cj0684 7lkDa Primosomal protein N' 45% Cj0685c CipA protein 54kDa No homology Cj0686 39kDa GCPE (Protein E) 62% CjO687c 3ctDa Flagellar basal body L-ring protein (FlgH) 39% Cj0688 S7kDa Phosphotransacetylase 44% Cj0689 44kDa Acetate kinase 53%

with a higher specificity toward the native CipA protein would allow for convenient

screening of a variety of C. jejuni ciinical isolates and laboratory strains to detemine

whether CipA expression could be comlated with the vinilence. In addition experiments

could be conducted to determine whether the presence of excess anti-CipA antibodies

would inhibit subsequent adherence or invasion of wild type C. jejuni into HEp2 cells

which would compfement the ni vitro findings in this study.

7.2 Future Directions

The functional characterization of CipA poses a challenge since the nucleotide

and amino acid sequences do not bear any striking homology to any sequences dcposited

within the NCBI databases. However, other avenues of investigation including detection

of protein-protein interactions, the use of reporter gene constructs and differential display

to detect host ce11 genes induced in response to infection could shed light on the

functional d e of CipA.

Pro tein- rotei in interactions

Affinity column chromatopph y and atZnity blotting techniques have been used

to identify protein-protein interactions between bacterial proteins and between

prokaryotic and eukaryotic proteins (Phizicky and Fields 1995; Rosenshine et al. 1996;

Sengupta et al. 1999; Zhou et al. 1999). Western immunoblot analysis performed by Dr.

A. Joe found that CipA was locaked to the cytoplasmic fraction of C. jemi cek. Since

the cipA mutant phenotype results in a reduced internalization within the HEp2 ceU

culture mode1 of infeçtion, it would be of interest to determine whether CipA is

associated with other C. jejuni proteins or more specifically, with membrane proteins. To

test whether CipA interacts with other C. jejuni proteins, wholeceil C. jejmi lysates

could be subjected to affinity chromatography using the GST-CipA fusion protein

immobilized ont0 Sepharose beads. The bound protein complexes could thm be

analyzed by SDS-PAGE (Phizicky and Fields 1995).

To specificaiiy invtstigate interactions with membrane proteins, Stngupta et al.

(1999) utilized biotinylation of whole cells to selectively label surface proteins foiiowed

by isolation of membrane fractions. The presence of protein complexes between the

CipA fusion proteins and biotinylated membrane proteins on an afflnity column could be

detected by immunoblotting. Altematively. C. jejuni celi lysates or subceuular fractions

could be separated by SDS-PAGE and transferred to nitrocellulose membrane. The

membrane would then be incubated with purifieci GST-CipA protein in a gel overlay

assay. Binding of the fusion protein to specific lysate proteins couId then be detected by

using monoclonal antibodies directed against the GST moiety. Control experiments

would employ the GST protein alone in the gel overlay assay. This technique was

utilized with success to demonstrate the binding of intimin, an outer membrane protein

from enteropathogenic E. col& to a protein originally designated as Hsp90 but later

renarned as Tir which is translocated h m the bacterium to the eukaryotic celi plasma

membrane (Rosenshine et al. 1996).

Cons tmction of a Cip A-GFP translational fusion

The use of the green fluorescent protein (gfp) h m Aeguorea v i c t o ~ as a reporter

gene has revolutionized the study of vinilence gene expression and potein localization

(Valdivia and Fallcow 1997). Construction of a protein fusion between gfp and the YopE

cytotoxin fkom Yersinia enierocoliîic~~ enabled the study of YopE expression under both

in vin0 and in vivo conditions (Jacobi et al. 1998). Similady, a gfpCipA translational

fusion present on either a low copy shuttle vector or, perhaps mort convtniently,

integrated into the C. jejuni chromosome would provide much idormation regardhg the

regulation of CipA producti~n. The gfpCipA producing straîn could be utilized to

investigate CipA expression and localization during adhmnce and intemalization of C.

jejuni into eukaryotic cells by employing confocal laser rannuig micmscopy. In

addition, the conaibution of CipA could be examined in the in vivo setting using anUnal

models of infection. Examination of intestinal sections for the presence of green

fluorescing organisms could be used to compare CipA protein expression using both

colonization models (chick, mouse) and models of carnpylobacteriosis (RITARD, ferret).

Differential d i s ~ l a ~ analvsis

Differential display PCR allows for the identification of induced mRNA

transcripts under defined environmental conditions (Liang and Pardee, 1992). This

technique has been employed to identify prokaryotic genes induced during infection of

host cells compared to organisms cultured in vitro (Abu Kwaik and Pederson 19%;

Zhang et al. 1996). At present, the identification of C. jejuni proteins induced during

interaction with host ceiis has relied upon two-dimensional protein gel electrophoresis

(Konkel and Cieplalc 1992a). The heightened sensitivity of differentiai display PCR

would allow for the detection of newly expresseci and dingenially expressed C. jejuni

genes which canno t be identifid using other s ystems. Altematively , di fferential display

PCR could also be used to examine the induction of host cell proteins in response to

infection with C. jejuni (Re- 1999) Since the disruption of the c@A d e l e results in a

reduced uptake of C. jejuni, cornparison of mRNA isolateci h m HEp-2 ceils infectecl

with wiId type C. jejuni or 901LK2 couid Iead to the identification of host ceIl rtsponsts

which are invasion-specific.

In conclusion, the study of the pathogenic mechanisms of C. jejmi has been

hampered in the past by the lack of mutagenesis strategies and difnculties associated with

molecular genetic techniques. However, with the completion of the C. jejrcni genome

sequencing projet coupled with novel methods to study differentid gene expression, it

should soon be possible to screen an array of genes comparing expression in the presence

of various stimuli (Pallen, 1999). In addition, as the number of sequenced microbial

pathogens increases, cornparisons between related organisrns or strains of the same

species wiil also contribute to studies of microbial pathogenesis (Alm et al. 1999; Behr et

al. 1999; Pallen, 1999).

Chapter 8

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