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THE ROLE OF CYTOLETHAL DISTENDING TOXIN IN
HELICOBACTER HEPATICUS INDUCED MURINE
GASTROINTESTINAL DISEASE

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Jason Samuel Pratt

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THE ROLE OF CYTOLETHAL DISTENDING TOXIN IN HELICOBACTER
HEPATICUS INDUCED MURINE GASTROINTESTINAL DISEASE

By

Jason Samuel Pratt

A THESIS

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

MASTER OF SCIENCE
Department of Microbiology and Molecular Genetics

2005

ABSTRACT

THE ROLE OF CYTOLETHAL DISTENDING TOXHNI IN HELICOBACTER
HEPATICUS INDUCED MURINE GASTROINTESTINAL DISEASE

By
Jason Samuel Pratt

The gram-negative bacterium, Helicobacter hepaticus, can trigger inflammatory
bowel disease (IBD) and hepatitis in mice. H. hepaticus expresses a member of the
cytolethal distending toxin (CDT) family of bacterial cytotoxins. CDT is a DNA
damaging toxin that causes cell cycle arrest in the G2/M phase, cellular enlargement and
eventually cell death in cultured mammalian cells. Persistent infection with Helicobacter
hepaticus can lead to chronic inflammation and neoplasia in mice. We explored the role
of CDT in the etiopathogenesis of H. hepaticus induced murine gastrointestinal disease
using isogenic CDT mutants. CDT was not required to initiate inflammatory bowel
disease (IBD), but led to an increase in disease severity. In long-term cancer studies,
CDT producing strains of H. hepaticus were able to cause an increased incidence of
colon tumorigenesis in an IBD model, and hepatic tumorigenesis in a hepatitis model
when compared to control mice and those challenged with a CDT deficient strain. CDT
production by H. hepaticus is required for long-term persistence in mice. Clearance of
the CDT deficient strain from mice led to a protective immunity upon rechallenge with
both wildtype and the CDT deficient mutant of H. hepaticus. These results suggest that
CDT plays two roles in the pathogenesis of H. hepaticus infection; a proinflammatory

role as well as an immunosuppressive role that allows persistence of the organism.

This work is dedicated to my family and friends

iii

ACKNOWELDGEMENTS

I would like to acknowledge my lab for all of their help in my tenure as a graduate
student. I would also like to give special thanks the USDA National Needs fellowship for
funding my research, my committee and Dr. Young for their guidance, and the support

staff that aided my work including the Histology Lab and NFSTC Staff.

iv

TABLE OF CONTENTS

LIST OF TABLES ........................................................................... viii
LIST OF FIGURES ......................... ix
INTRODUCTION ........................................................................... 1
MATERIALS AND METHODS .......................................................... 5
Mutant Construction ................................................................ 5
CDT Assays ......................................................................... 5
Bacterial Strains and Cell Lines ................................................... 7
Animals .............................................................................. 9
Murine Infection with H. hepaticus .............................................. 9
Detection of H. hepaticus in Mouse Feces and Tissues ........................ 10
Mouse Necropsy and Histology (IBD) ........................................... 12
Mouse Necropsy and Histology (Hepatitis) ..................................... 13
Preparation of H. hepaticus antigens ............................................. 14
Evaluation of Serum Antibody Responses to H. hepaticus .................... 14
DTH Response ....................................................................... 15
Statistical Analysis .................................................................. 1 5
SHORT TERM IBD STUDY ............................................................... 16
Rationale .............................................................................. 16
Results ................................................................................. 16
CDT Influences the Ability of H. hepaticus to Trigger Colitis ....... 16
Discussion ............................................................................ 20
LONG TERM IBD AND GASTROINTESTINAL CANCER STUDY ............... 22
Rationale .............................................................................. 22
Results ................................................................................ 22
Mice that Clear Infection Develop Minimal Typhlocolitis ............ 23
LONG TERM COLONIZATION AND PERSISTENCE STUDY .................... 27
Rationale .............................................................................. 27
Experimental Design ................................................................ 27
Results ................................................................................. 29
Antibody Responses Against H. hepaticus in IL-10'/' Mice ........... 29
Mice that Clear Infection are Protected from Rechallenge ............ 34
LONG TERM HEPATITIS AND HEPATIC CANCER STUDY ..................... 37
Rationale .............................................................................. 37
Results ................................................................................. 37
Discussion ............................................................................. 39

GENERAL DISCUSSION .................................................................. 43

REFERENCES ................................................................................ 49

vi

LIST OF TABLES

Table 1 ......................................................................................... 30

Table 2 ............................................................... ' .......................... 35

vii

LIST OF FIGURES

Figure 1 ........................................................................................ 6

Figure 2 ........................................................................................ 8

Figure 3 ........................................................................................ 11
Figure 4 ........................................................................................ 18
Figure 5 ........................................................................................ 19
Figure 6 ........................................................................................ 26
Figure 7 ........................................................................................ 28
Figure 8 ........................................................................................ 31
Figure 9 ........................................................................................ 33
Figure 10 ....................................................................................... 38
Figure 11 ....................................................................................... 40
Figure 12 ...................................................................................... 41

viii

Introduction

Helicobacter species are responsible for a wide range of acute and chronic human
and veterinary infections (25). A subset of this genus, the enterohepatic Helicobacter
species (EHS) encompasses a number of emerging pathogens that colonize the intestinal
tract and/or livers of humans, a variety of other mammals and birds (10, 22, 25). They
are the etiologic agents of gastroenteritis, hepatitis, and bacteremia. All Helicobacters
are gram-negative, microaerobic, motile, curved to spiral rod shaped bacteria (11). The
EHS have shown an ability to persist long—term in a chronic infection. These organisms
have been associated with chronic inflammation and neoplasia. A parallel can be drawn
with the EHS and the relationship between chronic gastritis and gastric cancer associated
with H. pylori infection in humans (1).

Helicobacter hepaticus is a murine pathogen that naturally infects the distal
gastrointestinal tract of mice, with high levels of colonization in the cecum. It was
originally discovered in control A/J Cr mice used as a part of a long-terrn cancer study.
These controls developed much higher rates of liver cancer and hepatitis than expected.
It was later discovered that H. hepaticus was the causative agent for the development of
the chronic hepatitis and hepatocellular cancer seen in these A/J Cr mice (11, 27). H.
hepaticus has since been associated with the development of inflammatory bowel disease
(IBD) in immune altered mice, and has been used as a model system for studying human
IBD (3, 5, 14). Long-term infection with H. hepaticus in these immune-altered animals

has been associated with the development of gastrointestinal neoplasias (7, 9).

In most strains of mice, H. hepaticus infection causes subclinical hepatic disease
and enteritis. Mice with an altered immune function develop a severe and progressive
typhlitis/colitis associated with H. hepaticus infection. This may lead to rectal prolapse,
adenocarcinoma, weight loss and death (2, 3, 5, 8, 19). As a result, this animal model has
been proposed as a model for studying human Crohn’s disease.

H. hepaticus and a number of other EHS have been shown to produce a cytotoxin
that is a member of the cytolethal distending toxin (CDT) family (4, 30, 32). Cytolethal
distending toxin is a virulence factor produced by a host of pathogenic bacteria, including
Campylobacterjejuni, and other Campylobacters, Haemophilus ducreyi, Actinobacillus
actinomycetemcomitans, Helicobacter hepaticus and certain strains of Escherichia coli
(reviewed in (6, 17, 21)). CDT causes cell cycle arrest in the G2/M phase, resulting in
cellular enlargement and eventually cell death in effected mammalian cells. CDT is an
AB toxin. The functional CDT holotoxin is composed of the product of three genes,
cth, cdtB, and cdtC (15, 20, 24) with CdtB constituting the active (A) subunit, and Cth
and CdtC combining to makeup the (B) subunit. The cdtB gene product is homologous to
type I deoxyribonucleases, while Cth and CdtC share no homology with other known
proteins. It has been hypothesized that Cth and CdtC are involved in delivering CdtB to
the cells (16). This working hypothesis states that CdtB forms a tripartite complex with
Cth and CdtC which allows the holotoxin to bind to the cell and facilitates entry of the
toxin (20).

Although the exact role of CdtC is unknown, it is suggested that this subunit is
required for the assembly of active holotoxin or that it may work in coordination with

Cth to bind to the outer membrane (1 3). The crystal structure of CDT has now been

solved, and the N terminus of CdtC appears to create a steric block of the active site of
CdtB. This inhibition of the CdtB DNase activity may be a self-regulatory mechanism
for CDT. It could prevent premature DNase activity, that could potentially damage the
bacterial cell producing the toxin (20). Cth possess a regiOn encoding for a signal
peptidase II recognition site that is characteristic of interaction with lipoproteins. This
provides evidence for an involvement in anchoring the holotoxin on the bacterial outer
membrane.

CdtB is the most highly conserved subunit of the protein, showing little variation
in its sequence across a ntunber of bacterial species. Studies have shown that transient
expression of CdtB in eukaryotic cells is sufficient to replicate the dramatic chromosomal
aberrations and cell cycle arrest that is observed with extracellular treatment with the
holotoxin. Also, microinjection of CdtB into the nucleus of a cell is sufficient to induce
DNA damage and cell cycle arrest (18).

CdtB possess a nuclear localization domain, in the N terminusof the protein, that
allows this subunit to target the nucleus where it can inflict damage on the DNA (16). It
has been proposed that this DNase activity is responsible for the cell cycle arrest that is a
key feature of CDT-mediated cytotopathic effect in vitro. The role of CDT in the in vivo
pathogenesis of organisms that elaborate this toxin has been investigated. Initial studies
using isogenic CDT mutants of Haemophilus ducreyi (26, 29) and C. jejuni (23) reported
minimal attenuation of virulence. A more recent report demonstrated that wild-type C.
jejuni, but not an isogenic mutant lacking CDT, triggered gastroenteritis in NF-KB-

deficient mice (12).

The purpose of these studies is to determine the role of cytolethal distending toxin
in murine gastrointestinal disease using isogenic CDT mutants of H. hepaticus in a model
of 1) Crohn’s and colitis that results from a TH] mediated immune response in C57BL6
IL-10 knockout mice and 2) hepatitis and hepatolcellular cancer in Al] mice. By
studying this murine model of inflammatory bowel disease, I hope to draw a correlation
between the role of the bacterial virulence factors and the triggers of human

gastrointestinal disease.

Materials and Methods

Mutant Construction

The cloned CDT gene cluster, as well as the surrounding region of the
chromosome, were mutagenized via transposon mutagenesis in vitro, and cloned in E.
coli. The transposon contained the gene chloramphenicol acetyl-transferase (cat), which
confers chloramphenicol resistance. The sites of transposon insertion were then
determined by restriction mapping and DNA sequence analysis. The isogenic mutants
were tested for CDT activity in vitro. pVBY9::Tn16, in which the transposon is inserted
outside of the CDT coding sequence was introduced into 3B1 (wildtype6 H. hepaticus)
by high voltage electroporation to produce the CDT positive isogenic mutant 3Bl ::Tn16.
pVBY9::Tn20, which contains the transposon inserted into the cth gene , was
introduced into 3B1 to produce 3B1::Tn20, a CDT negative isogenic mutant (See Figure
1).

CDT assays

H. hepaticus was harvested fi'om agar plates with a cell scraper and was
resuspended in Hanks' balanced salt solution. Total bacterial proteins were solubilized
with a nonionic detergent (bacterial protein extraction reagent [B-PER] according to the
recommendations of the manufacturer. Bacteria were pelleted by centrifugation at 2,040 x
g for 10 min, resuspended in 300 pl of B-PER, and vortexed. Insoluble material was
removed by centrifugation at 13,800 x g for 5 min, and soluble proteins contained in the
supernatant were collected and stored at —80°C. H. hepaticus grth from three 100-

mm-diameter plates was harvested into 1 ml of phosphate-buffered saline and disrupted

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by six 30-s pulses on ice. Debris was removed by centrifugation, and the cleared

sonicates were filtered through a 0.2-um-pore-size filter before being stored at —80°C.

HeLa cells were seeded onto 13-mm-diameter circular glass coverslips in 24-well
tissue culture plates or in 25-cm2 tissue culture flasks treated with B-PER extracts,
sonicates from wild-type H. hepaticus, or the isogenic CDT mutants at a density of
2 X 103 per well. Twenty microliters of bacterial sonicate was added to each well, and the
plates were incubated in 5% C02 at 37°C. CDT activity was determined by direct
microscopic examination of stainedmonolayers or cell cycle analysis by flow cytometry

(See Figure 2).

Bacterial Strains and Cell Lines

The wild type H. hepaticus strain 3B1 (the type strain, ATCC 51488) was
obtained from the American Type Culture Collection (Manassas, VA). The isogenic
mutants 3Bl ::Tn16 and 3B1::Tn20 were generated by transposon mutagenesis (42).
3B1::Tn20 has a transposon inserted near the start of cth and no longer produces
cytolethal distending toxin. 3B1::Tn16 has a transposon inserted downstream of CdtC in a
putative intragenic region and still produces functional cytolethal distending toxin (42).
Wild type H. hepaticus and isogenic mutant strains were grown at 37°C for 3 to 4 days in
a microaerobic environment, which was maintained in vented GasPak jars without
catalyst after evacuation to —20 mm Hg and equilibration with a gas mixture consisting of
80% N2, 10% C02, and 10% H2. H. hepaticus was grown on tryptic soy agar (TSA)
supplemented with 5% sheep blood and with 20 ug/ml chloramphenicol (all from Sigma,

St. Louis, M0) for the chloramphenicol-resistant transposon mutants.

I 331 (wildtype

Tn16 (CDT +)

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Figure 2. Cytopathic effect in HeLa cells72 hours afler treatment with
bacterial extracts. Compared to control cells, cells treated with detergent
extracts from wild type H. hepaticus strain 33] exhibit cytoplasmic and
nuclear enlargement along with nuclear abnormalities. Cell cycle analysis
demonstrated Gz/M arrest (Data not shown). The isogenic mutant
3Bl::Tn16 (with the insertion site downstream of the CDT gene cluster)
retained the ability to produce cytopathic effect and cell cycle arrest,
whereas 381::Tn20 (with the insertion site at the beginning of the cth
gene) lost both of these abilities.

Animals

All animal protocols were reviewed and approved by the Michigan State
University All University Committee on Animal Use and Care. Breeding pairs of
Helicobacter-free C57BL/6 IL-10-/- mice were obtained frOm the Jackson Laboratories
(Bar Harbor, ME) and housed with autoclaved food, bedding and water with cage
changes performed in a laminar flow hood. For the infection study, 4-6 week old
C57BL/6 IL-lO-l- mice were transferred to the University Research Containment Facility
(URCF) at Michigan State University and housed under the same conditions as the
breeding pairs. Animals were housed in groups of up to 5 animals per microisolator cage.

Specific pathogen free A/J mice were obtained from Jackson Laboratories (Bar
Harbor, ME). For the hepatitis study, 8 week old N] were housed at the University
Research Containment Facility (URCF) at Michigan State University and housed under

the same conditions as described above.

Murine Infection with H. hepaticus

H. hepaticus was harvested afier 48 h of growth on agar plates and
resuspended in a small volume of tryptic soy broth. The optical density (OD) at 600 nm
of the inoculum was measured and ten-fold serial dilutions of the inoculum plated to
quantify the CFU used for infection. Mice were inoculated with a single dose of a
suspension of bacteria with an OD of 1.0 at 600nm (~1 x 108 CFU) in a volume of 0.2-
0.3 mL. Bacteria were introduced directly into the stomach with a 24-gauge ball-tipped

gavage needle. Control mice were inoculated with sterile tryptic soy broth.

Detection of H. hepaticus in Mouse Feces and Tissues

Fresh fecal pellets were collected from each mouse. Culture for H.
hepaticus was accomplished by homogenizing feces in 0.5 mL of PBS and plating 50 uL
on TSA supplemented with 5% sheep blood, 20 ug/mL cefOperazone, 10 ug/mL
vancomycin and 2 ug/mL amphotericin B (TSA-CVA). DNA was isolated from fecal
pellets as described previously (36). DNA was isolated from gastrointestinal tissue
collected at the time of necropsy using a commercial kit (Qiagen Tissue Kit, Valencia,
CA).

Direct, single-stage PCR amplification to detect H. hepaticus was
performed using the primers 5’ GCA TTT GAA ACT GTT ACT CTG 3’ (B38) and
5’ CTG TTT TCA AGC TCC CC 3’ (B39) that produce a 417-bp amplicon (31).
PCR was performed using 5 uL of template at approximately 100 ng/ul of DNA
extracted from fecal or tissue samples. Each 25 uL PCR reaction contained 20
pmol of each primer, 200 uM of each dNTP, and 1.5 U of T aq DNA polymerase
in a final concentration of 10 mM Tris—HCl, 50 mM KCl, and 1.5 mM MgC12
(Ready To Go PCR beads; Amersham Pharrnacia Biotech, Piscataway, NJ).
Cycling conditions included 30 cycles of 30 seconds at 94°C, 45 seconds at 54°C and 45
seconds at 72°C. PCR products were visualized by agarose gel electrophoresis. To
provide a measure of relative levels of colonization with H. hepaticus, a nested PCR
system was developed (See Figure 3). The first stage PCR employs primers 5’ OCT ATG
ACG GGT ATC C 3’ (C97) and 5’ ACT TCA CCC CAG TCG CTG 3’ (C05) that
amplify the small-subunit rRNA gene from all known Helicobacter species (10), yielding

an approximately 1200 bp amplicon. PCR was performed using Ready To Go PCR

10

Nested PCR

 

338

 

Helicobacter

005/097 Amplicon (1190 bp) Specific

 

B39

 

838/339 Amplicon (417 bp) H. hepaticus Specific

 

Figure 3. Nested PCR used to detect the presence of H. hepaticus. Total DNA was extracted from fecal
pellets and cecal tissue. Helicobacter specific primers COS/C97 amplify a portion of the 168 gene. In
order to increase sensitivity and specificity, the COS/C97 amplicon is used as a template for H. hepaticus
specific primers B3 8/B39.

beads. Cycling conditions included 30 cycles of 30 seconds at 94°C, 60 seconds at 58°C
and 90 seconds at 72°C. The nested stage was performed using primers B38 and B39
described above using 1 uL of the reaction mixture from the primary PCR as template.
PCR products were visualized by agarose gel electrophoresis. In our experience, this
nested PCR has sufficient sensitivity to detect 25-50 genome equivalents of H. hepaticus

in a volume of luL (approximately lOOng) of DNA purified from mouse feces.

Mouse Necropsy and Histology (IBD)

Mice were euthanized by C02 asphyxiation at six weeks post infection. In order to
remove the ileocecocolic junction, the peritoneal cavity was opened and the cecum
externalized. The terminal ileum and proximal colon were then transected in order to
remove the cecum. The tissue was placed in a petri dish containing phosphate buffered
saline and its contents were expressed using the back of a razor blade. The tissue was
then placed into a histological tissue cassette in neutral buffered formalin (10%). Once
fixed, the tissue was processed and paraffin embedded. The tissue blocks were sectioned
so that the lumen of the ileocecocolic could be viewed. Tissue sections were placed on

glass slides and stained with hematoxylin and eosin.

Histologic section were encoded to prevent bias and scored for inflammation using
the following system: 0 normal; 1, small multifocal lamina proprial and/or transepithelial
leukocyte accumulations; 2, coalescing mucosal inflammation with or without early
submucosal extension; 3, coalescing mucosal inflammation with a prominent multifocal

submucosal extension with or without follicle formation; and 4, severe diffuse

12

inflammation of the mucosa, submucosa, and deeper layers (31).

Mouse Necropsy and Histology (Hepatitis)

Mice were maintained for 18 months to determine the effects of H. hepaticus on
long-term infection of wildtype mice. One mouse per cage was euthanized by C02
asphyxiation at 9 months post infection, while the remainder were euthanized at 18
months. The peritoneal cavity was opened in order to expose and extemalize the liver.
The liver was then observed to determine the presence or absence of noticeable tumor
formation. The liver was then removed with gall bladder attached and placed in neutral
buffered formalin (10%). Upon fixation, the lobes were separated using a razor blade.
The lobes were transected laterally and the sections placed in a histological tissue
cassette. The tissue was processed and paraffin embedded. Tissue sections were placed
on glass slides and stained with hematoxylin and eosin. In order to view H. hepaticus
within the tissue, a spirochete specific Steiner stain was also employed. Tissue sections

were blinded and scored by a veterinary pathologist.

Blood samples were also collected from the heart as follows: Upon opening the
peritoneal cavity, the diaphragm was punctured. The rib cage was transected using
scissors, exposing the heart. A 25-guage needle was inserted into the left ventricle of the
heart in order to extract a sample. The average sample collected ranged between 200-500
pl. Samples were then spun at 10,000 x g for 10 min to separate the serum. Serum

samples were removed and stored at —80 °C.

13

Preparation of H. hepaticus Antigens

H. hepaticus was cultured on TSA blood plates and suspended in PBS at O.D.600
1.5-3.0. The suspension was then frozen at -70°C, thawed, and pelleted at 5,000 RPM for
10 minutes. The pellet was resuspended in B-PER I reagent (Pierce Chemical Co.,
Rockford, IL, USA) then centrifuged at 13,000 RPM for 5 minutes. The protein
concentration of the and was measured by the Lowry technique (Bio-Rad protein assay,

Bio-Rad).

Evaluation of Serum Antibody Responses to H. hepaticus

At necropsy, blood was collected via cardiac puncture from C02 asphyxiated
mice. Blood was centrifuged at 10,000 rpm for 10 minutes, and the serum preserved with
15mM sodium azide. An enzyme-linked immunosorbent assay (ELISA) was used to
detect H. hepaticus-specific serum IgG and IgA. Nunc Polysorb 96-well plates (Nalge
Nunc International, Rochester, NY) were coated with 100 uL of a lO-ug/ml
concentration of H. hepaticus protein in carbonate buffer (pH 9.6) overnight at 4°C.
Plates were blocked with 1% bovine serum albumin in phosphate-buffered saline. Serum
was diluted 1/ 100 and biotinylated secondary antibodies included goat anti-mouse IgG]
(Sigma) diluted 1/5,000 and goat anti-mouse IgG2c (Southern Biotech, Birmingham, AL)
diluted 1/ 1,000. Incubation with extravidin-peroxidase (Sigma) at a dilution of 1/ 1000
was followed by incubation with 2,2’-Azine-di[3-ethy1benzthiazoline sulfonate] (ABTS)
horseradish peroxidase substrate (Pierce) for color development. Optical density at
405nm was recorded by an ELISA plate reader (VERSAmax; Molecular Devices,

Sunnyvale, CA).

14

DTH Response

To measure the development of delayed type hypersensitivity, mice were given 10
pg of H. hepaticus antigen in a volume of 50 pL (prepared in B-PER I Reagent as above
and diluted in PBS) by injection into the hind footpad. The opposite footpad was injected
with PBS alone. Twenty-four hours after injection, footpad thickness was measured with
a dial thickness gauge (Mitutoyo Corporation, Kawasaki, Kanagawa, Japan), and the

difference in thickness between the control and antigen-treated footpad recorded.

Statistical Analysis

Statistical analysis was performed using the IMP statistical package (SAS, Cary,
NC). Categorical inflammation scores were compared by the nonparametric Wilcoxon
rank-sum test with statistical significance set to a P value <0.05. ELISA and DTH data
were analyzed by analysis of variance. The Tukey-Kramer HSD test was used to identify

groups with significantly different means with an alpha level set to 0.05.

15

Short Term IBD Study

Published in Infection and Immunity as “In vitro and in viVo characterization of

Helicobacter hepaticus cytolethal distending toxin mutants.” 2004 May; 72 (5):2521-7.

Rationale:

H. hepaticus has been shown to cause inflammatory bowel disease in immune
altered mice. Specific-pathogen-free IL-10—/— mice develop severe typhlocolitis when
they are infected with H. hepaticus. In order to test the role of CDT in the pathogenesis
of disease caused by H. hepaticus, isogenic CDT mutants were created and tested for the
ability to cause IBD in C57BL/6 IL-lO -/- mice. Because they lack IL-10, a major signal
in the downregulation of the TH-l response, and upregulation of the TH-2 response
pathway, these mice are biased towards a TH-l inflammatory response. This response is
analogous to that seen in Crohn’s disease in humans. The goal of this experiment was to
establish a link between the presence of a function CDT holotoxin and the ability of H.

hepaticus to cause gastrointestinal disease in a murine model.

Results:
CDT Influences the Ability of H. hepaticus to Trigger Colitis

In this study, specific-pathogen—fiee (possessing no known Helicobacter spp.)
C5 7BL/6 IL-10 knockout mice, were orally challenged with either wild-type H.

hepaticus 3B1, the CDT positive isogenic mutant , or the CDT negative mutant. To serve

16

as a negative control, a group of animals was gavaged with sterile trypticase soy broth.
The colonization status of these animals was monitored via collection of fecal pellets by
culture, DNA extraction and PCR. Confirmation of the presence of the isogenic H.
hepaticus CDT mutants was accomplished by PCR with primers spanning regions of the
CDT locus. PCR performed on these mutants produced a larger amplicon when
compared to wildtype H. hepaticus. The difference in amplicon size corresponds with
the insertion of the transposon in and around the genes.

At six weeks post infection , animals were sacrificed and a necropsy was
performed. At this timepoint, all of the animals that received H. hepaticus remained
colonized as determined by PCR and culture. In all cases, only the specific challenge
strain was recovered from infected animals. By 6 weeks post infection, none of the
control animals developed significant typhlocolitis (See Figure 4A, Fig. 5). One control
animal had a small focus of chronic inflammatory cells at the ileocecal junction, a lesion
that can occur spontaneously in uninoculated animals. All of the IL 10 'l' animals in the
positive control group (infected with wildtype H. hepaticus) developed lesions, and
pathology of inflammatory bowel disease. These animals developed extensive mucosal
hyperplasia, accompanied by mucosal and submucosal inflammation (See Figure 4B, Fig.
5). IL-10—/— mice infected with the transposon-marked CDT—producing isogenic mutant
3Bl::Tn16 also developed typhlocolitis with a severity equivalent to that of mice infected

with wild-type H. hepaticus 3B1 (See Figure 4C, Fig. 5). IL-10—/— mice that were

17

 

 

 

 

 

Figure 4. Histolopathologic lesions in the cecae of IL- 10-/- mice 8 months after challenge with isogenic
strains of H. hepaticus. lL-lO-l— mice were sham infected (A), infected with wildtype H. hepaticus strain
381 (B), the CDT-positive mutant 3Bl::Tn16 (C), or the CDT-negative mutant 3Bl::Tn20 (D). Mice
challenged with CDT-producing strains exhibited marked inflammation and hyperplasia. Mice challenged
with the CDT-negative mutant, which was cleared by 4 months afier infection, developed diminished
disease.

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Figure 5. Categorical inflammation scores for uninfected IL-10-/- mice and mice infected with wild type
H. hepaticus strain 3B1 and the isogenic mutant strains eight months after challenge. Comparisons
between groups were performed using the Wilcoxon rank sum test.

19

infected with the CDT-negative mutant 3B1::Tn20, however, exhibited significantly less
severe typhlocolitis than mice infected with CDT-producing H. hepaticus strains.
Although all mice were infected with the mutant H. hepaticus, one mouse was
histologically normal, and the majority of animals exhibited only mild, patchy mucosal
inflammation (See Fig. 4D, Fig. 5). Although two mice infected with 3B1::Tn20 did
develop diffuse mucosal inflammation, none of the mice infected with the CDT-negative
mutant developed the submucosal inflammation encountered in a subset of the mice
infected with CDT-positive strains. Mesenteric perivasculitis was encountered in infected

animals with the most severe inflammation but never in uninfected controls.

Discussion:

The collected data indicates that H. hepaticus CDT may play a significant role in
the development of inflammatory bowel disease by infected immune-altered mice.
Isogenic CDT mutants developed less severe disease than mutants infected with the wild-
type strain of H. hepaticus. There was a drastic quantitative and qualitative difference in
the nature of the typhlocolitis. None of the mice infected with the CDT negative mutants
developed significant submucosal inflammation, unlike the CDT positive strains.

Cytolethal distending toxin activity in H. hepaticus is not required to initiate
inflammatory bowel disease in IL-lO-deficient mice, but influences disease severity.
Transposon mutants that lie in the coding sequence for the cthBC gene cluster were
disruptive enough to eliminate cytotoxic activity. The presence of the Chloramphenicol
resistance marker does not effect ability of the isogenic mutants to cause either IBD or

hepatitis. The CDT negative mutant is unable to colonize wildtype mice after 4 months

20

post infection, suggesting that CDT may be required for long-term colonization of

wildtype mice.

21

Long Term IBD and Gastrointestinal Cancer Study

Rationale:

After exploring the roles of CDT in the etiopathogenesis of murine IBD in a short
term study and hepatic disease in a long term study, role of CDT in a long term IBD
study needed to be elucidated. Since the CDT negative mutant was unable to persist in
NJ Cr mice with wildtype immune function, the question was raised as to whether the
mutant could persist in immune altered C57BL/6 IL-10 -/- mice. In this study, mice were
infected with wildtype H. hepaticus as well as the isogenic mutants over a 9 month study

to determine the role of CDT in a long term IBD and gastrointestinal cancer study.

Results

Our previous results show that infection of IL-10’/' mice with the CDT-deficient
H. hepaticus mutant is associated with decreased IBD pathology six weeks after
challenge compared to animals infected with wild-type H. hepaticus (31). Both the CDT-
deficient mutant and wild-type H. hepaticus were easily detectable by culture and PCR at
the end of this six-week infection study.

To determine the role of CDT in the development of long-term sequelae of H.
hepaticus infection including colonic neoplasia, additional infection studies were initiated
using isogenic CDT mutants. The H. hepaticus mutant 331::Tn20 is deficient in CDT-
production due to a transposon insertion in cth. 3B1::Tn16 is an isogenic control mutant

that has a transposon insertion downstream of cdtC in a presumed intergenic region (31).

22

Four to six week-old female C57BL/6 IL-10'/' mice were infected with wild-type
H. hepaticus strain 3B1 and the two isogenic mutant strains. An equal number of control
mice were sham-infected with sterile culture broth. There were ten animals in each
experimental group. Animals in all experimental groups were monitored for H. hepaticus
colonization by H. hepaticus-specific PCR amplification of DNA isolated fresh fecal
pellets and at certain time points, by selective culture for H. hepaticus from feces.

C5 7BL/6 IL-10'/’ mice were initially colonized by wild-type H. hepaticus 3B1
and by both isogenic mutants. Wild-type H. hepaticus and the CDT-positive isogenic
mutant 3B1 ::Tn16 colonized both strains of mice for the entire duration of each
experiment. Conversely, although the CDT-deficient mutant 3B 1 ::Tn20 was detectable at
45 days post infection (p.i.) in IL-10'/' animals, this mutant was not found by PCR or
culture at 115 days p.i. All mice challenged with 3B1::Tn20 remained negative for H.
hepaticus for the remainder of each experiment. At all times where colonization was
assessed by both culture and PCR for H. hepaticus, there was concordance between the

two methods.

Mice that clear infection develop minimal typhlocolitis

Histological examination was performed of the ileocecocolic junction of the IL-
10’/' mice challenged with wild-type 3B] and the two isogenic mutants 251 days post
infection. This time point was 190 days after the CDT-deficient mutant 3B1::Tn20 was
last detected in the feces of infected animals, and 136 days afier the animals were first
shown to be free of H. hepaticus. 0f the forty animals in this experiment, three had to be
sacrificed prior to this time. One animal infected with 3B1::Tn16 developed rectal

prolapse 59 days p.i., and another 331::Tnl6-infected animal had to be euthanized due to

23

severe dermatitis 160 days p.i. One animal infected with wild-type 3B1 developed rectal
prolapse 130 days p.i.

Nine of the ten control animals had no or minimal evidence of inflammation at the
ileocecocolic junction at the time of necropsy. One of the Control animals had significant
inflammation and hyperplasia. Feces and tissue from this animal were examined carefully
for H. hepaticus infection by culture and PCR, and H. hepaticus was not detected in this
outlier.

Animals with infected with either wild-type H. hepaticus or the CDT-positive
mutant 3B1 ::Tn16 developed moderate to severe typhlocolitis. These animals developed
marked mucosal hyperplasia with infiltration of the lamina propria with chronic
inflammatory cells. This disease was significantly different compared to uninfected
controls. IL-10'/ ’ mice that were initially infected with the CDT-deficient mutant
3B1::Tn20 but eventually cleared the infection between 61 and 115 days p.i. had minimal
signs of inflammation. Two animals had no visible inflammatory infiltrates, while the
majority of the remaining animals only had small, scattered aggregates of chronic
inflammatory cells with minimal hyperplasia. The scores of the animals infected with the
CDT-deficient mutant 3B1::Tn20 were not significantly different from the scores from

uninfected controls.

The CDT positive mutant,3Bl ::Tn16 and CDT negative mutant, 3B1::Tn20 were
both able to effectively colonize C57BL/6 IL-10 -/- mice, and were detectable for the first
sixteen weeks of the study by PCR of a fecal DNA prep. At 36 weeks post- infection,
none of the animals in the control group showed any signs of typhlocolitis. All of the IL

10 'l' animals in the positive control group (infected with wildtype H. hepaticus)

24

developed lesions, and pathology of inflammatory bowel disease. These animals
developed extensive mucosal hyperplasia, accompanied by mucosal and submucosal
inflammation, and some individuals developed adenocarcinoma (See Fig. 6). IL 10 4'
mice infected with the transposon marked CDT mutant 3B1 ::Tn16 also developed severe
typhlocolitis, equivalent to that seen in the wild-type infection. Mice infected with the
3B1::Tn20 mutant developed disease, however, it was much less severe than either of the
CDT (+) infected animals. 3B1::Tn20 infected mice also exhibited a significantly
decreased amount of mucosal hyperplasia than 3B1, or 3B1 ::Tn16. The 3B1::Tn20
mutant was unable to colonize IL-lO -/- mice after 16 weeks post infection. None of the

animals infected with 3B 1 ::Tn20 exhibit tumor formation in the cecum or colon.

25

 

Figure 6. Mucinous adinocarcimona in the proximal colon of a CS7BL/6 IL-lO knockout mouse infected
with wildtype H. hepaticus at 8 months post infection. Characteristic glandular invasion into the
submuscosa was observed.

26

Long Term Colonization and Persistence Study
Rationale:

Since 3B1::Tn20, the isogenic H. hepaticus mutant that lacked CDT production
was initially able to colonize C57BL/6 IL-lO'l' mice, but Was unable to persist, a long
term colonization and persistence study was established. The focus of this study was to
determine the colonization status of animals challenged with wildtype H. hepaticus as
well as 3B1::Tn20 over a long term study in order to pinpoint the window of clearance of
the CDT negative mutant, and to determine whether or not there was a protective immune
response upon reintroduction of the bacteria to animals that had previously cleared the
infection.

Experimental Design

For this longitudinal study, sixty C57BL/6 IL 10 -/- were housed in cages ranging
from three to five animals per cage. Ten mice served as uninfected controls and ten were
challenged with wildtype H. hepaticus. Forty mice were challenged with the CDT
deficient strain, 3B1::Tn20. Colonization status was monitored using fecal samples taken
aseptically approximately every three weeks throughout the duration of the study. Each
cage was considered a unit of infection because of the coprophagic nature of the mice.
Nested PCR assays were used to determine colonization status (See Fig. 7). A primary
PCR was performed with primers COS/C97 that amplify a region of the small 16S rRNA
gene of all known Helicobacter species. The product of this assay was used as the
template for a secondary PCR which bind to a region within the amplicon specific for H.
hepaticus. The nested PCR has the ability to detect between 25-50 genome equivalents

of H. hepaticus DNA in approximately 100 ng DNA prepared from a fecal pellet.

27

 

 

6O lL-10 KO
mice

 

 

//I\

 

 

 

 

40 mice
(11 cages)

 

 

10 mice
(4 cages)

 

 

10 mice
(4 cages)

 

 

 

 

 

I

Group 1

I

Group 2

I

Group 3

 

 

 

infect with
CDT-deficient

 

 

H. hepaticus

infect with
wild type
H. hepaticus

 

 

uninfected

 

 

 

 

I

I

I

 

 

monitor colonization by nested PCR assay every 21 days

 

 

I

 

 

terminate experiment

on day 225

 

 

 

 

10 mice (3 cages) from Group 1
with infection cleared by day 150

 

 

/I\

 

4 mice

 

 

 

 

 

 

I

3 mice

 

 

3 mice

 

 

 

I

 

 

rechallenge
with wild type
on day 190

 

 

 

rechallenge
with mutant
on day 190

I

 

 

 

rechallenge
with mutant
on day 190

 

 

 

 

I

I

I

 

 

monitor colonization by nested PCR assay
on day 197. 211 and 225

 

 

I

 

 

terminate experiment

on day 225

 

 

28

Figure 7. Design of longitudinal infection study to follow the loss of gastrointestinal

colonization by a CDT-deficient H. hepaticus strain. A) Overall experimental design. 60 lL-lO-l- animals
were divided into three groups. Group 1 was infected with the CDT deficient H. hepaticus strain, group 2
infected with wild type H. hepaticus and group 3 was sham-infected with sterile culture broth. Coloniza-
tion was monitored by a nested H. hepaticus-specific PCR assay. B) Design of nested experiment to test
if clearance of the CDT-deficient H. hepaticus mutant was associated with development of protection
against reinfection. Ten mice originally challenged with the CDT-deficient mutant were rechallenged
with H. hepaticus at least 40 days afier clearance of the mutant.

Results

All mice were initially colonized by wildtype H. hepaticus, as well as the CDT
deficient strain. Wildtype H. hepaticus was able to persist in the gastrointestinal tract,
and was detectable in the feces for the duration of the experiment. As indicated by our
previous studies, the CDT deficient mutant was not able to persist. At 75 days post
infection, all samples were positive for H. hepaticus colonization by primary PCR.
However, an increasing fraction of the cages were negative by primary PCR from this
timpepoint on. All cages challenged with the CDT deficient mutant remained positive by
the more sensitive secondary portion of the nested PCR until 117 days post infection (See
Table 1). At subsequent timepoints, colonization status by the secondary PCR assay
followed the form of the primary PCR, as more cages tested negative for the presence of
H. hepaticus. This demonstrated that the CDT negative mutant was unable to maintain
persistence in the mouse gastrointestinal tract for greater than four months post infection
(See‘Fig. 8).

CDT plays a significant role in the development and severity of inflammatory
bowel disease in H. hepaticus infected IL-10 knockout mice. Additionally, the presence
of a functional CDT holotoxin affects colonization, and is necessary for long-term

persistence of H. hepaticus in immune-altered mice.

Antibody Responses Against H. hepaticus in IL-lO‘I' Mice

Mice infected with H. hepaticus develop a significant specific antibody response
to H. hepaticus. None of the sera from uninfected C5 7BL/6 IL-lO'I’ animals contained
antibody reactive to H. hepaticus antigens measured by ELISA. All animals infected

with wild-type H. hepaticus and the CDT deficient isogenic mutant developed a robust

29

 

 

 

 

Challenge Strain PCR (culture)

9d. p.i. 32d. pl 45d. pt. 61 pi. 115d. p.i. 251d. p.i
Wild-type ‘ + + (+) + (+) + + (+) ‘ + (+)
381::Tn16 (CDT+) + + (+) + (+) + + (+) + (+)
331::Tn20 (cor) + + (+) + (+) + - H - (-)
uninfected - - (+) - H - - H - (-)

 

Table 1. Temporal monitoring of the colonization of IL-1 0-/- mice with H .

hepaticus by H. hepaticus-specific PCR and culture.

30

Fraction of cages PCR positive

 

1.0

 

 

0.9 -—
0.8 -
0.7 -
0.6 -
0.5 -
0.4 -
0.3 -
0.2 —

 

 

 

 

l—I 331::Tn20 infected (primary PCR)
H 3B1::Tn20 infected (nested PCR)
H 331 infected (primary PCR)

 

 

 

 

0.0

I I l I I I I
25 50 75 100 125 150 175 200 225

Days post infection

Figure 8. Monitoring the loss of colonization with a CDT-deficient H.
hepaticus mutant using a nested PCR assay. H. hepaticus could be
detected in the feces of animals challenged with wild type H. hepaticus
for the entire 225 duration of the experiment. There was progressive
loss of colonization with the CDT-deficient mutant starting 75 days
after infection, as judged by the primary stage of the PCR assay, and
starting at day 125, as judged by the nested assay. }

31

anti-H. hepaticus serum Th2 associated IgG1 and THl associated IgG20 specific humoral
immune response (See Fig. 9A,B). Serum samples collected at 225 days post infection
(the termination point of the experiment), animals infected with wildtype H. hepaticus
developed a greater IgG response than animals infected with the CDT deficient mutant.
Rechallenge of 3B1::Tn20 infected animals that had subsequently cleared infection
showed a boosted IgG20 response when compared to the uninfected animals, or animals
that cleared and were not rechallenged. The IgG2c response ranged from approximately
2.5 fold higher in 3B1 infect animals, to approximately 10 fold higher in rechalleneged
animals (See Fig. 9B).

Delayed type hypersensitivity (DTH) testing was used to measure the
development of a cellular immune response specific to H. hepaticus. Ten micrograms of
a BPER detergent protein extract of H. hepaticus in 50 microliters of PBS was injected
into the right hind footpad of the mice. The left hind footpad was injected with PBS
containing a diluted amount of BPER reagent to serve as a negative control. The
difference in footpad thickness was measured using a dial thickness gauge at 24 and 48
hours. Mice infected with wildtype H. hepaticus, as well as the CDT deficient mutant,
showed a significant DTH response to H. hepaticus antigen when compared to uninfected
control mice. Mice originally infected with wildtype H. hepaticus showed a greater DTH
than those that were originally infected with the CDT deficient mutant. There appeared
to be a booster effect similar to that seen in the ELISA in mice that cleared infection and

were rechallenged when compared to those that had cleared, but were not rechallenged

(See Fig. 9C).

32

 

 

 

 

 

 

 

 

 

A.
24 lgG1
20
16
DZ a
(D 12
08 ab
04 b b
b i i
24 IgG2c
a
20
ab
16 b
:5
o” 1.2 b
b
08
04
c
0
C. 04
ab DTH
a
13
E 0.3
0
.§
E’ ab
3102
'g ab ab
0)
U
§_0J
g b
OJ

 

 

 

b'\ '6 & §
0 s \ 0 6- 0'5 0-0 @.o
.9 is (65‘s? ,9?"th @330 636°C}
p «p
‘2’ 0‘2" 0" Q"
0 0

Figure 9. Development of IgG] (A), IgGZc (B) antibody responses and delayed type hypersensitivity (C)
to H. hepaticus. All animals infected with either wildtype H. hepaticus or the CDT deficient mutant
developed H. hepaticus-specific lgGl and IgG2c responses with a > 2:1 ratio of lgG20: IgG] consistent
with a THl response. For each panel, groups not connected by the same letter were significantly different
by Tukey-Kramer HSD with an alpha level set at 0.05. 0D. optical density.

33

Mice that Clear Infection are Protected from Rechallenge

Once it was established that C57BL/6 IL 10 -/- mice were able to clear infection
of the CDT negative strain of H. hepaticus, we wished to determine if clearance was
coupled with a protective immunity. Three cages of the mice that were initially
challenged with 3B1::Tn20, but had cleared infection were rechallenged. One cage
containing 4 mice were rechallenged with wildtype H. hepaticus while two cages (3
mice each) were rechallenged with the CDT negative isogenic mutant. One cage that had
cleared infection was not rechallenged to serve as a control. Colonization status was
monitored by nested PCR as previously described.

One cage of the infected animals rechallenged with 3B::Tn20 showed no
detectable levels of H. hepaticus at 7 and 21 days after the rechallenge by both the
primary and secondary PCR assays. The other cage of mice rechallenged with
3B1::Tn20 was positive by both primary and the secondary PCR on day 7, but was
negative by primary PCR on day 21. Animals rechallenged with wildtype H. hepaticus
were negative by primary PCR on days 7 and 21, but were positive by the nested PCR
(See Table 2).

At day 35 after rechallenge, fecal pellets were collected from each individual
mouse to determine if colonization status varied within each cage. Due to the
coprophagic nature of the mice, it was hypothesized that all animals within a cage would
have the same colonization status. Surprisingly, the PCR assay on the individual
samples revealed that there was animal to animal variation within a cage, as well as in

different cages subject to the same treatments (See Table 2).

34

 

Rechallenge Strain

 

wild-type H. hepaticus 381::Tn20 eggs #1 381 ::Tn20 cage #2
Primary PCR - + — - - + + - - —
Nested PCR - + - - + + + - + -

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2 Assessment of colonization status in mice that cleared previous infection
with the CDT-deficient mutant 3B1::Tn20, 35 days after re-challenge with wild
type or mutant H. hepaticus

35

Three of the four mice rechallenged with wildtype H. hepaticus showed clearance
and were negative by both primary and secondary PCR at 35 days after the rechallenge.
The one animal in this cage that remained positive was shedding a relatively large
amount of H. hepaticus as its levels were detectable by both primary and secondary PCR.
H. hepaticus was cultured from the feces of this animal, and was confirmed by PCR to be
wildtype H. hepaticus, rather than the 3B1::Tn20 by which it was originally challenged
(See Table 2).

The two cages of mice rechallenged with the CDT negative mutant showed
varying results. In one cage, there was minimal evidence of clearance, as two of the
animals were positive by both primary and nested PCR. The other animal was negative
by primary PCR, but positive by secondary PCR. In the other cage of animals
rechallenged with the CDT deficient strain, two of the three animals were negative for H.
hepaticus by both primary and nested PCR, while the other animal was negative by

primary, but positive by secondary PCR at day 35.

36

Long-Term Hepatitis I Hepatocellular Cancer
Rationale:

H. hepaticus has been linked to chronic active hepatitis and hepatocellular
carcinoma in wildtype A/JCr mice. Since strains lacking a functional CDT holotoxin
caused less disease in a murine IBD model, we wished to test the role of CDT in H.
hepaticus induced liver disease. Isogenic CDT mutants were tested for the ability to
cause hepatitis and hepatocellular cancer in NJ Cr mice in a long-term study. The goal of
this experiment was to establish a link between the presence of a function CDT holotoxin

and chronic hepatitis and hepatocarcinoma.

Results

In the hepatitis study, wildtype and the CDT positive mutant 3B1::Tn16 were able
to colonize A/J mice in a long-term study and were detectable at 18 months post infection
via PCR. The CDT negative mutant, 3B1::Tn20 was detectable by PCR at one month
and two months post infection in the Al] mice, but was undetectable in DNA from a fecal
prep at 4 months, 6 months, 11 months post infection and throughout the duration of the
study. The animals infected with CDT positive strains (3B1 and 3B1 ::Tn16) developed
hepatitis including a marked increase in inflammatory infiltrates located in the lobules
near the central vein in the portal tracts, as well as calcification in the parenchyma (See
Fig. 10 B, C). The control animals and those challenged with the CDT negative strain,
3B1::Tn20, showed little to no hepatitis (See Fig. 10 A, D). Helicobacter was observed

in tissue via Steiner stain and confirmed by PCR and recovered by plating on selective

37

>f

'f Tn16(CDT+) :4 I, - . Tn20(CDT-)

. . r

/

 

Figure 10. Representative histopathology of hepatitis with involvement of portal areas. Progressive
chronic inflammation centered around veins and bile ductules. Inflammation occured throughout the
liver and was associated with biliary epithelial hyperplasia and dysplasia. Inflammatory infiltrate consists
primarily of lympoctyes, including plasma cells, eosinophils and neutrophils and involves areas of
parenchyma. The 381 and 3Bl::Tn16 challenged mice showed a much more severe inflammation when
compared to the control and 3Bl::Tn20.

38

media in the CDT positive strains, but was undetectable by any method in the uninfected
control and the Tn20 infected animals (See Fig. 11).

At 18 months, the termination point of the experiment, the livers were extracted
and examined histologically for evidence of hepatitis and hepatocellular cancer. None of
the control animals, or animals infected with the CDT deficient strain of H. hepaticus
showed marked inflammation of the liver or hepatocellular tumorigenesis. However, The
animals infected with wildtype H. hepaticus as well as the CDT positive mutant both
showed marked inflammation of the liver. Over half of the animals sampled at this
timepoint from the two groups CDT positive groups developed noticeable tumor
formation grossly. Upon histological examination, all of the wildtype and CDT positive
mutant had developed adenomas or carcinomas, while none of the animals in the
uninfected control group, or the CDT negative group showed signs of tumorigenesis
(See Fig. 12).

Discussion

The in vitro effects of CDT on cells are most likely different than the effect seen
in vivo. Cell cycle arrest is seen in the G2/M transition of the cell cycle after exposure to
high levels of CDT due to the cell’s check point control. This leads to distension, nuclear
fragmentation and eventually cell death. It is likely that the damage caused by CDT in
this case overwhelms the checkpoint control. Low level exposure to CDT is often
insufficient to see cytotoxic effects, but it is likely that these cells that are able to bypass
the checkpoint by repairing damage have a greater likelihood of mutations that lead to

tumorigenesis. DNA damage caused by exposure to CDT should lead to an increase in

39

 

 

 

 

Figure 11. At 4 months post infection, two mice per cage were sacrificed and their livers removed. DNA
was extracted from liver tissue and H. hepaticus specific PCR was performed. Animals infected with the
CDT positive mutants showed a positive test for H. hepaticus by PCR and were also visible in the tissue
by the silver staining procedure, Steiner stain. Control animals were negative both by PCR and Steiner
stain. Surprisingly, the CDT negative mutant was unable to colonize and was not detectable by PCR or
Steiner stain.

40

 

Figure 12. Histological section of the left lobe from the liver of an animal infected with the CDT
positive mutant, 381 ::Tn16. The large adenoma, indicated by the arrow, could be observed
grossly at necropsy. Upon microscopic examination, there was evidence of a large inflammatory
infiltrate in the left lobe, but not in the tumor itself. Also, a lack of bile duct formation was
observed, a trademark of liver tumorigenesis.

41

tumorigenesis, as well as an increase in cytotoxicity, depending on the level of exposure
to the toxin.

Cytolethal distending toxin is a potent virulence factor produced by a number of
pathogenic bacteria in order to manipulate host cells. The active subunit of the toxin
produces double strand breaks in the DNA of intoxicated eukaryotic cells. The effects of
this damage elicit a checkpoint control response, leading to cell cycle arrest in the G2/M
transition. Cells that are unable to repair the damage caused by CDT remain viable for a
period of time, before undergoing apoptosis. This cell death is most likely the result of
catastrophic amounts of DNA damage conferred by high levels of toxin. These assays
are reproducible in vitro, but the level of exposure to the toxin in vivo is much tougher to
gauge. It is likely that low level exposure, perhaps as little as one molecule of toxin, is
sufficient to cause double strand breaks in the nucleus. Low-level exposure is unlikely to
overwhelm the host cell repair mechanisms, allowing them to function normally. These
cells will be able to pass the checkpoint, and continue through the cell cycle. This DNA
damage caused by CDT is similar to the effects of ionizing radiation, a known
carcinogen. As a result, treatment of eukaryotic cells with CDT employs the same DNA
repair mechanisms as ionizing radiation. Inefficient or' error prone repair of
chromosomal aberrations caused by DNA damaging agents has been implicated in the
formation of cancer cells.

We are unable to determine if the tumorigenesis observed in infected animals is a
result of DNA damage, or simply a byproduct of long-term inflammation. In some
studies, reactive oxygen species released as a result of chronic infections have been

shown to cause tumor formation.

42

General Discussion

The chronic nature of infection is a hallmark of Helicobacter infections. While
this feature may be of minimal significance to the host in Some cases, chronic
Helicobacter infections and the associated long-term inflammation can lead to the
development of neoplastic disease. Bacteria that are able to cause persistent infections
have developed varied strategies to evade immune surveillance (20, 29). H. pylori
produces the virulence factor vacuolating cytotoxin A (VacA) which has a theorized role
in the evasion of immune surveillance by H. pylori , thus allowing it to chronically
colonize the gastric mucosa (21). VacA has been shown to inhibit T cell activation, and
thus may interfere with the generation of a protective immune responseiby the host (1,
15). While H. hepaticus does not possess a homologue of vacA, the structural gene for
VacA, it is hypothesized that another toxin may play a similar role in this organism.
Cytolethal distending toxin, which has no known homologues in H. pylori, may serve as
an immunoregulatory toxin with similar function in H. hepaticus. CDT has been shown
to induce apoptosis in primary human peripheral blood mononuclear cells and cultured T-
cell lines (24, 30, 32). In addition to a direct effect on T-cells, CDT may be able to
influence the host immune response pathways by interfering with antigen presenting
cells. It was recently shown that primary human macrophages and dendritic cells treated
with CDT showed a decreased efficiency in cytokine production and the ability to
stimulation of T-cell proliferation.

In three independent studies, we provide evidence that CDT is necessary for long-

terrn colonization of the murine gastrointestinal tract by H. hepaticus. A CDT-deficient

43

isogenic mutant of H. hepaticus is able to maintain colonization of IL-lO-/— mice for at
least 61 days after its introduction to the animal, but is subsequently cleared by 115 days
following experimental infection. Questions may arise about the fitness of the organism
as a result of producing an extra protein, chloramphenicol acetyl-transferase, at relatively
high levels. This is however been remedied by construction of an isogenic mutant that
retains a functional CDT gene cluster. The inability of the CDT-deficient mutant to
persist is not an effect of the construction of the transposon mutant since the CDT
positive mutant maintained CDT expression andjwas able to maintain persistent
colonization. This suggests that IL-10- /- mice infected with the CDT-deficient mutant
are able to mount an immune response that results in clearance of the organism, a
phenomenon that is not apparent in animals challenged with wildtype H. hepaticus or the
CDT positive mutant.

At the end of the experiment (225 days after initial challenge with H. hepaticus)
the magnitude of H. hepaticus-specific humoral and cellular immune responses was
greater in animals infected with wild type H. hepaticus, as judged by ELISA reactivity
and development of DTH. The most likely explanation for this is that these responses
were measured at a time well after clearance of the CDT deficient mutant, and the loss of
colonization was responsible for waning of immunity. Our results are in general
agreement with Ge and colleagues, who reported that an independent CDT deficient H.
hepaticus mutant was unable to maintain persistent colonization of outbred Swiss
Webster mice (14). In their study, mice infected with either wild type H. hepaticus or
their CDT deficient mutant developed significant H. hepaticus-specific ELISA reactivity

compared to uninfected controls. However they noted that mice infected with the CDT

44

deficient mutant had decreased reactivity compared to animals infected with wild type.
This may reflect the time that passed between clearance of the organism and the
collection of serum at the end of the experiment.

In the current study, we have extended the finding that a CDT deficient H.
hepaticus mutant is unable to maintain long term colonization of the murine gut by
showing that animals that clear infection are provided with a degree of protection against
reinfection. Initial infection with the CDT deficient mutant 3B1::Tn20 is associated, in a
subset of individuals, with the development of a protective immune response against re-
challenge with either 3B1::Tn20 or wild- type H. hepaticus. The development protective
immunity against wild-type H. hepaticus is significant. It is possible that 331::Tn20 has a
defect (perhaps unrelated to lack of CDT) that is responsible for the long-term
colonization defect. This defect may be amplified by rechallenge of an animal that has
already encountered the organism, and thus loss of the organism upon rechallenge is not
necessarily reflective of protective immunity. However, the fact that animals that have
lost colonization with 3B1::Tn20 rapidly clear rechallenge with wild-type H. hepaticus
provides much stronger evidence that this is due to the generation of protective immunity.
In addition to an essential role in maintaining long-term intestinal colonization, CDT also
appears to have a proinflammatory role during H. hepaticus infection of IL-10-/- mice.
The mechanism of H. hepaticus clearance is yet to be determined. It remains unclear if
the lack of persistence in the mutant is due to an increase in the ability of the animal’s
immune response to clear the pathogen, or to the inability of the organism to compete
with the resident microbiota due to a lack of fitness. CDT has been shown to have an

immunomodulatory activity (28), and it is possible that the absence of a functional

45

holotoxin in the mutant renders it more susceptible to the host immune response. It is
likely that H. hepaticus CDT alters the nature and/or specificity of the host immune
response, allowing persistence.

In an earlier study, we noted that six weeks after infection, IL-10-/- mice
challenged with the CDT deficient H. hepaticus mutant developed significantly less
typhlocolitis than animals infected with wild- type H. hepaticus despite similar levels of
colonization (42). In the current study, we demonstrate that eventual clearance of the
CDT deficient H. hepaticus mutant results in almost complete resolution of typhlocolitis.
The combined roles of CDT, having both immunomodulatory activity (allowing escape
from immune surveillance and chronic colonization) and also proinflammatory activity
(leading to sustained typhlocolitis) again mirrors the VacA toxin of H. pylori (21). It
remains to be determined if similar roles will be found for other bacteria that elaborate
CDT.

IgG interactions stimulate ingestion of cells and foreign particles by macrophages
' and neutrophils. A recent study by Xu et a1 states that H. ducreyi CDT induces the
apoptosis of dendritic cells and hampers both the induction of cytokine production by
antigen presenting cells (APCs) and the ability of APCs to elicit T-cell activation. The
modulation of the host immune system by wildtype H. hepaticus CDT would allow for
greater persistence in spite of the presence of increasing IgG levels. The inability of the
mutant to produce a functional CDT holotoxin, and therefore, alter the immune response,
leaves it more susceptible to immune clearance.

Mice infected with the CDT (-) strain also showed a delay in the rise of the total

IgG in response to H. hepaticus than wildtype infected animals. The delay could be a

46

result in the inability of the CDT negative mutant to colonize as rapidly as wildtype
because it cannot modulate the immune response. The total IgG response to H. hepaticus
peaks at approximately 8 weeks, and appears to level off, maintaining a relatively
constant level. By altering the function of APCs, wildtype H. hepaticus is able to thrive
in the presence of an elevated IgG response, whereas mutants lacking CDT are unable to
persist, leading to lower levels, and eventually clearance of the organism. Mice infected
with the CDT negative strain also showed lower levels of total IgG in response to H.
hepaticus than wildtype infected animals. Although the results to date are not statistically
significant, there appears to be a trend to the decrease in colonization of 3B1::Tn20
infected animals. Whether this is due to an increase in the ability of the animal to clear
the pathogen, or to the inability of the organism to persist is still unclear. CDT has been
shown to have am immune-modulatory effect, and it is possible that the absence of a
functional holotoxin in an organism renders it more susceptible to the host immune
response. The initiation of clearance of the CDT negative mutant also seems to
correspond to the peak in the IgG response. It is possible that the elevated levels of IgG
that are maintained after the two-month time point aid in the clearance of the organism.

The collected data indicates that H. hepaticus CDT plays a significant role in the
development of inflammatory bowel disease by infected immune-altered mice.
Additionally, the presence of a functional CDT holotoxin affects colonization, and is
necessary for long-term persistence H. hepaticus in immune altered mice.

Mice infected with the CDT (-) mutant developed less severe disease than mutants
infected with the wild-type strain of H. hepaticus. There was a drastic quantitative and

qualitative difference in the nature of the typhlocolitis. None of the mice infected with

47

the CDT negative mutants develOped significant submucosal inflammation, unlike the
CDT positive strains.

Virulence factors, by definition, offer bacteria a distinct advantage in survival or
pathogenesis. It is likely that CDT exerts its cytotoxic effects on cells that are normally
undergoing continuous replication. These cells may include epithelial cell that line the
intestine, as well as immune cells. Due to the nature of the gut, intestinal epithelial cells
are constantly undergoing renewal and developmental maturation. This process is
initiated by the stem cells of the crypt, and proceeds throughout the entirety of the villus,
where they are eventually sloughed off at the tip of the villi. This would not provide a
suitable home for bacteria that require attachment to host cells in order to colonize as
these organisms would be sloughed off readily. By inducing cell cycle arrest, pathogenic
organisms such as C. jejuni or E. coli could produce CDT in order to facilitate intestinal
colonization. By inducing cell cycle arrest, an increase in the number of cells that would
be conducive to their attachment could occur. It is also probable that CDT interferes with
the developmental maturation of B cells and T cells. This would effectively dampen the

immune response and lead to increased survival of the pathogen (18).

48

10.

11.

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