denning the role of cipa in the · list of abbreviations cdt cm cho ct edta fcs gbs gst il-8 int407...
TRANSCRIPT
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
National Library 1+1 of,, Bibiiothèque nationale du Canada
Acquisitions and Acquisitions et Bibliographie Services services bibliographiques
395 Wellingbn Stremt 395. rue WeWlgtm OttawaON KIA ON1 OttawaON K 1 A W Canada canada
The author has granted a non- exclusive licence dowing the National Library of Canada to reproduce, loan, distn'bute or sell copies of this thesis in microform, paper or electronic formats.
The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts fiom it may be printed or otherwise reproduced without the author's permission.
L'auteur a accordé une licence non exclusive permettant à la Bibiiothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur papier ou sur format électronique.
L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la îhèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.
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
References
References
Abu-Kwaik, Y., Federson, LA. (19%) The use of differential display-PCR to isolate
and c haracterize a Legionella pneumophila locus induced during the intraceiiular
infection of macrophages. Mol. Microbiol. 21: 543-556
Adam, T., Arpin, M., Prevost, MX., Gounon, P, Sansonetti, P J. (1995) Cytoskeletal
rearrangements and the hinctional role of T-plastin dming entry of Shigellaflexneri into
HeLa cells. J. Cell. Biol. 129: 367-381
Aisen, P., Leibman, A. (1972) Lactofemn md transfemn: a comparative shidy. Biochim.
Bioph y S. Acta 257: 3 14-323
Alderton, M.R, Komlik, V., Coloe, P J., Dewhirst, FE. and Paster, B J. (1995)
Campylobacter hyczilei sp. nov., associatexi with porcine prouerative enteritis. Int. J. Syst.
Bacteriol. 45: 61-66
Alrn, RmA.9 Guerry, P., Trust, TJ. (1993) The Cmpylobucter 6* promoter is subject to
environmental regdation. J. Bacterior. 175: 44484455
A h , RA., Ling, LS., Moir, D.T, King, B.L., Brown, E.D., Doig, P.C., et al. (1999)
Genomic-sequence comparison of two unrelateci isolates of the human gastxic pathogen
Helicobacter pylori Nature 397: 176- 180
AUos, B.M. (1997) Association between Cumgylobucter infection and Guillain-Bam
syndrome. J. Infect. Dis. 176 (Suppl2): S 125-8
AUos, B.M. and Blaser, MJ. (1995) Campylobacterjejuni and the expanding spectnim
of related infection. Clin. infect. Dis. 20: 1092-1 101
Altekruse, SF., Cohen, ML. and Swerdlow, D.L. (1997) Ernerging foodborne diseases.
Emerg. Infect. Dis. 3: 285-293
Alteknise, S.F., Stern, N Je, Felds, Pd., Swerdlow, DL. (1999) CampyIobucterjejuni -
An emerging foodbome pathogen. Emerg. Infect. Dis. 5: 28-35
Arico, B., m e r , J.F., ROY, C., Stibitz, S m , MOL~LIC~, D, Faikow, S., G m , R.,
Rappuoli, R (1989) Sequences required for expression of Bordetella pertussis Wulence
factors share homology with prokaryotic signal tnuisduction proteins. Roc. Natl. Acad.
Sci. USA 86: 667 1-6675
Aspinall, G.O., Fujimoto S., McDonald, A.G., Pmg, E., Kwjanczyk, LA., Pemer,
J.L. (1994) Lipopolysaccharides h m Cmnpylobacier jejuni associateci with Guillain-
Barre syndrome patients mimic human gangliosides in structure. Infect. Immun. 62:
2122-2125
Babakhani, F.K, Bradley, GA., Joens, LA. (1993) Newborn piglet model for
Campylobactenosis. Infect. Immun. 61: 3466-3475
Baqar, S., Bourgeois, AL., Applebee, LA, Mourad, AS., Kkinosky, MX., M o h
Z., Murphy, J.R. (1996) Murine intranasal challenge model for the study of
Campylobacter pathogenesis and immunity. Infect. Immun. 64: 49334939
Barrett, J.F., Hoch, J.A. (1998) Two-component signal transduction as a target for
microbial anti-in& tive therapy . Antimicrob. Agents. Chemother. 42: 1529-1 536
Behr, M.A. W i i n , MA., GUI, WE, Saiamon, H., Schoolnilc, G.K., Ranc, S., SrnaIl,
P.M. (1999) Comparative genomics of BCG vaccines by whole-genome DNA
microarray. Science 284: 1520-1523
Bieber, D., Ramer, S.W., Murray, W J, TobqT, Feninndez, R., Schoolnik, G.K.
(1998) Type IV pili. transient bacterial aggregates, and virulence of enteropathogenic
Escherichia coli. Science 280: 21 14-21 18
Bjorn, Md., Sokol, PA., Iglewski, B.H. (1979) Muence of iron on yields of
extraceliular products in Pseudomonas aemgkusa cultures. J. Bacteriol. 138: 193-200
Black, RmE, Levine, M.M., Clements, M L , Hughes, T9, Biaser, MJ. (1988)
Experimental Cmnpyiobacter jejuni infections in humans. J. Inftct. Dis. 157: 472479
Blaser, M J., Parsons, RB., and Wang, W.L. (1980) Acute colitis caused by
Campylobucter feîw ss. jejmi. Gastroentedogy. 78: 448453
Blaser, M Je, Duncan, D J, Warren, G.H., Wang, W.L. (1983) Experimental
Campylobacter jejuni infection of adult mice. Infect- Immun. 39: 908-916
Bras, A.M., Chatterjee, S., Wren, B.W., Newell, DeGw, Ketley, J.M.B (1999) A novel
Cmpylobacter jejuni two-component regulatory system important for temperature-
dependent growth and colonization. J. Bacteriol. 181: 3298-3302
Calderwood, SB, MeLnlanos, J Jw (1987) Iron regulation of Shiga-like toxin expression
in Escherichia coli is rnediated by thelfur locus. J. Bactenol. 169: 47594764
Caldwell, M A , Waker, RI., Stewart, SB., Rogers, JE. (1983) Simple adult rabbit
mode1 for Campylobacrer jejuni enteritis. Infect. Immun. 42: 1 176- 1 182
Chan, V.L., Louie, H., Bingham, E L w (1995) Cloning and transcription regulation of
the femc uptake regulatory gene of Campylobacter je- TGII9011. Gme. 164: 25-3 1
Chan, V L , Kim, NwW, Bourke, B., Eani, E., Ng, D, Lombardi, R, Bingham, H,
Hong, Y., Wong, T, Louie, & Cmpylobacter jejzmi TGH9û11 (ATCC 4343). In: de
Bmijn, F.J., Lupski, J.R., Weinstock, GM. (eds) Bacterial genornes: physical structure
and analysis. New York, Chapman and Hall, 1998: 633-636
Chang, N., Taylor, D.E. (1990) Use of pulsed-field agarose gel electrophoresis to size
CampyIobacter spp. genomes and to constmct a Sal1 map of Campylobacter j e m i
UA580. J . Bactenol. 172: 521 1-5217
Chiba A.S., Kusunoki, Hm, Obata, R, Machinami, R and Kanazawa, 1. (1993) Serum
anti-GQI b a n t i i y is associated with ophthalmoplegia in Miller Fisher syndrome and
GuiIlahBarre syndrome: clinical and immunohistochemical studies. Neurology.43:
1911-1917
Cornelis, G.R., Boland, A., Boyd, A.P., Geuijen, C, Iriarte, M, Neyt, C, Sory, M-P,
S tainier, 1. (1998) The vùulence plasmid of Yersiniu, an antihost genome. Microbiol.
Mol. Biol. Rev. 62: 1315-1352
Cossart, P. (1997) Subversion of the mammaiian cell cytoskeleton by invasive bacteria.
J. Clin. Invest. 99= 2307-23 11
Cover, T.L., and Blaser, MJ. (1 989) The pathobiology of Campylabacter infections in
humans. Annu. Rev. Med. 40: 269-285
Crowe, S B , Alvarez, Lm, Dytoc, M., Hunt, R H , Mdler, M., Sherman, P, Patel, J,
Jin, Y., Ernst, P.B. (1995) Expression of interleukin-8 and CD45 by human gastric
epithelium after Helicobucterpylori infection in vitro. Gastroenterology- 108: 65-74
Daikoku, T., Kawaguchi, Mw, Takama, K., Suaikf, S. (1990) Partial purincation and
characterizatmion of the enterotoxin producd by Cmnpylobucter jejni. Infat. Immun. 58:
2414-2419
Darfeuille-Miehaud, A, Fomsalr, C, Joly, B, Cluzel, R (1986) Identification of a
nonfimbrial adhesive factor of an enterotoxigenic Escherichia coli strain. Infect Immun.
60: 44-55
De Melo, M.A., Gabbiani, G., Pechere, J-C. (1989) Cellular events and intraceiiular
suMval of CumpyIobacterjejmi during infection of HEp-2 ceils. infect. Immun. SI:
22143222
Doig, P., Yao, R, Bwr, D.H., Guerry, P, Tmst, TJ. (1996) An environmentally
regulated pilus-like appendage invoived in Campylobacter pathogenesis. Moi. Micmbiol.
20: 885-894
Donnenberg, MA, Nataro, J.P. (1995) Meihods for studying adhesion of diarrheagenic
Escherichia coli. Me&. Enzpol. 253: 324-336
Duffy, M C , Benson, J.B., Rubih, S J. (1980) Mucosal invasion in Campylobocter
ententis. Am. J. Clin. Pathol. 73: 706-8
Eckmann, L., Ka-, M.F., Fierer, Je (1993) Epithelial ceiis secrete the chemokine
interleukin-8 in response to bacterial entry. Infect. Immun. 61: 45694574
Elsinghorst, E.A. (1994) Measurement of invasion by gentamicin resistance. Meth.
Enzymol. 236: 405-420
Endtz, R.P., Ruijs, G J., van Küngeren, B., Jansen, W.H., van der Reyden, T. and
Mouton, R.P. (199 1) Quinolone resistance in Campylobactct isoiatcd h m man and
poultry following the introduction of fiuoroquinolones in veterinary medicine. J.
Antimicrob. Chemother. 27: 199-208
Everest, P.He, Goossens, H., Sibbons. P., Lloyd, D.R., Knutton, S., Leece, R, Ketiey,
J.M.9 W i , P.& (1993) Pathological changes in the rabbit ileal loop mode1 c a u d
by Campylabacter jejuni from human colitis. 1. Med. Mimbiol. 38: 3 16-321
Fauchere, J.L, Rûsenau, A., Veron, M.., Moyen, EN., Richard, S., Poster, A. (1986)
Association with HeLa cells of C'lobacter jejmi and Campylubacîer culi isolated
from human feces. Infect, Immun. 54: 283-287
Fauchere, JL, KeweUa, M., Rosenau, A., M o h a n ~ , K, Veron, M. (1989) Adhesion
to HeLa cells of Campylobacter jejuni and C. coli outer membrane components. Res.
Microbiol, 140: 379-392
Finlay, B.B., Ruschkowski, S., Dedhar, S. (1991) Cytoskeletal reanangements
accompanying Salmonella entry into epithelial cells. J. Cd. Sci. 99: 283-2%
Finlay, B.B., Falkow, S. (1997) Common themes in microbial pathogenicity revisited.
Microbiol. Mol. Biol. Rev. 61: 136-169
Fox, J.G., Ackerman, JJ., Taylor, N., Claps, M., Murphy, J.C. (1987) Campyllobacter
jejuni infection in the ferret: An animal mode1 of human campylobacteriosis. Am. J. Vet.
Res. 48: 85-90
Galan, JS., Collmer, A. (1999) Type IiI secretion machines: bacterial devices for
protein delivery into host celis. Science. 284: 1322- 1328
Gaudreau, C. and Gilbert, H. (1998) Antimicrobial resistance of clinical strains of
Cnmpylobacter jejmi subsp. jejuni isolated from 1985 to 1997 in Queùec. Canada.
Antimicrob. Agents Chemother. 42: 2106-2108
Gaunt, PA, Piddock, L J.V. (1996) Ciprofloxacin resistant CampyZobacter spp. in
humans: an epidemiological and laboratory study. J. Antimicrob. Chemother. 37: 747-
757
Goodwin, CS, Armstrong, J.A., ChSlvers, Tm, Peters, M., Collins, Mm., Sly, L,
Mccomeu, W. and Harper, WXS. (1989) Transfer of Campylobacterpylori and
Cmpylobaeîer mustelue to Helicobacter gen. nov. as Helicobacîer pylori comb. nov. and
Helicobcicter musrtelm comb. nov., respectively. Int J. Syst. Bacteriol. 39: 3 9 7 4 5
Goossens, &, Rummens, E., Cadranel, S, Butzier, JPP., Takeda, Y. (1985) Cytotoxic
activity on Chinese hamster ovary ceils in culture filtrates of Cmnpylobacter jejunkli.
Lancet ü: 203-215
Grant, C.C.R., Konkel, MX., CieplsL, W., T o m p k . LS. (1993) Role of flagella in
adherence, intemalization, and translocation of Campyllobacter jejuni in nonpolarized and
polarized epithelid ce11 cultures. Infect. Immun. 61: l764-l77l
Groisman, EA. (1998) The ins and outs of virulence gene expression: MC as a
regulatory signal. BioEssays. 20: %-IO1
Guerrant, RL, Wanke, C.A., P e d e , RA, Barrett, L J., Lima, AAM- O'Brien,
A D . (1987) Production of a unique cytotoxin by Cmnpylobacter jejuni. Infect. Immun.
55: 2526-2530
Guerry, P., Ah, A-R, Power, MA, Logan, SM., Trust, T J. (1991) Role of two
flagellin genes in Campylobacter motüity. J. Bacteriol. 173: 47574764
Guerry, P., Yao, R, Alm, RA., Burr, DI&, Trust, T J. (1994) Systems of
experimental genetics for Campyiobucter species. Methods Enzymol. 235: 474481
Guiney, DaGw (1997) Regulation of bacterial vuulence gene expression by the host
environment. J. Clin. Invest. 99: 565-569
Hani, EwK (1997) Hippurate hydrolase gene of CimpyZobucter jejMi. Pm. Thesis.
University of Toronto, Toronto, Canada.
Harvey, P., Battk, T, Leach, S. (1999) Different invasion phnotypes of
Campylobacrer isolates in Caco-2 ceil monolayers. J. Med. Microbiol. 48: 461-469
Henrich, B., FeldmPnn, RC., Haddùig, U. (1993) Cytoadhesins of Mycopl<rmio
hominis. Infect, Immun. 61: 2945-2951
Herrington, DA., Hall, RH., Losonsky, G., Mekalanos, JJ, Taylor, RK., Levine,
MaMW (1988) Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio
cholerae pathogenesis in humans. J. Exp. Med. 168: 1487-1492
Hersh, Dm, Monack, DM., Smith, MX., Ghori, N., Falkow, S., Z y c b k y , A. (1999)
The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1.
Roc. Natl. Acad Sci. USA %: 23%-2401
Hickey, T.E., Baqar, S., Bourgeois, A.L., Eaiag, CR, Guerry, P. (1999)
Campylobacter jejmi-stimulated secretion of interleukùi-8 by INT407 cells. Infect.
Immun. 67: 88-93
High, Na, Mounier, J , Prevost, M.-C, Spnsoneîü, P J. (1992) IpsB of Shigelkflaaen
causes entry into epithelial ceIls and escape h m the phagocytic vacuole. EMBO J. Il:
1991-1992
Hilbi, Hm, Moss, JE., Hersh, D., Chen, Y., Aronde], Je, Banerjee, S., Flavell, RA.,
Yuan, J., Sansonetti, P J., Z y c b k y , A. (1998) Shigella-induced apoptosis is
dependent on Caspase-1 which binds to IpaB. J. Biol. Chem. 273: 3289532900
Ho, T.W., Mishu, B., Li, C.Y., et ai. (1995) Guillain-Barre syndrome in northem China:
relationship to Cmpylobucteer jejuni infection and anti-glycolipid antibodies. Brain. 118:
597-605
Ho, T.W., W i n , HJ, Nacbsmkin, I., Li, C.Y, Veitch, J, Ung, EL, Wang, G.R.,
Liu, RC., Comblath, D.R., Asbury, AmK., Griffin, JmW., McRliann, G.M. (1999)
Anti-GDla antibody is associateci with axonal but not demyelinating forms of Guillain-
Barre syndrome. Ann. Neurol. 45: 168-173
Hodgson, A.E., McBride, B.W., Hudson, MJ., Hail, G., Leach, S.A. (1998)
Experirnental campylobacter infection and diarrhoea in immunodeficient mice. J. Med
Microbiol. 47: 799-809
Hoschutzky, Ha, Niauaich, W , Lttspeich, F., J ~ M , K. (1989) Isolation and
characterization of the non-fimbnal adhesin NFA4 from uropathogenic Eschekhia coli
07: K98: H6. Microb. Pathog. 6: 351-359
Hueck, C J., Han-, M J., Bajaj, V., Johnston, C., Lee, A., Miller, S.I. (1995)
Salmonella typhimuriwn secreted invasion determinants are homologous to Shigella Ipa
proteins. Mol. Microbiol. 18: 479490
Hueck, CJ. (1998) Type IIï protein secretion systems in bacterial pathogens of animals
and plants. Microbiol. Mol. Biol. Rev. 62: 379433
Ireton, K., Payrastre, B., Chap, II, Ogawa, W., Sakaue, E, Kasqa, M, Cossart, P.
(1996) A role for phosphoinositide-3-kinase in bacterial invasion. Science. 274: 780-782
Iriarte, M., Sory, M-P., Boland, A., Boyd, AS., Ming SB., Lambermont, M,
Corneiis, G.R. (1998) TyeA, a protein involved in control of Yop release and in
translocation of Yersinia Yop effectors. EMBO J. 17: 1907-1918
Jacobi, C.A., Roggenkamp, A., Rakin, A., Zumbihi, R., Leitritz, L., Heesemann, Jm
(1998) In vitro and in vivo expression studies of yopE from Yersihiu enterocolitica using
the gfP reporter gene. Mol. Microbiol. 865-882
Joe, A, Bourke, B., Hong, Y, Louie, H, Sherman, P., Chan, V.L. (1998)
Identification of a Campylobucter jejuni gene @@A) associated with adherence and
invasion of human intestinal cells. In: Lastovica, A.J., Newell, D.G, Lastovica, E. E.
(eds), Campylobacter, Helicobacter and nlated organisms: Proceedings of the 9*
International Workshop. Institute of Child Health, Cape Town. pp. 290-295
Johnson, W.M., Lior, Ho (1984) Toxins produced by Campylobacterjejuni and
Campylobacter colt'. Lancet. i: 229-230
Johnson, W.M., Lior, Ho (1986) Cytotoxic and cytotonic factors produced by
Campylobacter jejuni and Campylobacter coli. J. Clin. Microbiol. 24: 275-28 1
Johnson, WmMm, Lior, H. (1988) A new heat-Iabile cytolethal distendhg toxin (CLDT)
produced by Campyfobmter spp. Microb. Pathog. 4: 1 15-126
Kagnoff, M.F., Eckmann, La (1997) Epithelial ceils as sensors for microbial infection. J.
Clin, Invest. 100: 6-10
Kaniga, K., Tucker, S., Troliinger, Dm, Galan, JE (1995) Homologs of the Shigelh
IpaB and IpaC invasins are required for Salmonella typhimunUm entry into cultured
epithelial cells. J. Bacteriol. 177: 3965-39'71
Kaper, J.B, Glenn Morris JR, J, Levine, MM. (1995) Cholera. Clin. Microbiol. Rev.
8: 48-86
Keile, K., Pages, J M , Bolla, JmMo (1998) A putative adhesin gene h m Cumpyloboctcr
jejuni. Res. Microbiol. 149: 723-733
KerveMa, M., Pages, J.M., Pei, 2, Grollkr, G , Bisser, W.., Fauchere, J L (1993)
Isolation and characterktion of two Campyfobafler glycine-extracted proteins that bind
to HeLa ce11 membranes. Infect. Immun. 61: 3440-3448
Ketley, J.M. (1995) Virulence of Campylobocter species: a molecular genetic approach.
J. M d . Microbiol. 42: 3 12-327
Ketley, JoMm (1997) Pathogenesis of enteric infcctioa by Compylobacter. Micmbiology.
143: 5-21
Khawaja, R, Neote, K., Bingham, H.L., Pemer, JL., Chan, VL. (1992) Cloning and
sequence analysis of the flagellin gene of Campylobucter jejuni TGH9û11. Curr.
Microbiol. 24: 213-221
Kiehlbauch, J A , Albach, RA, Baum, L L , Chang, K.-P. (1985) Phagocytosis of
Cmpylobacrer jejuni and its intracellular survival in mononuclear phagocytes. Infect.
Immun. 48: 446451
Kim, N.W., Bingham, H., Khawaja, Re, Louie, H., Hani, E., Neote, K., Chan, VA.
(1992) Physical map of Cmpylobacter jejuni TGH9ûl1 and the 1ocaIization of ten
genetic markers using pulsed-field gel electrophoresis. J. Bactenol. 174: 3494-3498
Kim, N.W., Lombdi , H., Biagham, &, Hani, E, Louie, H., Ng, D., Cban, V L
(1993) Fine mapping of the three rRNA operons on the updated map of Campylobacter
jejuni TGH9û 1 1 (ATCC 4343 1) J. Bacteriol. 175: 7468-7470
Klipstein, FA, Eagert, R.F. (1984) Properties of crude Cmnpylobacter jejuni heat-
Iabile enterotoxin. Infect. Immun. 45: 3 14-3 19
Klipstein, FA, Eogert, R.F. (1985a) Imrnunological relationship of the B subunits of
Campylobacter jejuni and Escherichia coli heat-labile enterotoxins. Infect Immun. 48:
629-633
Klipstein, FA, Eogert, R.F., Short, Hm, Schenk, EAm (198%) Pathogenic properties of
Cmpylobacter jejuni: assay and comlation with clinicaI manifestations. Infect. Immun.
58: 43-49
Konkei, MX., Joens, LA. (1989) Adhesion to and invasion of HEp-2 ceils b y
Campylobacter spp. Infect. immun. 57: 29842990
Konkel, MX., Babakhani, Fm, Joens, LA. (11990) Invasion-dated antigens of
Campylobacter jejuni. J. infect. Dis. 162 888-895
Konkel, M E , Cbplak Jr, W. (1992a) Altered synthetic response of Ccunpylob~~~ter
jejuni to cocultivation with human epithelial ceiis is associated with enhanced
internaiization. Infect. Tmmun. 60: 49454949
Konkel, MmE.9 Corwin, MD., Joens, LA., Cieplak Ji., W. (1992b) Factors that
influence the interaction of Compylobacter jejuni with cultwed mammalian cells. J. M d
Microbiol. 37: 30-37
Konkel, MX., Hayes, SB, Joens, L A , Cieplak Jr., W. (1992~) Characteristics of the
intemalization and intracellular sunrival of Cmpylobacter jejuni in human epithelial ceU
cultures. Microb. Pathog. 13: 357-370
Konkel, MX*, Lobet, Y., Cieplak, W. (1992d) Examination of multiple isolates of
Campylobacter jejuni for evidence of cholera toxin-iike activity. In. Nachamkim, 1..
Blaser, MJ., Thompkins. L.S., (eds), Cmnpyloboctcr jejuni: current status and future
trends. Amencan Society for Microbiology , Washington, D.C pp 193- 198
Konkel, MX., Mead, DJ., Hayes, S J., Ciepiak Jr,W. (1992e) Translocation of
Campylobacter jejuni across human polarized epithelial cell monolayer cultures. J. uifect.
Dis. 166: 308-3 15
Konkel, MX., Mead, D J., Cieplak Jr. W. (1993) Kinetic and antigenic characterizatiou
of altered protein synthesis by Ccuttpylobucter jejwu' during cuitivation with human
epithelid cells. J. Infect. Dis. 168: 948-954
Konkel, M.E, Cieplak Jr., W. Molecular pathogenesis of Cmnpyllobacter j e m i
enteritis. In: Paradise, L.J. Bendinelli, M., Friedman, H. (eds) Enteric Infections and
Immunity. New York, Plenum Press. 19%: 133-147
Konkel, M.E., Garvis, S.Gq Tipton, S.L., Anderson Jr, D.E., Cieplak Jr, W. (1997)
Identification and molecular cloning of a gene encoding a fibronectin-binding protein
(CadF) fiom Campybbacter jejuni. Mol. Microbiol. 24: 953-963
Konkel, MX., Kim, BJ., mena, J.D., Young, C.R., Ziprin, R (1998) Characterïzation
of the thermal stress response of Cumpybbacterjejuni. Infect. Immun. 64: 3666-3672
Konkel, M.E., Gray, S.E., Kim, B J., G(LfViS, S.G. Yoon, J. (1999a) Identification of
the enteropathogens Campylobacrer jejuni and Campylobacter coli based on the cadF
virulence gene and its product. J. Clin. Microbiol. 37: 510-517
Konkel, MX., Kim, B J., Rivera-Amill,V., Garvis, SIG. (1999b) Bacterial secreted
proteins are required for the intemalization of Campylobacter jejuni into culnired
mammalian cells. Mol. Microbiol. 3% 691-701
Korlath, J.A., Osterholm, M.T., Judy, LA., F o r f ~ g , JeC, R o b i i n , RA. (1985) A
point-source outbreak of campylobacteriosis associateci with the consumption of raw
rniik. J. Infect. Dis. 152: 592-596
127
Kuusela, P., Moran, AR., Vartio, T., Ko~unen, T.U. (1989) Interaction of
Campylobacterjejzmi with extracellular matrix components. Biochem. Biophys. Ac=
993: 297-300
Labigne-Roussel, A., H a d , J., TompLLag L. (1987) Gcne trausfer h m Escherichiu
coli to Campylobacfer spcies: development of shuttle vecton for genetic analysis of
Campylobacîer jejuni. J. Bacteriol. 169: 5320-5323
Labigne-Roussel, A., Courcow P., Tompicias, L. (1988) Gene disruption and
replacement as a feasible approach for mutagenesis of Cmpylobacter jejmi. I. Bacteriol.
170: 1704-1708
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature. 227: 680-685
Lai-King, No, Sherburn, R, Taylor, DE., Soks, MX. (1985) Morphological forms and
viability of CampyZobacter species studied by electron microscopy. J. Bacteriol. 164:
338-343
Lee, A, 09Rourke+ J.L., Barrington, P JO, Tnist, T Je (1986) Mucus colonization as a
determinant of pathogenicity in intestinal infection by Cmpylobacter jejuni: A mouse
cecal model. Infect. Immun. 51: 536-546
Lee, V.T., Schneewind, O. (1999) Type III machines of pathogenic Yminiae secrete
vinilence factors into the extracellular milieu. Mol. Mimbiol. 31: 1619-1629
Liang, Pm, Pardee, AB. (1992) Diffe~ntial display of eukaryotic messenger RNA by
means of the polymerase chah reaction. Science. 257: 967-971
Lindsay, J.A. (1997) Chronic sequelae of foodbome disease. Emrg. Infect Dis. 3: 443-
452
Lior, H., Woodward, DmL., Edgar, JA, Laroche, L J, GUI, P. (1982) Semtyping of
Campylobacter jejwii by slide agglutination based on heat-labile antigenk factors. J.
Clin. Microbiol. 15: 76 1-768
Litwin, C.M., Caidernood, SB. (1993) Role of iron in regulation of virulence genes.
Clin. Microbiol. Rev. 6= 137-149
Mangm, D.F, Wahi, SmMe (1991) Differential regulation of human monocyte
programmeci cell death (apoptosis) by chernotactic factors and pro-inflammatory
cytokines. J. Immunol. 147: 3408-3412
McCardell, B A , Madden, J.M., Stnnneld, JmTm (1986) Production of cytotoxins by
Campylobacter. Lancet. i: 1031
McCleUaad, Ma, Jones, R, Patei, Y., Nelson, M. (1987) Restriction endonucleases for
pulsed-field mapping of bacterial genomcs. Nucl. Acids. Res. 15: 59856005
Meinersmaon, R., Pei, Z., Blaser, M J. (1996) Capacity of a pebla mutant of
Campylobacter jejiur to colonize chickens. In: Newell, DG., Ketley, J.M., Feldman,
R.A. (eds) Campylobacter, Helicobacter and Related Orgnnisms. (eds) New York:
Plenum Press
Mekalanos, J J. (1992) Environmental sipals controlling expression of Wulence
determinants in bacteria J. Bacteriol. 174: 1-7
m e r , J.F., Dower, W J., Tompldns, LSm (1988) High-voltage electroporation of
bacteria: Genetic transformation of Cmngylobocreer jejuni with plasmid DNA. Roc. Natl.
Acad. Sci. (USA) 85: 856-860
Miller, J.F., Mekalanos, J Je, Fakow, S. (1989) Coordinate regulatim and sensory
transduction in the control of bacmiai vinilence. Science. 243: 916-922
Miller, S.I., Kukral, AM., Mekalanos, J J. (1989) A twocomponent regdatory system
@hop phoQ) conîrols Salmonella typhirnurium vinilence. Proc- Natl. Acad Sci. USA 86.
5054-5058
Mishu, B e , Iiym, AA, Ko~ki, CL, Vriesendorp, F, Cook, SDm, Mithen, FA,
Blaser, M J. (1993) Serologic evidence of previous Cmpylobucter jejuni infection in
patients with the Guillain-Barre syndrome. Ann. I n m . Mecl. 118: 947-953
Moran, AP., Penner, J.L. (1999) Seroîyping of Campylobacterjejuni bascd on htst-
stable antigens: devance, mokcular basis and implications in pathogenesis. J. Appl.
Microbiol. 86= 36 1-377
Moser, I., Schroeder, W., Salaikow, J. (1997) Cmpylobucter jejmi major outer
membrane protein and a 59 kDa protein are involved in binding to fibmnectin and INT
407 ce11 membranes. FEMS Microbiol. Lett. 157: 233-238
Nachamkin, 1.9 AUos, BMm and Ho, Tm (1998) Cmnpylobc~cter species and Guillain-
Barre Syndrome. Clin. Microbiol. Rev. 11: 555-567
Neisser, A., Bernheier, E, Berger, Te, Moran, APm, Schwerer, B. (1997) Senun
an ti bodies agains t gangliosides and Chpylobacrer jejuni lipopol ysaccharides in Miller
Fisher syndrome. Infect, Immun. 65: 4038-4042
Neweii, D.G., McBride, He, Dolby, J.M. (1985) Investigations on the mle of flagella in
the colonization of infant mice with Cmnpylobacter jejlmi and attachent of
Campylobacterjejuni to human epitheiial cell iines. J. Hyg. (Camb). 95: 217-227
Nuijten, P J.M., Barteis, C., Bleumink-Piuym, NMeC., Gaastra, W., van der Zeiist,
BAM. (1990) Size and physical map of the Campylobacter jemi chromosome. Nucleic
Acids Res. 18: 621 1-6214
Oelschlaeger, TA., Guerry, P, Kopedro, D J. (1993) Unusual microtubule-dependent
endocytosis mechanisms ûiggered by Campylobacter jejuni and Ci~obacterfreundn.
Proc. Nad. Acad Sci. USA 90: 6884-6888
Olsvk, O., Wachsmuth, K., Morris, G , Feeley, J-C. (1984) Genetic probing of
Cmpylobacter jejrcni for cholera toxin and Escherichia coli heat-labile enterotoxin.
Lancet. ii: 449
On, SaLaW. (1996) Identification methods for Campyiobacters, HeIicobacters, and rdattd
organisms. Clin- Microbiol. Rev. 9: 405422
Pallen, M J. (1999) Microbial genomes. Mol. Microbiol. 32: 907-912
Panigrahi, P., Losonsky, G., DeToiia, L J, Glenn Morris Jr., J. (1992) Human
immune response to Campylobacter jejmi pmteins expressed in vivo. Infect. immun 60:
49384944
Pei, Z., Ellison, KT., Blaser, M J. (1991) Identification, pudication, and
characterization of major antigenic proteins of Cmnpyfobacter jejuni. J. Biol. Chem- 26ik
16363-16369
Pei, Z., Blaser, M J. (1993) PEB 1, the major cell-binding factor of Campylobacîer
jejmi, is a homolog of the binding component in Gram-negative nutrient transport
systems. J. Biol. Chem. 268: 18717-18725
Pei, Z., Bumcoa, Ca, Grignon, B., Baqar, S., Huang, X., Kopecko, J., Bourgeois,
A-L., Fauchere, JL, Blaser, M J. (1998) Mutation in the peblA locus of
Campylobacter jejuni reduces interactions with epithelial ceUs and intestinal colonization
of mice. Infect. Immun. fi 938-943
Pemer, JL, Hennessy, J.N., Congi, RV. (1983) Semtyping of Campylobucter jejrmi
and Cumpylobacter coli on the basis of thermostable antigens. Eur. J. Clin. Microbiol. 2:
378-383
Penner, JmL- (1988) The genus CampyZobocter. a decade of progress. Clin Microbiol.
Rev. 1: 157-172
Pennisi, E. (1999) First food-borne pathogen sequenced. Science. 283: 1243
Phizicky, EX, Fields, S. (1995) Protein-protein interactions: methods for de-on and
analysis. Microbiol. Rev. 59: 94-123
Pickett, CL., P e i , EmC, Cottle, D.L., RusseU, G., Erdem, NA., Zeyth, EL (1996)
Prevalence of cytolethal distending toxin production in C-obacter jejwi and
relatedness of Compylobucfer sp. CM genes. Infect, Immun. 64: 2070-2078
Prescott, J.F., Barker, I.K., Manninen, K.I., Miniats, O.P. (1980) Campytobacter
jejuni colitis in gnotobiotic dogs. Can. J. Comp. Med 45: 377-383
Ricciareüi, M.G., F i y , B.B (1994) Polarized epithelial monolayers: Mode1 systems to
study bacterial interactions with host epithelial cells. Meth. Enymol. u6: 438-447
Purdy, Dm, Park, SS. (1993) Heterologous gene expression in Cumpylobacter coli: the
use of bacterial luciferase in a promoter probe vector. FEMS Microbiol. Leit Ill: 233-
238
Rabinowitz, R.P., D~niienborg, MS. (19%) Escherichio coli. Paradise, LJ. Bendinelli.
M., Friedman, H. (eds) Enteric Infections and lmmunity. New York, Plenum Press. 19%:
pp. 101- 131
Rees, J.H., Soudain, S.E., Gregwii, NA. and Hughes, RA. (1995) Campytobacter
jejuni infection and Guillain-Barn syndrome. N. Engl. J. Med 333: 1374-1379
Relman, D.A. (1999) The search for unrecognized pathogens. Science 284: 1308-1310
Rosenshine, 1, Ruschkowski, S., Stein, M., Reinscheid, DJ., Mills, Sm., F ' i y , B.B.
(1996) A pathogenic bacterium triggers epitheliai signais to form a functionai bacterial
receptor that mediates actin pseudopod formation. EMBO J. 15: 2613-2624
Ruiz-Paiacios, G.M., T o m , J., Torres, NJ., Escamilla, E., Ruiz-Palacios, B.R.,
Tamayo, J. (1983) Cholera-like enterotoxin produced b y Cmnpylobacter jejuni. Lancet.
ü: 250-253
Russen, RG., O'Domoghue, M., Blake, D.C, Zulty, J , DeTolls, L J. (1993) Early
colonic damage and invasion of Cmpylobucter jejuni in experirnentally challenged
infant Macaca mulana. J . Infect. Dis. 16û: 210-215
Russell, R.G., Blake, D.C. Ji. (1994) Ce11 association and invasion of Cam-2 cells by
Campybbacter jejuni. Infect. Immun. 62: 3773-3779
Sambrook, J., Fritsch, ES., Maniatis, T. (1989) Molecular cloning: a laboratory
manual, 2nd ed. Cold Spring Harbor Laboratory Ress, Cold Spring Harbor, New York
Sanchez, R, Fernandez-Baca, V., Diaz, Mm., Munoz, P., Rodrigue-Creixems, M,
and Bouza, E. (1994) Evolution of susceptibilities of Campyloacter spp. to quinolones
and macrolides. Antimicrob. Agents Chemother. 38: 1879-1882
Sengupta, S., Tripathi, J , Tandon, R, Raje, M., Roy, W., Basu, S.&,
Mukhopadhyay, A. (1999) Hemoglobin endocytosis in Leishamrmia is mediateci through
a 46kDa protein located in the fiagellar pocket. J. Biol. Chem. 274: 2758-2765
Schroder, W., Moser, 1. (1997) Rimary structure analysis and adhesion studies on the
major outer membrane protein of C~tpylobucter jejmi. FEMS Microbiol. Lett. 150:
141-147
Sharetzsky, C, Edlind, T.D., LiPum, JL., Stull, T L (1991) A novel approach to
insertional mutagenesis of Haemophilus i n f i m . J. Bacterioi. 173: 156 1-1564
Sharma, S.A., Tummuru, M.K.R., Miller, G.G., Blaser, M J. (1995) Interleukin-8
response of gasaic epithelial cell lines to Helicobucter pylon stimulaîion in viîro- Infect.
Immun. 63: 1681-1687
Shaul, P.W, Anderson, R G W . (1998) Rule of piasmalemmal caveolae in signal
transduction. Am. J. PhysioI. 275: Lû43-L85I
Shenker, BJ., McKay, Tm, Datar, S., Miller, M., Chowhia, R, Demuth, Da (1999)
Actinobacillus actinomycetemcomitans immunosuppressive protein is a member of the
family of cytolethal distending toxins capable of causing a G2 a m s t in human T ceiis. J.
Immunol. 162: 4773-4780
Shigematsu, M, Umeda, A., Fujimoto, S., Amako, K. (1998) Spirochaete-likt
swimming mode of Cmpylobacter jejmi in a viscous environment. J. Me& Microbiol.
47: 52 1-526
Skirrow, M.B. (1977) Cumpylobacter enteritis: a new disease. BMJ. 2: 9-1 1
Smith, K.E., Besser, J.M, Hedberg, C.W., Leaw, F.T., Bender, J.B., Wicklund,
J.H., Johnson, B.P., Moore, ICA., Osterholm, M.T. (1999) Quinolone-resistant
Cmpylobacter jejuni infections in Minnesota, 1992-1998. N. Engl. J. M d 340: 1525-
1532
Smith, D.E., Fisher, PA. (1984) Identification. developmental regulation, and response
to heat shock of two antigenicdy related forms of a major nuclear envelope protein in
Drosophila embryos: application of an improved method for affinity purification of
antibodïes using polypeptides immobilized on nitroceiiulose blots. J. CeU. Biol. 99: 20-
28
Soto, GE., Hultgren, S JO (1999) Bacteriai adhesins: common themes and variations in
architecture and assembly. J. Bacteriol. 181: 1059-1071
St, Geme, J.W. (1997) Bacterid adhesins: detenninants of microbial colonization and
pathogenicity. Adv. Pediatr. 44: 43-72
Steiner, T.S, Lima, A.A.M., Nataro, J.P., Guerrant, RL. (1998) Enteroaggregative
Escherichk coli produce intestinal inflammation and growth impairment and cause
interleulàna release fiom intestinal epitheliai ceils. J. Infect. Dis. 177: 88-96
Stern, N J. (1992) Reservoirs for Cmpylobacter jejuni and approaches for intervention
in poultry In: Nac hamkin, I., Blaser, M. J., Tompkins, L.S. (eds) Campylobucter jejuni:
Current Status and Future Trends. Washington DC, American Society for Microbiology.
pp. 49-60
Szymanski, CM., King, M, Haardt, M., Armstrong, G a . (1995) Campylobacrer
jejuni motility and invasion of Caco-2 cells. Infiit. Immun. 63: 42954300
Tauxe, RV. (1992) Epidemiology of Campylobacter jejwi infections in the United
States and other industrial nations In: Nachamkin, I., Blaser, M.J., Tompkins, L.S. (eds)
Campylobacter jejuni: Current S tatus and Future Tmds. Washington DC, American
Society for Mimbiology. pp. 9-12
Taylor, D.E. (1992) Genetics of Campylobacter and Helicobacter. Annu. Rev.
Microbiol. 46: 35-64
Taylor, D.N. (1992) Campylobacter jejuni infection in developing countries. In:
Nachamkin, I., Blaser, MJ., Tompkins, L.S. (eds) Cartlpylobacter jejunR Cumnt Stanis
and Future Trends. Washington DC, Amencan Society for Mimbiology. pp. 20-30
Thomas, C., Hiil, D Je, Mabey, IW (1999) Morphological changes of synchronized
Campylobacter jejwri populations during growth in single phase liquid culture. Lem
Appl. Microbiol. 28: 194-198
Thompson, L.M., Sniibert, RM. Johnson, J.L. and Krieg, N.R. (1988)
Phylogenetic study of the genus Campylobacter. Int. J. Syst Bacteriol. 38: 190-200
Towbin, H , Staehelin, Ta, Gordon, JO (1979) Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Roc. Natl.
Acad- Sci. USA 76: 4350-4354
Valdivia, RH., Faikow, S. (1997) Fluorescence-bascd isolation of bacterial genes
expressed wi thin host cells. Science. 277: 2007-20 1 1
Van Spreeuwel, J S , Duursma, G.C., Meijer, C J.LM.t Bnq R, Rosekrans, P.C.M.,
Lindeman, J. (1985) Campylobacter colitis: histologicd immuno-histochemical and
uitrastructural findings. Gut. 26: 945-951
van met, A.H.N., Wooldridge, KG., Ketley, JoMa (1998) Mn-responsive gene
regulation in a Canpylobacter jejwi fur mutant. J. Bactenol. 180: 5291-5298
Vandamme, P. and De Ley, Jo (1991a) Proposa1 for a new family, Cmnpylobc~cteraceae.
Int. J. Syst. Bacteriol. 41: 451455
Vandamme, P., Falsen, E., Rossau, R., Hoste, B., Segers, P., Tytgat, R a d De Ley,
J. (199 1b) Revision of Campylobacter, Helicobacter. and W o l ~ l l a taxonomy:
emendation of genenc descriptions and proposal of Arcubacter gen. nov. Int. J. Syst
Bacteriol. 40: 88-103
Vandamme, P., Daneshvar, M.I., Dewhirst, FE. Paster, B J, Kersters, K.,
Goossens, H. and Moss, CW. (1995) Chemotaxonomic analysis of Bacteroides grocilis
and Bacteroidès uerolyticus and reclassifïcation of B. gracils iis Campylobacter gracilis
comb. nov. Int. J. Syst. Bacteriol. 45: 145-152
Veion, M. and Cbatelain, R (1973) Taxonomie study of the genus Compylobacter
Sebald and Veron and desipation of the neotype strain for the type specics
Cnmpylobacter fetw (Smith and Taylor) Sebald and Vemn. Int. J. S yst. Bacteriol. 23:
122-134
Wang, Y., Taylor, D.E. (1990) Natural aansfonnation in Campylobucter species. J .
Bactenol. 172: 949-955
Wassenaar, TOM, Bleuminlt-Pluym, MC., van der Wst, B.A.M. (1991) Inactivation
of Cmnpylobacter jejuni flagellin genes by homologous recombination demonsûates that
flaA but notflaB is required for invasion. EMBO 1. 10: 2055-2061
Wassenaar, T.M., Fry, BN., van der Zeijst, B.A.M. (1993a) Genetic m5UÙpdation of
Cmpylobacte,: evaluation of naturai transformation and electmporation. Gent. 132:
131-135
Wassenaar, T.M., van der WJst, B.A.M., Ayling, R, NeweU, D.G. (1993b)
Colonization of chicks by motility mutants of Ccunpylobucter jejuni demonstrates the
importance of flagellin A expression. J. Gen. Microbiol. 139: 1171-1 175
Wassenaar, T.M. (1997a) Toxin production by Cmpyiobacter spp. Clin. Microbiol.
Rev. 10: 466476
Wassenaar, TmMœ, Engeiskirchen, M, Park, S., Lastovica, A. (199%) Differential
uptake and killing potential of Cmpylobucter jejuni by human peripheral
monocyteslmacrophagcs. Med. Mimbiol. Immunol. 186: 139-144
Westerlund, B., Korhonen, Tm& (1993) Bacterial proteins bindulg to the mammalian
extracellular matrix. Mol. Microbiol. 9: 687494
Whitehouse, C A , Baibo, P.Bq P d , ES, Cottle, DL, Mirabito, PM., Pickett,
C.L. (1998) Cantpylobacter j e jn i cytolethal distendhg toxin causes a Grphase cell
cycle block. Infect. Immun. 66= 19341940
W i n , M., Seymour, R, Henderson, B. (1998) Bacterial perturbation of cytokine
networks. Infect Immun. 66= 2401-2409
Witte, W. (1998) Medical consequences of antibiotic use in agriculture. Science. 279:
996-997
Wooldridge, KG., Williams, P.H. (1993) I m uptake mechanisms of pathogenic
bacteria. FEMS Microbiol. Rev. 12: 325-348
Wooldridge, KG., Wüliams, Pa., Ketley, J.M. (1994) bn-responsive genetic
regdation in Cmnpylobacter j e j w cloning and characterization of afir homolog. J.
Bacteriol. 176: 5852-5856
Wooldridge, K G , Wiams, P.H., Ketky, J.M. (1996) Host-signal transduction and
endocytosis of Cmpytobacter jejuni. Mimb. Pathog. 21: 299-305
Wosten, UMSmMm, Boeve, M, Koo~, MmG& VM Nuenen, A C , v i n der ?djrt,
BA-M. (1998) Identification of Compylobucter jejuni promoter sequences. J. Bacteriol.
180594-599
Yanisch-Perron, C., Vieira, J., Messing, J. (1985) Improved Ml3 phage clonïng
vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene
33: 103-119
Yao, R, A h , RA., Trust, T J., Guerry, P. (1993) Construction of new Cmpyiobmer
cloning vectors and a new mutational cm cassetîe. Gene. 130: 127-130
Yao, R., Burr, D.H., Do& P., Trust, T J., Niu, Hm, Guerty, P. (1994) Isolation of
motile and non-motiIe insertional mutants of Ciuqylobucterje~C the role of motility m
adherence and invasion of eukaryotic celis. Mol. Microbiol. 1 4 883-893
Yao, R, Burr, D.H., Guerry, P. (1997) CheY-mediated modulation of Cunpylobacter
jejuni virulence. Mol. Microbiol. 23: 102 1-103 1
Young, G.M, SchmieI, DJL, Miller, VA. (1999) A new pathway for the secretion of
virulence factors by bactena: the flagellar export apparatus functions as a protein-
secretion system. Roc. Natl. Acad Sci. USA. 96: 6456-6461
Yuki, N. (1997) Molecular mimicry behueen gangliosides and lipopolysaccharides of
Campylobocter jejmi isolated h m patients with Guillain-Barn syndrome and Miller
Fisher syndrome. J. Infect Dis. 176 (Suppl2): S150-153
Yuki, No, Takahashi, M., Tagawa,, Y., Kasbiwase, K., Tadokoro, K., Salto, K, (1997)
Association of C'fobacter jejuni wotype and antiganglioside antibody in Guillain-
Barre syndrome and Fisher's syndrome. Ann. Neurol. 42: 28-33
Zhang, J P . 9 Normarck, S. (19%) Induction of gene expression in Escherichia c d i a f k
pilus-mediated adherence. Science. 273: 1234-1236
Zhou, D., Mwseker, MA, Gaian, J.E. (1999) Role of the S. fyphünurizun actin-bindùig
protein SipA in bacterial intemalization. Science 283: 2092-2095