Genetic Regulation of Virulence Factors Contributing to Colonization and Pathogenesis of Helicobacter Pylori

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GENETIC REGULATION OF VIRULENCE FACTORS CONTRIBUTING TO COLONIZATION AND PATHOGENESIS OF HELICOBACTER PYLORI

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Patrick E. Baker, B.S.

* * * * *

The Ohio State University

2003

Dissertation Committee:

Dr. Kathryn A. Eaton, Adviser Approved by Dr. Yasuko Rikihisa

Dr. G. Steven Krakowka ______Adviser Dr. Michael Oglesbee Molecular, Cellular, and Development Biology Program

ABSTRACT

Helicobacter pylori is a gram-negative, microaerophilic bacterium that colonizes the human stomach. It is the causative agent of gastritis, gastric ulcers, and stomach cancer. Elucidation of the mechanisms by which H. pylori is able to colonize and persist in the stomach is the scope of much of the research being done concerning H. pylori. The central focus of the research presented in this dissertation involves the study of two factors implicated in colonization: the urease enzyme and the lipopolysaccharide (LPS) of H. pylori.

Urease catalyzes the degradation of urea into ammonia and carbon dioxide.

Urease is believed to function in protecting the bacterium from the acidic environment until it crosses the gastric mucosa. Our lab has demonstrated that urease is essential for colonization. The second chapter of this dissertation seeks to establish a link between the level of urease expression with the loss of colonization potential of H. pylori isolated from infected piglets and subjected to rounds of in vitro passage. Analysis of total RNA obtained from the H. pylori isolates was performed using the slot blot technique, the Northern ELISA method and reverse transcription - polymerase chain reaction (RT-PCR) using a thermocycler with real-time analysis capabilities. The results from both the slot blot and RT-PCR anaylsis indicated that urease RNA is

ii constitutively expressed in a 26695 strain and in piglet- recovered isolates of this strain

regardless of the number of in vitro passages.

Because RNA expression of a given gene may not necessarily reflect the amount

of the gene product or its activity due to posttranscriptional or posttranslational

regulation, urease activity was determined for the 26695 strain and the recovered pig isolates. Urease activity was measured by the phenol-hypochlorite assay. In this assay ammonia generated from degradation of urea by H. pylori urease was used to convert phenol in the presence of sodium hypochlorite into indophenol. Indophenol can be quantified spectrophotometrically at a wavelength of 625 nm. Using this assay, it was demonstrated that urease activity of the 26695 isolates of varying in vitro passages was similar to the strictly lab-passaged 26695 strain. This was consistent with the slot blot and RT-PCR data. Taken together these results of the these RNA expression experiments and this urease activity assay suggest that urease is constitutively expressed during sequential rounds of passage in vitro and that colonization in the pig does not alter urease expression levels upon recovery and culturing in the laboratory.

LPS is a complex glycolipid found on the surface of gram-negative bacterium. It is involved in bacterial interactions with the host. An interesting and unique feature of the LPS of H. pylori is the expression of Lewis blood group antigens, mostly Lewis X

(Lex) and Lewis Y (Ley), on the terminal region of the O-chain of LPS. The Lewis antigens are also expressed on the surface of gastric epithelial cells. Therefore, the function of the Lewis antigens of H. pylori may involve camouflaging the bacterium from the host immune system or induction of autoimmunity in the host.

iii Isolates of H. pylori from a single gastric biopsy have been shown to exhibit

different Lewis antigen phenotypes when grown under in vitro conditions. The rate of

this phase variation is between 0.2% and 0.5%. Recently, this phase variation has been

attributed to the fucosyltransferase genes, futA and futB. These genes function in the last step of Lewis antigen synthesis to add fucose to C-3 of N-acetyl glucosamine of the carbohydrate backbone of LPS. FutA and futB contain poly-cytidine (poly(C)) tracts that may regulate expression of the genes, and therefore phase variation, via a slip- strand mechanism. Although futA and futB appear to have redundant functions, it has been proposed that they exhibit specificity in regards to the Lewis antigen phenotype expressed. Hence, the hypothesis of the third chapter is that the on/off status of futA and futB determines the pattern of expression of Lex and Ley antigens.

In order to determine the relationship between the expression of the futA and

futB status and the Lewis antigen phenotype, the length of the poly(C) region of H.

pylori isolates was analyzed by polymerase chain reaction (PCR) of their genomic

DNA. The resultant PCR products were sequenced in forward and reverse directions

using the primers used for PCR.

In order to determine the Lewis antigen phenotype, proteinase K-treated whole

cell preparations of the H. pylori samples were separated by electrophoresis on a SDS-

PAGE gel followed by electroblotting on a nitrocellulose membrane. The blot was

x y probed with anti-Lewis x (Le ) and anti-Lewis y (Le ) monoclonal antibodies. In

addition, the phenotype of the isolates was determined by enzyme-linked

immunoabsorbent assay (ELISA) using the same antibodies as in the immunoblots.

iv FutA and futB expression did not correlate with Lex expression, but because both

futB and Ley were expressed in all strains, no relationship could be determined. When isolates of the same strain were compared, there was no relationship between futB expression and animal virulence. However, futB appears to be sufficient for Lewis x/y synthesis. Furthermore, in this study neither futA nor Lex determines level of virulence

of an individual strain for its host.

The fourth chapter of this dissertation evaluated the ability of H. pylori LPS and

O-antigen of LPS to modulate the CD4+-dependent host immune response to H. pylori.

This study was performed by using an M6 strain and a SS1 strain in which the gene

encoding β-1,4-galactosyltranserase (HP0826) had been inactivated by insertional

mutagenesis. Galactostransferase is necessary for synthesis of Lewis antigens, and

inactivation results in truncated O-antigen lacking Lewis antigens. Splenocytes

isolated from uninfected and SS1-infected C57Bl/6 mice were cultured and were

stimulated with sonicate preparations of M6 H. pylori strain and M6:0826 mutant

strains and LPS samples of SS1 strain an SS1:0826. Stimulation of the splenocytes

was determined by measuring IFNγ secreted into the culture media using ELISA.

To evaluate the role of CD4+ cells on the stimulatory effects of LPS O-antigen,

transgenic mice either homozygous or heterozygous for the gene encoding the T-bet

transcription factor were used for additional splenocyte stimulation studies. T-bet is

required for expression of IFNγ in CD4+ cells and natural killer (NK) cells but not CD8+

cells; therefore, antigenic stimulation of T-bet-deficient cells would be reduced.

In this study, the absence of O-antigen in bacterial sonicate reduced IFNγ

induction of splenocytes from infected wild type mice. However, loss of O-antigen in

v LPS samples (50 µg/mL) increased IFNγ induction compared to LPS from the SS1 strain, although H. pylori LPS with or without Lewis antigens induced high levels of

IFNγ in splenocytes from infected and uninfected mice. Absence of IFNγ production by CD4+ cells reduced IFNγ levels although at a level higher than M6 sonicate stimulation of infected wild type splenocytes. This suggests that CD8+ cells may be the source of IFNγ production by H. pylori-stimualted splenocytes. IFNγ production by T- bet-negative splenocytes was non-specific since both infected and uninfected splenocytes were stimulated at nearly the same level.

The data presented in Chapter Four suggest that O-antigen cannot alone account for the strong stimulatory effect of the host immune system and may even downregulate the inflammatory response to H. pylori based on the higher stimulation of IFNγ

SS1:0826 LPS compared to SS1 LPS. However, further studies must be done to determine which cell population in the splenocytes cultures is stimulated and whether

LPS levels used in this assay are biologically significant.

This is dedicated to my parents, Herbert and Delois Baker.

vii

ACKNOWLEDGMENTS

I would first of all like to thank my parents and my grandmother Annie Bell

Baker, a retired educator, for always emphasizing that a key to obtaining my dreams

was a good education. I would also like to express my gratitude to my late

grandmother Mabel Ward for teaching me that earthly pursuits without spiritual pursuits

are fruitless. I greatly appreciate my sister Shunder for her unyielding support and my brother Ezra for critiquing the rough draft of my disseratinon. Also, I must thank my extended family for their vigorous encouragement even though they were not quite sure exactly what it was I was doing!

It is with tremendous pleasure that I am also able to acknowledge my advisor,

Dr. Kathryn Eaton, for her earnest desire to ensure that I grow as a scientist as I pursued my degree. I am also thankful for the comarderie and the insightful advice from the members of the Eaton Lab: Steve Dannon, Ray Mankowski, Meghan Mefford, Richard

Peterson, Jonathon Rosenberg, Tracy Thevenot, and Amy Wanken.

I consider myself extremely fortunate to have made so many great friendships while in Columubus. I would like to thank Michelle Gray for those late-night study sessions; Lawerence “LD” Dearth for his unique sense of humor and his genorisity;

Matt Hartman for making life outside of lab enjoyable whenever he was around; and

viii Dan and Jamie Sanford for being there when I was in a time-of-need. I am also grateful for these friends who made life in Columbus enjoyable: Stacey Dearth, Tiffiney “Little

T” Roberts, Dahai Tang, Matt Anderson, Ellen Nixon, Matt Buccellato, Melinda

Butsch, Bridgite Arduini, Yingjie Zhang, and Drew Dangle. I would be remiss without mentioning the “Gene Jocks” - Lawerence Dearth, Daniel Sanford, Matt “Primetime”

Hartman, Matt Anderson, Matt “Matty Basement” Buccelato, Matt Sivko, Gloria Sivko,

Murli Narayan, Brian “Silky Smooth” Davies, Naveen Dakappagari, Al Kovacic, Virgil

Richard, Jeff Voorhes, Drew Dangle, Tom Carsillo, Yasuro Sugimoto, and Ihab Younis

- for making softball a great distraction from labwork!

VITA

November 28, 1972...... Born – Quitman, Georgia

1995...... B.S. Biology, Albany State University

1995 - 1997 ...... Laboratory technician,

University of Virginia

Department of Molecular Physiology and

Biological Physics

1997 - present...... Graduate Teaching and Research

Associate, The Ohio Stae University

PUBLICATIONS

Research Publications

1. Variable Expression of Redundant α-3-Fucosyltransferase Genes futA and futB

Lacks Correlation with Phase Variation of Lewis x and Lewis y antigens in

Helicobacter pylori (in preparation)

2. Eaton, KA., Gilbert, JV, Joyce, EA, Wanken, AE, Thevenot, T, Baker, P, Plaut,

A, Wright, A. (2002). In vivo complementation of ureB restores the ability of

Helicobacter pylori to colonize.. Infection and Immunity, 70:771-778

FIELDS OF STUDY

Major Field: Molecular, Cellular, and Developmental Biology

TABLE OF CONTENTS

Page Abstract...... ii

Dedication...... vi

Acknowledgments...... vii

Vita...... x

List of Tables ...... xiv

List of Figures...... xv

Chapters:

1. Introduction...... 1

2. Expression pattern of the urease enzyme of Helicobacter pylori during in vitro passage following isolation from infected piglets Abstract...... 30 Introduction...... 31 Materials and Methods...... 34 Results...... 44 Discussion...... 52

3. Expression of Redundant α-3-Fucosyltransferase Genes futA and futB Lacks Correlation with Phase Variation of Lewis x and Lewis y antigens in Helicobacter pylori Abstract...... 56 Introduction...... 57 Materials and Methods...... 62 xii Results...... 65 Discussion...... 72

4. The Role of the Lewis Histo-blood group Antigens Expressed on the Lipopolysaccharide of H. pylori in Modulating Host Immunity

Abstract...... 76 Introduction...... 78 Materials and Methods...... 82 Results...... 86 Discussion...... 96

5. Future Work...... 102

Bibliography ...... 107

xiii

LIST OF TABLES

Table Page

3.1 Primer sequences used in this study ...... 62

3.2 Correlation between genotype and phenotype of H. pylori strains ...... 72

xiv

LIST OF FIGURES

Figure Page

1.1 Lewis antigens are defined by different fucosylation patterns ...... 24

1.2 Core oligosaccharide...... 25

2.1 RNA slot blots were performed ...... 45

2.2 Slot blots were analyzed by densitometry ...... 46

2.3 The Northern ELISA technique ...... 47

2.4 Real-time RT-PCR analysis ...... 49

2.5 Urease activity was determined by modification ...... 50

2.6 Urease activity was determined by the phenol-hypochlorite assay ...... 51

3.1 Lewis Antigen Chains...... 59

3.2 Nucleotide sequence and the resultant amino acid sequence of the poly(C) region of futA...... 66

3.3 Gene sequence alignment of the poly(C)region of futB ...... 68

3.4 Predicted amino acid sequence alignment of futB ...... 69

3.5 Western blot analysis of lipopolysaccharide (LPS) of H. pylori strains...... 70

3.6 Expression of Lex and Ley by H. pylori as measured by ELISA ...... 71

4.1 ELISA analysis of IFNγ secretion in uninfected C57BL/6 mouse splenocytes ...... 87

4.2 ELISA analysis of IFNγ secretion by splenocytes derived from infected C57Bl/6 mice ...... 87

4.3 ELISA analysis on IFNγ production by splenocytes derived from uninfected T-bet (+/-) mice...... 90

4.4 ELISA analysis of IFNγ secretion by T-bet (-/-) splenocytes...... 91

4.5 Stimulation of splenocytes derived from T-bet (+/-) mice infected with SS1 ...... 92

4.6 Stimulation of splenocytes derived from T-bet (-/-) mice infected with SS1 ...... 92

4.7 LPS samples were treated with 20 µg/ml of Proteinase K prior to addition to splenocyte culture ...... 94

4.8 Delayed-type hypersensitivity ...... 95

xvi

CHAPTER 1

INTRODUCTION

Background and Significance

Helicobacter pylori is a gram-negative bacterium that has adapted to colonize the human stomach. Since its initial isolation and characterization by Barry Marshall and Robin Warren (162), H. pylori has been associated with numerous gastric maladies ranging in severity. The hallmark of H. pylori infection is inflammation of the gastric mucosa. While gastritis is typically sub-clinical, more severe consequences of infection include mucosa associated lymphoid tissue (MALT) lymphoma, gastric carcinoma, and peptic ulceration.

As early as 1892, spiral bacteria were reported in the stomachs of mammals (32,

229); however, it wasn’t until 1906 that spiral bacteria were described in the human stomach (132). For many years afterwards these spiral bacteria were thought to be either postmortem contaminants (206) or Pseudomonas aeruginosa (246, 247) (245).

Despite reports of urease activity in mammalian gastric mucosa (152), the disappearance of gastric urease activity upon treatment with tetracycline (141), and the eventual discovery that animals do not express urease, the suggestion that gastric urease was bacterial in origin (63) was slow in gaining acceptance.

1 In 1984, Warren and Marshall published their findings that H. pylori can be isolated from the human stomach and that infection from this bacterium is associated with active chronic gastritis and gastric or duodenal ulceration (162). H. pylori was originally classified as a member of the Camplyobacter genus and was referred to as

Camplyobacter pyloridis ( and later Camplyobacter pylori). This seemed logical since, like the members of Camplyobacter spp., H. pylori is microaeophilic and spiral, and the mole percent of guanine and cytosine (35-38%) is similar to Camplyobacter spp (27,

161). However, H. pylori lacks the electron carrier thermoplamaquinone which is commonly used to identify Camplyobacter spp. (248). Also, comparison of the genomic sequences (42) and the fatty acid composition (248) of H. pylori and

Camplyobacter spp. further indicated that they were unrelated. Consequently, a completely different genus, Helicobacter, was created for H. pylori and H. mustelae isolated from the stomach of ferrets (95). Since that time, a number of Helicobacter spp. from various vertebrate hosts have been named, such as H. bilis, H. hepaticus, H. rodenium.

In developed countries, 20-50% of the population is infected with H. pylori

(257). Epidemiological studies from Japan, the United States, and Europe report a drop in infection rate of greater than 25% every decade (209, 221, 225, 242). This may be the result of better levels of public hygiene, access to proper indoor plumbing that separates sewage from drinking water, and/ or reduction in crowded housing, which all may present barriers to H. pylori transmission via a fecal-oral or person-to-person route

(208). In contrast to developed countries, 70-90% of the population of undeveloped

2 countries is infected with H. pylori, and H. pylori is considered one of the most common chronic pathogens in humans.

H. plyori infection can be eliminated through the use of antibiotic regimens that may include a single antibiotic, a proton pump inhibitor with two antibiotics, or, more commonly, a bismuth salt with two antibiotics (108, 109, 111). The most commonly used antibiotics are clarithomycin, metronidazole, and amoxicillin. Once the bacterium is eradicated, gastritis and ulceration are resolved. Although eradication rates from initial treatment can exceed 85%, 21-25% of cases may require a second phase of treatment and in some cases infection remain resistant to eradication (108).

The prevalence of H. pylori infection and recrudescence after initial treatment due to bacterial resistance or patience non-compliance to drug therapy has prompted the search for more effective treatments of H. pylori infection. In addition, the cost of current antimicrobial drugs, especially in developing countries may prohibit treatment.

Therefore, further research must be done to elucidate how the bacterium is able to establish colonization and cause disease.

Host Immune Response to H. pylori Infection

The consequence of H. pylori infection appears to stem, at least in part, from the nature of the host immune response induced by the bacterium. The immune response to pathogenic invasion is classified into two arms of defense: the cell-mediated response

+ and the antibody response. CD4 helper T (TH) lymphocytes are central mediators of both arms. However, the TH cells can be sub-divided into populations of TH1 cells that secrete cytokines, such as interferon-γ (IFNγ), interleukin (IL)-2, and tumor necrosis

3 factor-β (TNFβ) that promote a cell-mediated response and TH2 cells that secrete IL-4,

IL-5, IL-6, IL-9, IL-10 that specify an antibody response (183, 184, 224).

This dichotomy is actually an oversimplification as several TH cell subsets have been described that have cytokine profiles that defy strict TH1/TH2 categorization (84,

128, 154, 205, 251). Also, there is evidence of two regulatory subsets of TH cells.

Several studies have described the TH3 cell population that produces TGF-β and

suppresses local inflammation to persistent infection (90, 113, 192). Another regulatory

TH cell subtype is the Tr1 cells that produce IL-10 and inhibits antigen-specific TH cell

High proliferation and colitis in SCID mice injected with TH CD45RB cells (97).

Therefore, classification of TH cells based on their cytokine pattern is problematic in

that subsets of these cells may not express the full range of cytokines characteristic of

TH1 or TH2 cell types, their cytokine profile may be heterogeneous, or these cells may

function to regulate the local immune response.

Despite the complexity of TH classification, TH1/TH2 distinction is useful in characterizing the immune response induced by H. pylori. Gastric TH cells isloated from H. pylori-infected human subjects (23, 125) and from H. felis infected mice (173) showed elevated levels of IFNγ, a hallmark of a TH1 response, but not of the TH2 cytokines IL-4 and IL-5 indicating that response to H. pylori primarily involves TH1 type cells and is, therefore, a cell-mediated response.

In addition to IFNγ, the upregulation of other proinflammatory cytokines, such as TNF-α, Il-8, Il-6, and IL1-β have also been shown in individuals infected with H. pylori (185, 194, 285). The induction of these proinflammatory cytokines along with the lack of or low level expression of cytokines of TH2 type cells, which are important in

4 downregulating chronic inflammatory reactions (235), are in accordance with the consensus that H. pylori is the causative agent of inflammation of gastric mucosa.

It is important to note that although H. pylori elicits a strong TH1-type immune reaction, this response is not sufficient to clear H. pylori infection. H. pylori is primarily an extracellular pathogen, therefore a TH2 should be effective to clear infection. Because of the success of vaccination studies using either recombinant

Helicobacter proteins or whole cell sonicate in clearing H. pylori and H. felis in the stomach of mice (46, 61, 64, 130, 143, 151, 219), H. pylori may select for TH1 and suppress TH2. TH2 can be inhibited by IFNγ which is upregulated during H. plylori infection.

While it is unclear how inflammation benefits Helicobacter colonization, inflammation has been linked to pathogenesis. Epidemiological studies have shown that Helicobacter-induced gastritis is associated with the development of gastric cancer

(45). Furthermore, a study with Mongolian gerbils infected with H. pylori has shown that chronic gastritis can eventually lead to the development of gastric cancer (272). A possible explanation for this linkage is that chronic inflammation generates reactive oxygen and nitrogen species that can cause DNA damage which could contribute to tumoriogenesis (22, 158).

Host Genetic Risk Factors

Because only 10-20% of infected individuals develop disease, host risk factors that may predispose individuals to clinical manifestations are also the focal point of H. pylori research (33). As discussed earlier environmental factors, such as sewage- contaminated water and overcrowding, can be important host risk factors contributing to

5 disease(208). Also, diet plays a significant role in disease. In some studies it was

shown that risk of chronic atrophic gastritis and duodenal ulceration due to H. pylori

infection is significantly decreased by an intake of sufficient amounts of antioxidant

micronutrients such as vitamin C (86, 191, 238) and vitamin A (5, 238) as well as the

non-antioxidant micronutrient vitamin B12 (240). On the other hand, in the Korean

population, a diet high in salt has been positively correlated with gastric cancer and H.

pylori infection (117, 139, 140) and this correlation has also been demonstrated in

Mongolian gerbils (196). However, smoking and high consumption of alcohol and

caffeine, which were long-suspected of being risk factors for gastric ulcers, were found

to have little association with susceptibility to gastric disease (4).

In addition to environmental and dietary factors genetic host factors have also

received keen interest. Genetic factors associated with H. pylori infection and

pathogenesis are typically involved with the host immune response. These include

cytokine polymorphisms. Polymorphisms have been found within the promoter region,

the first intron and the 3’ untranslated region of the mouse tumor necrosis factor-alpha

(TNF-α) (30, 116). A number of polymorphisms in the TNF-α gene have been

correlated with differential expression of TNF-α among strains of mice. TNF-α is a central mediator of inflammation and, therefore, modulation of TNF-α expression level can alter immune function. In mice infected with Toxoplasma gondii, TNF-α

polymorphisms appear to determine the susceptibility and severity of the disease due to

the pathogen (89). Also, the polymorphisms have been associated with development of

an autoimmune disease analogous to human systemic lupus nephritis in NZB/NZW F1

(B/W) mice.

6 Gastric diseases in humans associated with H. pylori infection have also been

correlated with TNF-α polymorphisms. The human TNF-α gene possesses two single

nucleotide polymorphism (SNP) sites, TNF-α-308 and TNF-α-238, within the promoter have been associated with differential TNF-α expression and susceptibility to disease.

TNF-α-308 and TNF-α-238 are located 308 and 238 base pairs, respectively, upstream of the transcriptional start site of the gene and are subject to a guanine to adenosine substitution (G/A). The TNF-α-308 A allele is associated with duodenal ulcers (134).

The expression of TNF-α-238 A allele has been correlated with a decreased incidence of gastric carcinoma (114, 288)

The anti-inflammatory cytokine IL-10 has three SNP’s in its promoter at the –

1082 (G/A), -819 (C/T), and –592 (C/A) positions (55, 79). The high IL-10-producing alleles of these SNP’s have been shown to be good markers for the severity and the susceptibility to gastric carcinoma (282). This was in concordance with previous data that have demonstrated a linkage between high expression of IL-10 and occurrence of gastric carcinoma (60, 182). However, the functional consequence of the high expression of IL-10 is currently unknown.

Another cytokine that is a potential host factor for Helicobacter-related disease is the cytokine IL-1β. IL-1β is a potent activator of inflammation during H. pylori infection. As in TNF-α and IL-10, IL-1β has 3 SNP’s that contribute to pathogenesis; however, only two of the SNP’s, -511 and –31, are within the promoter. The third SNP is within the gene itself at position +3954. A switch from cytosine to thymine at these positions has been previously associated with increased production of IL-1β (24, 80, 81,

123, 194). Because an elevation in IL-1β has also been observed in patients with gastric

7 cancer (124), the thymine alleles of these SNP’s could be determinants for

carcinogenesis.

The development of cancer is typically a multifactorial event. Therefore,

infection with H. pylori along with the possession of any or a combination of the alleles

of TNF-α, Il-10, and IL-1β associated with tumoriogenesis could increase the likelihood of the development of gastric carcinomas. Although the role of increased levels of IL-

10 in tumoriogenesisis still unclear, the function of enhanced levels of proinflammatory cytokines in tumoriogenesisis is better understood. Initially, high levels of IL-1β and TNF-α may serve to eliminate H. pylori infection. However, elevated levels of IL-1β have also been shown to inhibit acid secretion (80, 81). Prolonged acid inhibition can result in the destruction of parietal cells and irreversible hypochlohydria

(80, 81). This condition can facilitate the spread of localized inflammation in the antrum of the stomach to the corpus due to the decreased flow of gastric secretions and the resultant accumulation of bacterial toxins (80, 81). Deleterious by-products of inflammation include reactive oxygen and nitrogen oxide species that can cause tumoriogenesis. Furthermore, H. pylori-induced achlohydria has been known to allow superinfection of other bacteria that produce N-nitroso compounds that are also mutagenic (249, 250).

Bacterial Colonization Factors

An attractive goal of microbial research is to define those aspects of a pathogen that enable it to colonize the host. The virulence determinants that are essential for the bacteria to establish colonization in a host are termed colonization factors. Once identified these factors could be possible targets of chemotherapy or vaccines to block

8 colonization and subsequent pathogenesis. Animal models and targeted DNA

mutagenesis have proven to be invaluable tools in this endeavor. Studies utilizing these

techniques have led to the classification of the urease enzyme and motility-related genes

as colonization factors. Since its identification as a colonization factor, urease has been

used in in several vaccine studies (61, 130, 143, 151, 219)

Urease

+ The urease enzyme of H. pylori catalyzes the hydrolysis of urea into NH3 and

+ carbamate, which is further degraded to CO2 and another molecule of NH3 in aqueous

environments. Urease activity observed in animal gastric tissues was initially thought

to emanate from an enzyme in the mucous secretions since it could not be separated

from gastric epithelial cells (152). Because this study pre-dated the discovery of the

urea cycle, these early workers theorized that gastric urease activity of mammals was

necessary to degrade urea in the stomach into CO2 and ammonia that could be excreted

by exhalation and in the vomitus, respectively (152). It was not until 1950, that urease

activity was localized to sites of gastric ulceration (85). The first evidence that urease

activity is bacterial in origin emerged eighteen years later when it was found that the

stomachs of germ-free animals lacks urease activity (63). In 1990, the urease enzyme

was characterized and isolated from H. pylori (65, 107).

H. pylori urease is a nickel-containing heterodimer consisting of subunits UreA

(66 kDa) and UreB (29.5 kDa) (107). The apoenzyme (550 kDa) is believed to be

composed of 12 UreA/UreB heterodimers with a 1:1 UreA to UreB subunit ratio. The

heterodimers are arranged in a four-ring stack with each ring being 13 nm in diameter

9 and composed of 3 heterodimers (21, 98). In addition, the urease apoenzyme has six

nickel binding sites, and each site can hold two Ni2+ ions.

The ureA and ureB genes are part of a gene cluster composed of the accessory genes ureE, ureF, ureG, ureH that are responsible for nickel acquisition and insertion into urease. Gene deletion studies in Klebsiella aerogenes have shown that ureF, ureG, and ureD (homologous to ureH in H. pylori) are essential to urease function (174, 175).

UreE forms a homodimer with histidine-rich, nickel-binding sites in urease suggesting

that UreE has a role in delivery of nickel ions to the urease metallocenter (36). This is

supported by the finding that insertional inactivation of the ureE gene in H. pylori

resulted in loss of 99% of urease activity compared to wild type H. pylori strains (269).

Yeast two-hybrid analysis has shown that UreE forms a heterodimer with UreG, which

carries a GTP-binding site (65). In the same study, UreH and UreF also were shown to

form a heterodimer in vitro (65). These heterodimers interact directly with the urease

apoenzyme via UreB binding sites to transfer nickel to urease (269).

Urease production by the bacterium accounts for 6-15% of its total protein

production (107, 234). This investment of cellular resources for urease production

suggests the importance of urease to H. pylori. This is further underscored by the

finding by this laboratory and others that virulent H. pylori strains that lack a functional

urease gene are unable to colonize animal hosts (7, 67, 83, 195, 263, 280). Despite

much that has been learned about urease, the function of urease in colonization and

pathogenesis has fueled much debate.

There are several theories concerning the significance of urease activity. The

prevailing theory is that urease protects H. pylori during early colonization by

10 + generating ammonia (NH3 ) which can neutralize local gastric acid until the bacterium

can burrow in the gastric mucous where it is shielded from the acid. The mechanics

behind this acid resistance has also been subject to much controversy. Initially, it was

envisioned that urease created a cloud of ammonia that buffered the gastric

microenvironment of the bacteria. This concept was supported by the observation that

urease could be found on the cell membrane of H. pylori as well as extracellularly when

grown in liquid culture medium (212). Also, electron microscopy has been used to show

surface urease activity by treatment of bacteria with sodium tetraphenlyborate (STPB) the precipitates with ammonia. Using this technique, it has been shown that urease activity is localized within the cytoplasm and is associated with the cell membrane (35,

66, 101, 213). Because the urease enzyme is not targeted to the cell membrane nor is it secreted, it was believed that membrane-attached urease and extracellular urease came from lysed bacteria (35, 159, 213). This so-called altruistic lysis theory also was the basis of speculation that extracellular urease could also serve as decoys in order for H. pylori to evade the host’s immune surveillance.

In order to test the altruistic lysis theory, Scott et al incubated a urease-negative mutant strain with cell lysate from a wild-type strain to determine if free urease binds to whole cells as the theory purports (233). After centrifugation of the mutant strain through a Ficoll step gradient to get rid of lysate, no urease activity was detected; therefore, urease from lysed cells did not adhere to the whole urease-negative mutants

(233). This finding was in opposition to the altruistic lysis theory. To explain the detection of extracellular urease activity, it is has been proposed that the centrifrugation step of the water extraction technique (213) (65) used to isolate extracellular urease

11 caused lysis of the cells resulting in release of intracellular urease that was mistaken for

extracellular urease. Even the validity of the electron microscopy data used to identify

surface-absorbed urease is now subject to debate, in part, because ureolytic bacteria

Staphylococcus aureus, Klebsiella aerogenes, and Proteus mirabilis that are known to

have only cytoplasmic urease exhibit the same distribution of urease activity as H.

pylori when using STPB (169, 170, 187). Therefore, the urease enzyme and urease

activity are believed to be confined to the cytoplasm

The recent study of the ureI, another gene of the urease gene cluster, has helped

immensely to shape a new theory to explain the significance of urease in colonization of

H. pylori. UreI is a polytopic, integral membrane protein (approximately 21 kDa) with homology to amide transporters amiS2 from Rhodococcus sp., amiS from

Psuedomona aeruginosa, and ORFP3 of Mycobacterium smegmatis (41). However, several lines of evidence indicate that UreI is a H+-gated, urea transporter.

SDS-polyacrylamide electrophoresis of hydrophobic segments of UreI has

shown that it is composed of six transmembrane regions connected by alternating

periplasmic and cytoplasmic hydrophilic loops (275). At the periplasmic surface of

UreI there are 6 histidines that are possible proton acceptors during acid activation of

UreI. At least one of these histidines, histidine 123 in the second periplasmic loop, has

been shown by site-directed mutagenesis to be essential for urea transport (275).

Urease activity of intact H. pylori appears to be upregulated at pH 6 and reaches

a maximum at pH 5 that is maintained until pH 3 where urease activity ceases (38, 275).

In another study it was shown that this activation of urease at low pH is inhibited by

permeablization of the cell membrane (220) indicating that passive diffusion of urea

12 disrupts regulation of urease activity. Also, UreI expressed in Xenopus oocytes via

cRNA injections resulted in the uptake of urea in a urea-specific, nonsaturable,

temperature independent, pH-dependent manner (275). Furthermore, the acid

activation profile of the urease enzyme in H. pylori paralleled the profile of the pH-

dependent entry of urea into the oocytes injected with urease cRNA. This provided

further evidence that UreI modulates urease activity by allowing urea into the cell.

Also, H. pylori strains in which ureI is inactivated by insertional mutagenesis

looses acid resistance when grown below pH 4 compared to wild-type strains grown at

the same pH (220). There is little or no urease activity of intact ureI- mutants at any pH

range, but when homogenized the urease activity of the mutant is detectable above pH 5

and reaches a maximum at pH 7.5-8 (275). This indicated that the optimum pH of

urease is at pH 7.5-8 and that free and membrane-bound urease could not be functional in the human stomach where the median pH is 1.4. Also, it suggested that UreI was essential for urease activity of H. pylori in acidic conditions.

The current hypothesis of the mechanism behind acid protection of H. pylori

incorporates what is known about the UreI channel. It proposes that UreI upon acid

activation transports urea into the cytoplasm where is utilized by urease to produce

NH3+ that passively diffuses into the periplasm where it buffers the periplasmic space

(275). Acidification of the periplasmic space results in the disruption of the proton

motive force (126). The proton motive force is the sum of the differences in proton

concentration and electrical charge across the inner membrane of the bacteria (190).

The proton motive force is used to drive the synthesis of ATP from ADP by F1F0

ATPase as well as other energy-linked processes vital to cell survival, such as flagella

13 movement, transport of substances into the cell against the concentration gradient, and

maintenance of the cell’s turgor (190). Below pH 4.0 in the absence of urea, proton

motive force of H. pylori is irreversibly lost; therefore, it is important that the

periplasmic space maintains a near neutral pH for cell survival (171). A pH-sensitive

fluorescent dye, 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), has

been used to analyze changes in periplasmic pH in the presence of urea at low pH.

BCECF increases its fluorescence in response to an in increase in pH. When visualized

under a confocal microscope, BCECF is confined to the periplasmic space of H. pylori

due to its inability to cross the inner membrane of H. pylori, though it can transverse the

outer membrane via porins that can accept negatively-charged molecules like BCECF.

Although there is no change in fluorescence of BCECF at neutral pH in the presence of

urea, at low external pH fluorescence increases in the periplasmic space upon addition

of urea either directly into the periplasm or into the media of the bacteria (20). This

demonstrates the cell’s attempt to buffer the periplasmic space by increasing the pH in response to the low, acidic pH of the culture medium. However, in UreI- mutants

BCECF in the periplasm does not show changes in intensity in response to acidic medium indicating that the UreI channel mediates regulation of urease activity, which in turn modulates the pH of the periplasmic space (20).

Urease regulation is essential when the bacterium reaches the gastric mucus layer where it is shielded from gastric acidification. Unbridled urease activity at this condition could cause alkalization and death to the bacterium; therefore, a mechanism must be in place to down-regulate urease activity at neutral pH. Because H. pylori is grown in the laboratory in broths and on culture plates at neutral pH, it should be

14 expected that such a regulatory mechanism exists. While UreI is believed to regulate

urease activity at the post-translational level, there is evidence of regulation of the

urease gene cluster at the post-transcriptional level in response to pH.

Furthermore, acid protection may be only one aspect of the benefits of urease.

It has been shown that urease negative mutants are unable to colonize achlorohydric pig

stomachs (71). Urease may provide the bacteria with a source of nitrogen in the form

of ammonia; therefore, lost of urease activity may severely hamper survival of H. pylori

in the stomach. Furthermore, urease mutants have been shown to have reduced

chemotactic motility compared to wild type strains, and urease inhibition by

acetohydrocamic acid also inhibited motility (188). This suggests that urease is

essential to chemotactic motility, another important factor in colonization.

Motility

Motility is a common feature of all gastric Helicobacter species and is essential

for colonization of the stomach for at least three gastric Helicobacter species: H. pylori,

H. felis, and H. mustelae (6, 7, 75, 77, 119). It is hypothesized that motility is necessary during initial colonization in order to convey the bacteria through the acidic environment of the gastric lumen to the safe haven of the mucosa layer. As is typical in many other motile bacteria, motility of the three gastric Helicobacter sp. is driven by flagella. H. pylori has a unipolar tuft of flagella, H. felis has a bipolar tuft and H. mustelae is peritrichous (121, 252). The differences in the organization of their flagella also reflect differences in the manner in which they propel themselves and in the region of the stomach they are found (58). For instance, H. mustelae utilizes spinning and burrowing motions to navigate through the gastric mucous (58). These 15 movements are more characteristic of the “random walk” of E. coli (29) than the

corkscrew motion of H. felis (136). Also, H. pylori (102) and H. felis can tract along mucous strands while H. mustelae is only rarely seen swimming in the mucous (58).

The flagellar tuft of H. pylori is composed of 4-7 flagella of 3 µm in length (93).

As with the other two gastric Helicobacter sp., the flagellum of H. pylori is encased in a sheath that extends into a bulb-like structure at the distal tip of the flagellum (93). The sheaths of the flagella are continuous with the cell membrane and are believed to protect flagella from the acidic contents of the stomach (93). This was demonstrated by Geis et al using flagellar sheaths isolated using a sucrose gradient that were resistant to acid treatment (93). In contrast, the flagellar filaments, the structural components of the flagellum, were completely degraded upon acid treatment (93).

The flagellar filaments are composed of FlaA and FlaB subunits. These subunits are quite distinct, only sharing 50% amino acid identity. FlaA is the most predominant and FlaB is localized primarily at the hook, the flagellar structure that is juxtaposed between the filament of the flagella and the basal body that is embedded in the cell membrane (131). However, both FlaA and FlaB are essential for full motility (120).

Also, when either flaA or flaB is inactivated by insertional mutagenesis, colonization of gnotobiotic piglets is weak and does not persist compared to wild-type strains (77).

The hook of the flagella connects the flagellar filament to the basal body. FlgE protein is the structural element of the hook (202). The basal body is comprised of the rod, the P-ring, and the L-ring. The rod links the basal body to the hook. The basal body is anchored to the periplasm and the outer cell membrane by the P- and L- rings.

In addition to the filament, the hook, and the basal body, the other main component of

16 the flagellum is the motor-switch complex (MSC). The MSC generates the rotational force of the flagellum. This force is derived from the proton motive force at the cell membrane. As stated earlier urease is believed to play an important role in the maintenance of the proton motive force at low pH. In agreement with this hypothesis, chemotaxis studies of H. pylori demonstrated that isogenic, urease mutants have reduced chemotactic motility in response to urea and sodium bicarbonate in viscous solutions (188).

The majority of the machinery of the flagellar apparatus is located outside of the cytoplasm. Two export mechanisms have been found to deliver the components of the flagellar apparatus for assembly. The signal-peptide-dependent Sec pathway secretes the P- and L-rings to the cell membrane. A flagellum-specific pathway secretes the rod of the basal body, the hook, and the FlaA and FlaB flagellin subunits. It has been deduced from studies of homologues in Escherichia coli (157), Salmonella typinmurium

(172, 268), and Caulibacter crescentus (157) that flbA, flbB, fliH, fliI, fliQ, fliL, fliP, and fliR H. pylori genes encode proteins that make up the flagellar export mechanism.

Insertional mutagenesis of fliP (118), fliI (115, 214), fliQ (88, 214), flbB (88), and flbA

(168, 232) in H. pylori showed that they were essential for flagellum formation and, hence motility. The protein product of the fliI gene is an ATPase that provides the energy for the export mechanism. The protein encoded by fliQ is believed to be involved in the transport of an unidentified adhesion factor, as the knockout mutants of these genes do not adhere to AGS cells in culture (88). The homologue of FlbB protein in S. typhimurium is involved in the assembly of the rod structure of the basal body

(172). Also in S. typhimurium, loss of the flbB gene has been associated with reduction

17 of the length of the FglE polymer that makes up the hook of the flagellum (202). The

flbA gene product is a cytoplasmic membrane protein that has been shown not only to

be essential for motility but to also modulate urease activity by some, as of yet,

undefined mechanism (168, 232). However, the other putative flagellar export

apparatus genes in H. pylori have not undergone similar characterization.

In S. typhimurim, the expression of the motility genes is controlled by a regulon

comprised of three components. One is the master regulatory operon flhCD which

responds to global signals such as adenosine 3’,5’-monophosphate to transcriptionally

activate the second component, the sigma factor sigma28 (σ28). σ28 is a subunit of the

RNA polymerase that specifically recognizes the majority of late flagellar genes in S.

typhimurim(110, 121). The final element of the motility regulon is the σ28 inhibitor

FlgM that, in effect, serves as a negative regulator of the late flagellar genes (110, 121).

H. pylori regulation of flagellar synthesis differs from that of S. typhimurim. H. pylori lacks homologues to the flhCD operon and to flgM. In addition, the promoters of the late flagellar genes of H. pylori are not solely recognized by σ28 as in S. typhimurim

(121). In fact, the majority of these genes are regulated by sigma54 (σ54), and most of

the other motility genes contain recognition sites for sigma70 ( σ70) (121). Another

distinctive feature of H. pylori flagellar regulation compared to other bacteria with

flagella is that some of the motility genes of H. pylori are co-regulated with

housekeeping genes (25, 214, 244). For instance, the flagellar export genes fliI and fliQ are found within the same operon as the housekeeping genes ileS and murB (214).

As stated earlier, H. pylori does not have a homologue to the negative regulator

FlgM, but the flagellar regulatory mechanism of H. pylori has other negative regulators

18 of its own. There is some evidence that supercoiling of DNA may inhibit transcription.

The gene that encodes topoisomerase I which relieves negative supercoiling and gyrA gene that encodes for the enzyme gyrase which introduces negative supercoiling are found proximal to the flagellin subunit flaB and to the flagellar transcriptional activator gene flgR, respectively (244, 253). Also, flagellar export gene fliP is subject to spontaneous inactivation. This inactivation is traced back to a repeat of eight cytidines

(or, alternatively, CCCCACCC) within the coding region of the gene (118). During replication, this polycytidine region is vulnerable to slip-strand mispairing that may cause the addition or the deletion of cytidines. This can result in the alteration of the reading frame which can produce a truncated, non-functional protein; thus, inactivating the gene (118). Therefore, slip-strand mispairing serves as a regulatory mechanism of fliP.

Virulence Factors Contributing to Disease

CagA

H. pylori is organized into a dichotomy of type I strains, which are associated with gastric cancer and inflammation and ulceration of the duodenum of the host, and type II strains, which result in more asyptomatic infections (99). Furthermore, colonization by type I strains achieve a higher bacterial density than type II strains (19).

The cytotoxin-associated gene A, or cagA gene, is found in type I strains but not in type

II strains. This made cagA an excellent marker for disease-causing H. pylori strains and a candidate for virulence.

Early in the study of CagA, there was some evidence that CagA was a factor in the cytoxicity of the VacA protein and the induction of epithelial cells to produce IL-8 , 19 an neutrophil activator and chemoattractant (47, 53, 264). However, disruption of the

cagA gene by insertional mutagenesis did not affect VacA activity (265, 283) nor IL-8

induction (54). However, it was found that cagA is part of a 40kb DNA fragment of

about 30 genes that is found in type I strains but not in type II strains (40). Also, it

was found that this region was associated with Il-8 induction and the phosphorylation of

an unidentified 145 kDa protein in gastric epithelial cells. This DNA region is referred

to as the cag pathogenicity island (cag PAI). Inactivation of a number of these genes,

including cagC, cagD, cagE, cagH, cagI, cagL, and cagM, results in the loss of IL-8

induction and tyrosine phosphorylation of the epithelial protein (40, 216, 236, 266).

However, cagA is not merely a marker for the cag PAI, and more recent data

indicates that it plays a significant part in pathogenesis. CagA was identified as the

145 kDa protein in epithelial cells that is phosphorylated upon attachment of H. pylori

in cell culture (236) (16, 199, 237)and in vivo (286). CagA is translocated into the gastric epithelial cells via a type IV secretion system. Type IV secretion mechanisms are utilized by Agrobacterium tumefaciens (48, 228), Bordetella pertussis (37), Brucella suis (87, 197), Escherichia coli (278), Legionella pneumophila (267), and in Anaplasma

(Ehrlichia) phagocytophila and Ehrlichia chaffeensis (200) to export various macromolecules from the cell to the extracellular space or into another cell (37, 43, 228,

277). H. pylori possess six genes, including cagE (also known as picB) and cagT and open reading frames 528, 527, 525, and 524, in the PAI that are homologous to type IV genes identified in the other bacteria utilizing Type IV secretion systems (48).

Once across the cell membrane of the epithelial cells, CagA is phosphorylated at any of three putative tyrosine sites (Y-122, Y-899, Y-1039) (82). Within the

20 epithelial cells at the site of adherence, CagA forms cylindrical structures that are associated with actin mobilization and the formation of microspikes and ruffles by the epithelial cells (the hummingbird phenotype) (236). This hummingbird phenotype is similar to the response of a kidney epithelial cell line to scatter factor/hepatocyte growth factor (SF/HGF) via the Ras-signaling pathway (223). In addition, phosphorylated

CagA has been shown to interact with the SRC homology 2 domain-containing tyrosine phosphatase (SHP-2), which plays a major role in mediating the SF/HGF-mediated response (105, 286). SF/HGF is believed to function in vivo to induce epithelial and endothelial cell motility for tubule formation (276), wound healing (243), and blood vessel formation (39, 96). During H. pylori colonization, CagA may disrupt normal

SHP-2 regulation causing the cells to take on a cellular-transformed phenotype that may represent an early stage of gastric carcinogenesis (105, 286).

VacA

In the presence of H. pylori or its purified vacuolating cytotoxin, or VacA, enzyme, HeLa cells in culture undergo cytoplasmic vacuolation (49, 50). Other mammalian cell lines have been also found to be subject to VacA-induced vacuole formation, including gastric mucous cell line HM02 (26), gastric carcinoma cell line

KATO III (204), gastric epithelial cells RGM1 (203), human larynx carcinoma cell line

HEp-2 (222), and human kidney tumor cell line G401 (284). In addition, purified VacA has been shown to cause disruption of the membrane potential of the mitochondria of gastric eptithelial cell line AZ-521 and pHEp-2 cells resulting in reduction of ATP synthesis, consumption of O2 (129), and, in the case of pHEp-2 cells, cytochrome c

21 release (92). Also, there is evidence that VacA induces apoptosis in AGS cells, a human gastric epithelial cell line (133).

VacA is translated as a 140 kDa protein precursor, but is later processed into an

88 kDa mature protein upon proteolytic cleavage at its carboxy- and amino- termini (49,

52, 231, 258). The VacA protein is a monomer that assembles into a hexameric structure that pairs with a another VacA hexamer to form a dodecamer complex of approximately 900 kDa (1, 49). The VacA complex is frequently described as having a flower-like structure because of its central pore surrounded by 6-7 petal-like arrangements of the VacA monomers (1, 51, 135, 153).

Although the VacA has been shown to be non-esseential for colonization in the gnotobiotic piglet animal model (68), this toxin has been shown to be important in enhancing initial colonization of VacA+ strains in the mouse (227). Despite the significant amount of research on VacA, there is still considerable effort to determine the relevance of the toxin during infection. One hypothesis proposes that VacA functions to permeabilize the gastric epithelelial cell membrane allowing the bacterium access to nutrients. This is supported by a study demonstrating that the electrical resistance across a polarized monolayer of canine kidney epithelial cells is reduced by adherent H. pylori or purfied VacA protein (207, 210). A more recent study demonstrated that VacA selectively allows urea to passively cross the epithelial cell membrane (261). This was determined to be specific for urea by observing that glycerol and mannitol are not permeable and that the urea transporter inhibitor phloretin blocks the efflux of urea from the epithelial cells(261). As discussed earlier, H. pylori

22 transports urea into its cytoplasm via the UreI pH-gated urea channel where the urease

enzyme breaks it down to form CO2 and NH3, a function essential for colonization.

Lewis Antigens and Lipopolysaccharide

Workers in the field of H. pylori research became interested in Lewis antigens when they discovered these epitopes on the lipopolysaccharide (LPS) of H. pylori.

Lewis antigens were initially identified humans and, along with A, B, H (O) antigens,

comprise the histo-blood group antigens. The histo-blood group antigens are a family

of carbohydrates forming the terminal structures of glycan chains. They were originally

identified on red blood cells but are now known to be also expressed on tissues and

secretions such as saliva and gastric fluids (144, 160, 274). In contrast to the other histo-blood group antigens, Lewis antigens expressed on red blood cells are not actually

synthesized by red blood cells and transported to the cell membrane but are actually

absorbed to the cell surface from the secretions (274).

Histo-blood group antigens are characterized as mucin structures having a

composition of L-fucose, D-galactose (Gal), N-acetyl-D-glucosamine (GlcNAc), N- acetyl-D-galactosamine, and a protein moiety consisting of mainly proline, threonine, and serine (145-149, 218). The synthesis of histo-blood groups starts from a common precursor, Galβ-3/4GlcNAc. The further addition of fucose, GlcNAc or Gal to the precusor, and the nature of the linkage between GlcNAc and Gal in the precusor give rise to the formation of different Lewis antigen structures (Figure 1.1). The Lewis

23 Ga l1 4Gl cNA- Gal1 3Gl cNA- 2 2

Fuc1 Fuc1 H type H type

Gal1 4Gl cNA- Gal1 3Gl cNA- 3 4

Fuc1 Fuc1 Lewis x Lewis a

Gal1 4Gl cNA- Gal1 3Gl cNA- 2 3 2 4

Fuc1 Fuc1 Fuc1 Fuc1

Lewis y Lewis b

Gl cN Ac1 3 Ga l1 4GlcNA- GlcNA1 3Gal1 3Gl cNA- 2 2

Fuc1 Fuc1

A type A type

Ga l1 3 Ga l1 4GlcNA- Gal1 3Gal1 3Gl cNA- 2 2

Fuc1 Fuc1

B type 2 B type 1

Figure 1.1: Lewis antigens are defined by different fucosylation patterns (either fucose linkage to GlcNAc only [as in the case of Lea and Ley] or to both GlcNAc and Gal [Leb and Lex]) and Gal linkage to GlcNAc of the precursor (either a 1,3 linkage [Lea and Leb] or a 1,4 linkage [Lex and Ley]) .

24 antigen phenotypes that have been identified on the LPS are Lewis x (Lex), Lewis y

(Ley), Lewis i (Lei), Lewis a (Lea), and Lewis b (Leb) antigens (Fig. 1.1). LPS is found

at the outer cell wall of gram-negative bacteria. The LPS typically consists of three

tructures: lipid A, the core oligosaccharide, and the O-antigen. In H. pylori, Lewis

antigens are expressed on the O-antigen, the outermost moiety of the LPS. The O-

Figure 1.2: The core oligosaccharide of LPS. “R” is the point of attachment of Lewis antigens.

antigen is bound to a chain of 8-10 sugar residues in the core oligosaccharide. Keto-

deoxyoctonic acid (KDO) is found on the proximal end of the oligosaccharide chain.

The KDO is in turn attached to lipid A, a glycolipid that anchors the LPS to the outer

cell membrane of the bacteria. The O-antigen in some bacterial strains are absent, in

which case, they are referred to as rough form or low molecular weight LPS.

Conversely, strains in which O-antigen is present are called smooth or high molecular

weight LPS. The “smooth” and “rough” designations refer to the effect the presence or absence of the O-antigen has on the surface texture of the bacterial colonies.

From the vantage point of the outer cell membrane, the LPS of some gram- negative bacterium can elicit a strong host immune response that, if unchecked by

25 bacterial clearance, could possibly result in excess cytokine production causing septic

shock and endotoxemia. The endotoxic effect of LPS has been attributed to lipid A (91,

289). However, in a monocyte activation assay, 1000-fold more LPS of H. pylori was required to achieve the same level of activation as LPS of Salmonella minnesota (34).

This is consistent with other studies that have concluded that the LPS of H. pylori produces a reduced endotoxic response compared to LPS of other gram-negative bacteria such as E. coli and S.minnesota (31, 57, 59, 166, 193, 211). The low potency

of the LPS seems to downplay its role in the pathogenicity of H. pylori. However,

recent work concerning the Lewis antigens of the LPS has rekindled efforts to establish

LPS as an important modulator of the immune system of the host.

Lewis antigens are also expressed on gastric epithelial cells. Therefore, it is quite possible that the Lewis antigen phenotype of an infecting H. pylori strain could be identical to that of the host. This could have at least two effects on the immune system of the host resulting in two opposing consequences for the host.

Firstly, Lewis antigens identical to that of the host could lower the antigenicity of H. pylori allowing the bacterium to escape detection from the host’s immune system promoting persistence of infection. This could account for the low immune response of

H. pylori LPS. Studies have shown that Lewis antigen phenotypes of individual bacteria of a colony derived from a single isolate can undergo spontaneous switching at a mutational rate of 0.2% - 0.5% (12). Therefore, infection with a H. pylori strain with a Lewis antigen phenotype different from the host during early colonization can result in a persistent infection of bacteria expressing the same phenotype of the host. This would mean that bacteria that have the host’s Lewis antigen phenotype via phase

26 variation have a better chance of survival than those bacteria that have the same phenotype as the parental strain. In support of this hypothesis, it has been shown that

H. mustelae isolates from ferrets possess the same Lewis antigen phenotype as its host

(56). Also, H. pylori was recovered from Rhesus monkeys possessed the same Lewis phenotype of the host but different from the parental strain (279).

Some studies have demonstrated that the polycytidine regions of futA and futB, the α-3-fucoslytransferase genes, may regulate the expression of the functional enzymes encoded by these genes (10-12). FutA and FutB enzymes both function to add the sugar fucose to GlcNAc (106, 163). The polycytidine regions in both genes are vulnerable to slip-strand mispairing during replication resulting in addition or deletion of the cytidines in these regions(10). Slip-strand mispairing could possibly alter the reading frame of the genes creating premature stop codons that result in truncated, nonfunctional gene products. The inactivation of any one of these genes or both could cause the Lewis antigen phase variation. This implies that there is a mechanism in place for H. pylori to modify the Lewis antigen phenotype of its LPS to better suit survival in the host.

The similarity of the bacterium’s and the host’s epitopes could also lead to the development within the host of an autoimmune response against the host’s own gastric cells. Combined with chronic gastritis in the host, this could worsen an already bad prognosis. Several clinical studies have observed anti-Lex and anti-Ley antibodies in the sera of H. pylori- infected patients (14, 104, 239). Also, mice infected with H. pylori have been shown to produce autoantibodies against the gastric mucosa (189).

27 Alternatively, the Lewis antigens could function to enable H. pylori to adhere to

the gastric mucosa. This would allow the bacterium to resist the churning motion of the

stomach that would otherwise sweep it into the lower gastrointestinal tract. Several

studies have been done with the H. pylori surface-expressed proteins BabA and SabA demonstrating their binding to Leb and sialyl-Lex antigens, respectively, expressed on gastric epithelial cells (94, 112, 156, 215, 259). However, adhesin molecules on gastric epithelial cells that bind to Lewis antigens on H. pylori have yet to be identified.

The focus of this dissertation is the virulent factor urease and the putative virulence factor Lewis antigens. The 2nd Chapter of this dissertation will concern the

determination of the expression patterns of the urease gene across differing strains,

animal passages, and lab passages of H. pylori. In this chapter I intend to demonstrate

that loss of virulence of subsequent rounds of lab passage can be attributed to decrease

in transcription from the urease operon.

The 3rd chapter seeks to determine whether slip-strand mispairing of the polcytyidine region of the redundant fucosyltransferase genes, futA and futB, is the mechanism responsible for the phase variation of the Lewis antigens of H. pylori. The hypotheses tested are that the length of the poly(C) region can be used to determine whether futA and futB gene products are expressed; that FutA and FutB can direct the

Lewis phenotype expressed because of slight functional differences of the

fucosyltransferases described in Appelmelk et al; and that the on/off status of futA and

futB as determined by the length of poly(C) region can be used as a predictor of the

Lewis antigen phenotype of the bacterium.

28 The 4th chapter will describe how O-antigen interacts with the host’s immune system to mediate a Th1 type response. Specifically, this study will seek to 1) determine whether H. pylori LPS alone can stimulate mouse splenocyte cell cultures in a similar manner as H. pylori sonicate, 2) to determine if loss of O-antigen in H. pylori

sonicate abrogates stimulation of mouse splenocytes, and 3) to evaluate whether the

effect of O-antigen on the immune system includes CD4+ T-cell involvement.

CHAPTER 2

Expression pattern of the urease enzyme of Helicobacter pylori during in vitro passage

following isolation from infected piglets

ABSTRACT

Urease catalyzes the degradation of urea into ammonia and carbon dioxide.

Urease is believed to function in protecting the bacterium from the acidic environment until it crosses the gastric mucosa. The second chapter of this dissertation seeks to establish a link between the level of urease expression with the loss of colonization potential of H. pylori isolated from infected piglets and subjected to rounds of in vitro passage. Analysis of total RNA obtained from the H. pylori isolates was performed using the slot blot technique, the Northern ELISA method and reverse transcription - polymerase chain reaction (RT-PCR) using a thermocycler with real-time analysis capabilities. The results from both the slot blot and RT-PCR anaylsis indicated that urease RNA is constitutively expressed in a 26695 strain and in piglet- recovered isolates of this strain regardless of the number of in vitro passages.

30 Because RNA expression of a given gene may not necessarily reflect the amount

of the gene product or its activity due to posttranscriptional or posttranslational

regulation, urease activity was determined for the 26695 strain and the recovered pig isolates. Urease activity was measured by the phenol-hypochlorite assay. In this assay ammonia generated from degradation of urea by H. pylori urease was used to convert phenol in the presence of sodium hypochlorite into indophenol. Indophenol can be quantified spectrophotometrically at a wavelength of 625 nm. Using this assay, it was demonstrated that urease activity of the 26695 isolates of varying lab passages was similar to the strictly lab-passaged 26695 strain. This was consistent with the slot blot and RT-PCR data. Taken together the results of these RNA expression experiments and this urease activity assay suggest that urease is constitutively expressed during sequential rounds of passage in vitro and that colonization in the pig does not alter urease expression levels upon recovery and culturing in the laboratory.

INTRODUCTION

The urease enzyme of the gastric pathogen Helicbacter pylori catalyzes the

breakdown of urea into CO2 and NH3. This simple and straightforward function belies

the controversy concerning its significance. In recent years urease has been implicated

in the colonization ability and in the pathogenicity of H. pylori. The importance of

urease in colonization is now well established. Several studies have shown that H.

pylori strains in which urease is inactivated by insertional mutagenesis are unable to colonize the stomach in several animal models (7, 67, 83, 195, 263, 280). Moreover,

31 the mechanism by which urease promotes colonization and the elucidation of its

importance in causing disease has received intense study. The current, prevailing

hypothesis is that the NH3 by-product of urea degradation is utilized by the bacterium to

counter the acidic environment of the stomach. This protects H. pylori as it reaches the gastric mucosa where it is shielded from the acid.

Several hypotheses have been proposed to explain urease protection. An early hypothesis asserted that extracellular urease from lysed H. pylori cells adhered to the cell surface of live bacteria (35, 159, 213). Urease activity at the cell surface

produced ammonia that neutralized the acidic microenvironment of the bacteria

allowing for survival (35, 66, 100, 213).

However, data has emerged that contradicts this theory and points to a new

theory to explain urease protection. One crucial study demonstrated that urease is not

functional in at the low pH range of the stomach; therefore, free and membrane-bound

urease could not possibly play a role in acid protection (275). Also, this study found

that ureI encodes an acid-activated urea transporter that is able to regulate urease

activity post-transcriptionally (275). Another study has shown that urease found on the

cell surface in previous reports were artifacts from the preparation procedures for

measuring urease activity (233). These findings help shape the current hypothesis that

proposes that UreI upon acid activation transports urea into the cytoplasm where is

utilized by urease to produce NH3+ that passively diffuses into the periplasm where it

buffers the periplasmic space.

Another aspect of urease that has received some study is the regulation of urease

expression. The ureA and ureB genes encoding the subunits of urease are located in a

32 gene cluster. The urease gene cluster is comprised of the accessory genes, ureE, ureF, ureG, and ureH that function in the acquisition of nickel and the assembly of the nickel complex in the apoenzyme of urease (174, 175, 269). Also, among the accessory genes is the ureI gene. Unlike the other genes, ureI is not essential to the function of

urease.

Northern blot analysis of RNA of H. pylori revealed that transcription from the

urease gene cluster is under a pH-dependent regulatory mechanism that degrades urease

transcripts at pH 8, upregulates urease transcription at pH 6, and stabilizes transcription

at pH 5.5 (2, 233). This suggests that H. pylori is able to modulate urease activity at

the transcriptional level in response to its environment. This mechanism would allow

H. pylori to decrease urease production at high pH when alkalization by NH3 is not desired and, conversely, to increase urease production at low pH where urease is needed

to buffer acidic conditions.

Urease production by the bacterium accounts for 6-15% of its total protein

production (107, 234). Because of this large investment of cellular resources in the production of urease, it is logical that H. pylori has evolved a mechanism to tightly control urease production. If, as suggested by some, a role of urease is to protect against gastric acid, control of production would allow increased expression during colonization (i.e. transiting across the gastric lumen) and downregulation when H. pylori is burrowed within the neutral environment of the mucosa or when it is outside of the host. Therefore, the purpose of this chapter was to test the hypothesis that urease is differentially expressed in H. pylori isolates and that this disparity in expression levels reflect differences in virulence and in differences in pH modulation by the isolates.

33 This was done by comparison of the RNA level of isolates differing in passages in vitro and in vivo.

MATERIALS AND METHODS

Bacteria

The H. pylori clinical isolate 26695 and variants of this strain recovered from infected pigs, 26695 PP2, 91-124, 96-1106, 99-2310, were used in this study. Variant

22695 PP2 was passaged twice in germ-free piglets. Strains 91-124, 99-2310, and 96-

1106 were recovered from infected piglets and, subsequently, passaged in vitro 2, 4, and 14 times, respectively. Bacteria were grown overnight in Brucella Broth (Becton

Dickinson, San Diego, CA) with 10% fetal calf serum (FCS) (Gibco, Carlsbad, CA) or two to five days on trypticase soy agar (TSA) plates with 5% sheep blood (Fisher

Scientific, St. Louis, MO) under microaerobic conditions at 37oC. Microaerobic is defined as having an atmospheric composition of 85% N2, 10% CO2, and 5% O2.

RNA Isolation

Overnight cultures of Helicobacter colonies in Brucella broth were harvested by centrifugation at 4000 RPM. The supernatant was removed using a sterile pipette and the remaining pellet was resuspended in diethyl pyrocarbonate (DEPC)-treated phosphate-buffered saline, pH 7.2 (PBS) (Gibco). The pellet was washed twice in PBS before RNA isolation.

Bacteria grown on TSA-blood plates were either scraped from the plate using ethanol-flamed glass microscope slides or lifted from the plate using sterile inoculation

34 loops and resuspended in DEPC-treated PBS. The colony suspension was sedimented

and resuspended as described above.

Bacteria were either immediately used for RNA extraction or were stored in

RNALater® Stabilization Reagent (Ambion, Austin, TX) at - 20oC or - 70oC. RNA

was isolated by one of two methods: isolation with Trizol reagent (Gibco), a

phenol/guanidine isothiocyanate based procedure, or Rneasy® Mini Kit (Qiagen,

Valencia, CA), in which nucleic acid is extracted by absorption onto a silica-gel

membrane. RNA was stored in DEPC-treated water or RNAse-free water supplied with

the RNeasy Mini Kit. It was quantified using the GeneQuant® spectrophotometer

(Pharmacia, Kalamazoo, MI) at a wavelength of 260 nm.

Slot Blot

In order to synthesize digoxigenin (DIG)-labeled probe to detect urease and r16S RNA, polymerase chain reaction (PCR) was performed to amplify DNA segments from the ureA subunit of urease and from r16S. For r16S DNA, the forward and reverse primers were 5’-GCTCAGAGTGAACGCTGGCGGCGTGCC-3’ (HP16S-3) and 5'-

ACGAGCTGACGACAGCCGTG-3’ (HP16S-4), respectively. For ureA DNA, the forward and reverse primers were 5’-GCCAATGGTAAATTAGTT-3’ (URA-F) and 5’-

CTCCTTAATTGTTTTTAC-3’ (URA-R). For amplification of r16S DNA the PCR conditions were 35 cycles of 94oC for 1 minute, 55oC for 1 minute, and 72oC for 2 minute. The ureA gene fragment was amplified with 35 cycles of 94oC for 1 minute,

50oC for 1 minute, and 72oC for 2 minute. The PCR products were purified with the

QIAquick PCR Purification Kit (Qiagen) and were used as templates for the synthesis

of DIG-labeled probes. 1-3 µg of the templates in 16 µL of sterile water were denatured

35 by heating in boiling water for 10 minutes followed by immediate chilling in an

ice/water bath. 4 µL of the DIG-High Prime reagent, a component of the DIG-High

Prime® DNA Labeling and Detection Starter Kit I (Roche, Indianapolis, IN), was added

to the templates. The labeling reaction was allowed to occur for 1 hour or overnight at

37oC.

For analysis of urease mRNA and 16S ribosomal RNA (r16S) expression, RNA isolated from bacterial strains was applied to Trans-Blot® nitrocellulose membranes

(BioRad, Hercules, CA) using the Bio-Dot SF® (slot format) microfiltration apparatus

(BioRad) according to the manufacturer’s protocols. RNA was cross-linked to the

membrane using the UV Stratalinker® 2400 (Stratagene, La Jolla, CA). For each bacterial sample 10, 5, 1, and 0.5 µg of RNA was blotted onto the membrane.

The membrane was blocked with 1x blocking solution provided with the DIG-

High Prime DNA Labeling and Detection Starter Kit I (Roche) for 3 hours at 50o C for

3 hours while shaking. Afterwards, 20 ng/mL of ureA probe and 7.5 ng/mL of r16S probe were set in a boiling water bath for 5 minutes and then immediately cooled in an ice/water bath. The probes were then applied to duplicate slot blots in heat-sealable pouches. The pouches were heat-sealed and incubated overnight while shaking at 50o C for ureA probe and 55o C for r16S probe. Afterwards, the membranes were washed

twice for 15 minutes in 100mL of 2 X sodium chloride/ sodium citrate (SSC) solution,

0.1% sodium dodecyl sulfate (SDS) at room temperature (RT). This was followed by

two further washes in 0.1x SSC, 0.1% SDS at the hybridization temperatures for 15

minutes with constant shaking.

36 In order to detect the probes, the membranes were first rinsed in washing buffer

(0.1 M maleic acid; 0.15 M NaCl, pH 7.5; 0.3% (v/v) Tween-20) for 5 minutes. This

was followed by blocking with 100 mL of 1x blocking solution. Afterwards the blots

were incubated with the anti-DIG antibody conjugated to the alkaline phosphatase

enzyme at a concentration of 1:5000 in 20 mL of blocking solution for 30 minutes at

RT. The membranes were washed twice in 100 mL of washing buffer for 15 minutes.

The probes were detected in 10 mL of nitroblue tetrazolium (NBT)/5-bromo-4-chloro-

3-indolyl phosphate (BCIP) solution for 2-5 minutes. The blot was scanned using the

Alpha Imager and densitometric analysis was done using the Imagequant software.

Northern ELISA

Bio-Chem-Link reagent from the Northern ELISA Kit (Roche) was used to label

5.9 µg of total RNA from 26695 and 5.1 µg of total RNA from 91-124 with biotin. 6 µL and 5 µL of Bio-Chem-Link were added to the RNA, and this was allowed to incubate for 60 minutes at 65oC. To purify labeled RNA, the reactions were ethanol precipitated

by adding 0.1 volumes of 3 M sodium acetate and 2.7 volumes of ice-cold absolute

ethanol and incubating at –70oC for 30 minutes. Afterwards, the samples were

centrifuged at 15,000xg in an Eppendorf 5145C centrifuge for 30 minutes. The

supernatants were decanted and 200 µL of 70% ethanol was added to the resultant

pellets. The samples were centrifuged at 15, 000xg for 15 minutes. After decantation of

the ethanol, the pellets were resuspended in 9 µL of Rnase-free water. The biotin-

labeled RNA was quantified by a spectrophotometer at a wavelength of 260 nm. The

concentration of the RNA was adjusted to 0.5 µg/µL.

37 UreA and r16S DIG-labeled probes (synthesized as described earlier) were

diluted in 160 µL of hybridization buffer (supplied in kit) for a final concentration of 2

ng/µL, were set in boiling water for 10 minutes, and were immediately set in an ice-

water bath. 3 µL of biotin-labeled RNA was added to two each probe. Five-step serial

dilutions (1/10) were made from the hybridization solutions resulting in RNA

concentrations of 85.7 ng/µL, 8.57 ng/µL, 0.857 ng/µL, 85.7 pg/µL, 8.57 pg/µL, and

0.857 pg/µL for each of the probes. This was done for both 26695 and 91-124 RNA.

As a negative control RNA was omitted from two tubes of 2ng/µL ureA and r16S

probes and these solutions were diluted as described above. Hybridization was done at

50oC for 180 minutes on an orbital shaker at 400 RPM. Hybridization solutions are

transferred to streptavidin-coated microtiter plates (supplied in kit) and incubated at

50oC. The solutions were decanted and the plates were washed six times in 250 µL of washing buffer (supplied in kit) at room temperature. 100 µL of anti-DIG antibody coupled to horseradish peroxidase enzyme (1:30) was added to the wells and incubated at 37oC for 30 minutes on an orbital shaker at 400 RPM. Afterwards, the antibody

solution was removed and the wells were washed with 100 µL of washing buffer four

times. 100 µL of tetramethly benzidine (TMB) was added to the wells and incubated at

room temperature on an orbital shaker at 400 RPM until color development. After

adding 100 µL of stop reagent (supplied in the kit) to each wells, the plates were

analyzed by a spectrophotometer at a wavelength of 492 nm.

In vitro Transcription

In order to provide template DNA for the in vitro transcription of ureB and r16S

RNA PCR was performed using the primers 5’-

38 GCATTTAGGTGACACTATAGGCTAAGAGATCAGCCTATGTCC-3’

(HP16S3/Sp6-F) and HP16S for r16S and 5’-

GCATTTAGGTGACACTATAGGCGATAAAGTGAGATTGGGC-3’ (UreB5/Sp6-F)

and UreB4-R primers for ureB. The PCR resulted in DNA fragments with the Sp6

promoter at the 5’ end. The PCR conditions for r16S and ureB were the same as those

used for the PCR reactions used to synthesize template for r16S and ureB probe

synthesis.

In vitro transcription was performed with the r16S/Sp6 and ureB/Sp6 PCR

products using the Riboprobe® In Vitro Transcription Systems (Promega, Madison,

WI). 1 µL of the PCR products (1µg/µL) was added to the reaction mixture of 4 µL of

Transcription Optimized 5x Buffer, 2 µL of 100 mM dithiothreitol (DTT), 1 µL of 40

U/µL of recombinant Rnasin® Ribonuclease Inhibitor, 4 µL of 2.5 mM rNTP’s, 1 µL of

Sp6 polymerase (15U/µL), and 7 µL of Rnase-free water. The reaction mixture was

incubated for 1.5 hours at 37oC. 2 µL of RQ1 Rnase-free DNAse was added to the

reaction and incubated for 15 minutes at 37oC. All components were provided in the

Riboprobe kit.

A phenol/chloroform extraction was done on the reaction mixture to purify the

RNA. The RNA was extracted with 22 µL (1 volume) of a solution of TE-saturated

phenol:chloroform:isoamyl alcohol (25:24:1). This was mixed for 1 minute using a

vortexer and sedimented by centrifugation at 12,000 RPM for 2 minutes in an

Eppendorf 5415C microtube centrifuge. The upper aqueous phase was removed and

transferred to a sterile tube and the extraction procedure was repeated. Afterwards, 2.5

µL (approximately 0.1 volume) of 3M ammonium acetate (DEPC-treated) was added

39 and mixed by using a vortexer. 61.5 µL (approximately 2.5 volume) of absolute ethanol was added and mixed using a vortexer. This solution was incubated at –70oC for 30 minutes and afterwards sedimented at 12,000 RPM for 30 min with an Eppendorf mirocentrifuge. The supernatant was removed, and the pellet was resuspended in 500

µL of 70% ethanol and sedimented at 12,000 RPM for 15 minutes. The supernatant was removed, and the pellet was resuspended in 40 µL of DEPC-treated water. The RNA was quantified using the GenQuant spectrophotometer.

Real–time reverse transcriptase-polymerase chain reaction (RT-PCR)

The primers used for detection of r16S DNA were the HP16S3 and HP16S4 primers from the PCR reaction used to generate template DNA for the synthesis of the

DIG-labeled probes for the slot blot analysis. Primers specific for the ureB gene were utilized to determine urease RNA expression. The forward primer was 5’-

GCTCAGAGCGGCTGAAGAATAT-3’ (UreB4-F) and the reverse primer was 5’-

CGGGAAGAATGTTGTGTTCACC-3’ (UreB4-R).

RT-PCR was performed using the LightCycler RNA Master SYBR Green I kit

(Roche). Along with the RNA from the bacterial samples a set of RNA standards (100 ng, 10 ng, 1ng, 100 pg, and 10 pg) of ureB or r16S RNA was included in the assay. 2

µL of RNA and a solution of 7 µL of Rnase-free water, 1.5 µL of 50 mM manganese acetate, 7.5 µL of RNA Sybr Green I, and 4 µL of each of the 2.5 mM forward and reverse primers were placed in LightCycler capillary tubes (Roche). The capillary tubes were loaded into the LightCycler apparatus (Roche). The reaction steps for the RT-

PCR for ureB and r16S were programmed as follows:

40 UreB

RT Phase: 61oC for 20 min

Amplification Phase: 95oC for 5 seconds; 65oC for 50 seconds; 72oC for 13

seconds for 30 cycles

Denaturation Phase: 95oC for 2 minutes

Melting Curve analysis Phase: 95oC for 5 seconds; 65oC for 15 seconds; 95oC

for 0 seconds

r16S

RT Phase: 61oC for 20 min

Amplification Phase: 95oC for 5 seconds; 55oC for 50 seconds; 72oC for 13

seconds for 30 cycles

Denaturation Phase: 95oC for 2 minutes

Melting Curve analysis Phase: 95oC for 5 seconds; 65oC for 15 seconds; 95oC

for 0 seconds

To calculate the actual number of transcripts of urease or r16S, standard curves were generated from analysis of the known amounts of ureB and r16S RNA . The

LightCycler® PCR machine and its accompanying software calculated the amount of

PCR product yielded from the bacterial RNA samples, and this was in turn used to calculate the copy number of urease or r16S RNA via the standard curve.

41 Urease Activity

Two methods were used to determine the urease activity of the bacterial samples. One was a modification of the Urea/ammonia test kit (Boehringer Mannheim)

(72). Overnight Brucella broth cultures of H. pylori were harvested by centrifugation in the Beckman Model TJ-6 (Beckman Coulter, Fullerton, CA) centrifuge at 3,000 RPM for fifteen minutes. The supernatant was removed and the pellet was resuspended in 2 ml of 1x PBS and sedimented as before. This was repeated twice. The concentration of the colony suspension was adjusted to 1.0 x 109 colonies/mL. Ten µL of the suspension for each of the bacterial samples was added to the urease assay reaction solution of 83

µL of 80 µg/mL of reduced nicotinamide adenine dinucleotide phosphate (NADPH)

(Sigma), 39.5 µL of 3.7 µg/µl 2-oxoglutarate (included in test kit), 12 µL of 833 U/mL glutamate dehydorgenase (included in test kit), 55.5 µL of triethanolamine buffer, pH

8.0 (included in test kit), and 10 µL of 4M urea (Sigma). Immediately upon addition of the bacterial samples to the reaction mix, the continuous change in absorbance over 1-2 minutes was determined with a Beckman DU-600 (Beckman Coulter) spectrophotometer at a wavelength of 340 nm. The accompanying computer software plots the net absorbance vs. time. The linear phase of the graphs was used to calculate the rate (net absorbance/minute) of the reaction for each sample. The activity of each sample was expressed as the net absorbance/minute/ mg of protein.

The second method was the phenol-hypochlorite urease assay. The bacterial cells were prepared the same as in the first method except that ice-cold 50mM N-2- hydroxyethly- piperazine-N’-butanesulfonic acid, pH 7.5 (HEPES) was used instead of

1 x PBS. Fifty and 100 µL of the bacterial samples and 30 µL of the urea standards

42 (4000, 2000, 1000, 500, and 250 µg/mL) were added to 50 mM HEPES to a final

volume of 1 mL. Afterwards, they were incubated for 20 minutes at 37oC. Following the incubation 100 µL of each reaction was removed and added to 1.5 mL of Solution A

(106 mM phenol, 16.8 nM sodium nitroprusside) and then mixed in a 3 ml cuvette. 1.5 ml of Solution B (5 mg/mL NaOH, 0.44% [v/v] hypochlorite) was added next. The mixture was incubated for 30 minutes at 37oC and afterwards was read on a spectrophotometer at a wavelength of 625 nm to determine the activity. The concentration of protein in the bacterial samples was determined by using the BCA assay. Forty µL of 4 % (w/v) copper II sulfate (pentahydrate) (Sigma) was added to

1.6 mL of bicinchoninic acid (Sigma). Fifty µL and 100 µL of the bacterial samples were added to this solution. The BCA assay was determined. Protein concentration was calculated by comparison to a standard curve of bovine serum albumin (BSA) standards

(Sigma) are performed alongside the bacterial samples. The reaction was incubated at

37o C for 30 minutes and then analyzed using a spectrophotometer at a wavelength of

550 nm.

Statistics

Statistical analysis of data was performed using one-way ANOVA with the

Tukey-Kramer multiple comparison post-test (Instat Graphpad Software, San Diego

Ca).

RESULTS

Determination of Urease RNA in Several 26695 Isolates

Slot blots

This study used isolates of H. pylori 26695 strain that had been recovered following infection in the pig and subsequently passed in the laboratory. The isolates are described in the Material and Methods. Briefly, the isolates were 22695 PP2 which had been passaged in the pig twice; 99-2310 which after recovery from an infected piglet was lab passed four times; and 96-1106 pig isolate which was lab passed 14 times. RNA from the 22695 isolates was affixed to a nylon membrane by a slot blot apparatus and hybridized with DIG-labeled probes to ureA RNA and r16SRNA (Fig

1.1). Detection of r16S was included as a reference transcript since r16S should be expressed at the same level by all strains. Therefore r16S was an internal control to account for miscalculation in total RNA quantitation and possible errors in loading. The resultant bands were quantified by densitometry, which generates values corresponding to pixel intensity (Fig. 2.1). For each sample, the ratio of r16S: ureA was calculated and

44 this value is used to compare samples (Fig. 2.2). Data genereated from the slot blots indicated that the urease gene was expressed at the same level in all strains (p>0.05).

r16S ureA

99-2310 (Pig islotate; lab-passed 4 times)

26695 PP2 (Pig isolate; lab-passed 3 times)

26695 (Strictly lab-passed)

96-1106 (Pig isolate; lab-passed 14 times)

Figure 2.1: RNA slot blots were performed with 1µg and 0.5 µg of RNA from the 26695 isolates. They were hybridized with digoxigenin (DIG)-labeled DNA probes complementary to sequences in ureA transcripts and ribosomal 16S (r16S)

45 1.4

1.2

0.8

0.6

0.4

0.2

0 26695 PP2 99-2310 26695 96-1106

Figure 2.2: Slot blots were analyzed by densitometry using the Imagequant software. Values are given in pixel intensity of the bands on the slot blot. UreA expression is compared between isolates by the ratio of ureA:r16S . (n=2)

Northern ELISA

An alternative method to the detection and the quantification of mRNA,

Northern ELISA, was also explored. As it name implies this technique blends aspects of Northern blotting with the enzyme-linked immunosorbent assay (ELISA). Total

RNA from H. pylori labeled with biotin is hybridized in solution with the ureA and r16s

DIG-labeled probes used in the slot blots. The hybridization solution is applied to microtiter plate wells coated with streptavidin. The streptavidin tightly binds the biotin of the labeled RNA/DIG probe duplexes while unhybridized probe is removed in subsequent washes. The biotin-labeled RNA/DIG probe duplexes are detected with anti-DIG antibodies coupled to horseradish peroxidase that reacts with 3, 3’, 5, 5’-

46 tetramethlybenizidine (TMB). The horseradish peroxidase/TMB reaction can be

detected spectrophotmetrically and can be used to determine the amount of mRNA. As

0. 3

0. 25 s 6 1 r f 0. 2 o s. b A / nm)

A 0. 15 e r 450 ( u f o

s. 0. 1 b A

0. 05

0 91-124 26695

Figure 2.3: The Northern ELISA technique is used to determine the relative expression of ureA. Biotinylated RNA from 22695 and the 91-124 isolate are hybridized to DIG-labeled r16S and ureA DNA probes, then applied to streptavidin-coated wells of microtiter plates. After washing away unhybridized probe, an anti-DIG antibody conjugated to horseradish peroxidase is pipeted into the wells. Addition of tetramethylbenzidine causes a reaction with peroxidase that can be measured with a spectrophotometer at a wavelength of 450 nm. Expression of ureA transcripts is reported as the ratio of absorbance of ureA:absorbance of r16S. (n=3)

in the slot blots the amount of ureA RNA is expressed as the ratio of ureA RNA to r16S.

For this assay wild type 26695 strain and the 26695 isolate 91-124 were analyzed. The pig isolate 91-124 was lab passed twice. Interestingly, Northern ELISA analysis indicated that ureA in 26695 was transcribed nearly twice as much as in the 91-124 isolate; however, this was not statistically significant (p=0.792) (Fig. 2.3).

47 Real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

One-step RT-PCR using a real-time Lightcycler® apparatus was used to determine relative expression of urease transcript compared to r16S RNA. Unlike the other two methods, this assay is able to calculate the copy number of mRNA providing a more accurate calculation of urease expression. Another difference is that quantification was based on amplification of ureB instead of ureA. Since ureA and ureB are transcribed on the same RNA strand (2), quantification of the PCR product from primers within ureB should still represent amplification of urease RNA. As in the other two methods, r16S is used as reference RNA to ensure that comparisons between isolates reflect actual urease RNA levels rather than differences in the amount of total

RNA.

Real-time RT-PCR is able to calculate of the copy number of RNA transcripts because a standard curve of in vitro transcribed urease RNA of known copy numbers is used along with total RNA from the bacterial isolates. The LighCycler® machine and its accompanying software can determine the amount of replicons generated by each sample by measuring the incorporation of the fluorescent SybrGreen® molecule during the cycles representing the linear phase of the amplification. The slopes of the linear phase for each sample are compared to the slopes generated by the standards to determine the concentrations of the RNA of the samples originally applied to the RT-

PCR reaction.

Real-time RT-PCR was done using 26695, 96-1106, and 91-124. The results indicated that in the three isolates urease is expressed at approximately the same level

(p= 0.5975). The data from this assay is in agreement with data from the slot blots.

1.20E+02 ) r be 1.00E+02 num

opy 8.00E+01 (c B e

6.00E+01 ):ur r be

4.00E+01 num opy 2.00E+01 (c S 6 1 0.00E+00 26695 99-2310 96-1106 (p4) (p14)

Figure 2.4: Real-time RT-PCR analysis of 26695 and the two pig isolates 99-2310 and 96-1106. The P value is 0.5975, considered not significant. (n=3)

Determination of urease activity

Post-transcriptional and post-translational regulation of the urease transcript has been previously described for urease of H. pylori (38, 220, 275); therefore, analysis of urease activity of the isolates must be done to ensure that transcriptional activity detected in the previous experiments reflects enzyme activity for urease. Two methods for detection and quantification of urease activity were examined.

The first method for measuring urease activity was based on a commercially available urea detection kit. This kit was adapted for determining urease activity by

49 substituting the urease enzyme supplied in the kit with H. pylori. The following reaction allowed detection of urease activity:

Urea + H2O + H. pylori 2 NH3 + CO2

+ GlDH + 2-Oxoglutarate + NADH + NH4 L-Glutamate + NAD +H2O

+ The kit enzyme glutamate dehydrogenase (GlDH) uses the NH4 from the degradation of urea reacts and NADH to convert 2-oxoglutarate to L-Glutamate. As a result,

NADH is oxidixed to NAD+. Because NADH can be measured with light absorbance at a wavelength of 340 nm, the rate of reduction in absorbance as NADH is oxidized can be use to calculate the activity of urease. Using this procedure the high lab-

25 abs./min/µg) ∆ 20

5 Urease activity (x10E-3

0 99-2310 26695 96-1106 91-124 Strains

Figure 2.5: Urease activity was determined by modification of a commercially- available urea detection kit. The urease activity of 99-2310 is nearly three times that of 26695, 96-106 and 91-124. (n=2)

50 passaged 96-1106 (14 passes) and the low passaged 91-124 (2 passes) exhibited urease

activity comparable to 26695. However, 99-2310 (4 passes) had a urease activity

around 3 times as much as the other three (p<0.05).

Urease activity was alternatively measured by the phenol-hypochlorite assay.

This method is a more direct measure of urease activity in that it is not dependent on the activity of a second enzyme as in the first assay. The following is the reaction that takes

place:

NH3 + NaOCl + phenol + NaOH Indophenol

Using the phenol-hypochlorite assay, the urease activity of the isolates were similar

(Fig. 2.6). This is consistent with the RNA results of the slot blot and real-time RT-

PCR procedures.

0.450

0.400

0.350

0.300

0.250

0.200

0.150

0.100

0.050

0.000 26695 26695 99-2310 96-1106 PP2 (pp4) (P14) Strains

Figure 2.6: Urease activity was determined by the phenol-hypochlorite assay. The urease activ ities of the samples were comparable indicating that urease is constitutively expressed.

DISCUSSION

The data presented here suggest that urease is constitutively expressed in the

26695 isolates either passaged in the lab or recovered from the pig. Passage in vitro did

not appear to effect urease expression. Pig isolate 96-1106 was passaged in the lab 14

times after recovery from the pig. However, urease RNA and urease activity was

similar to 99-2310, which was only passaged four times in the lab after recovery.

Urease expression was measured by three methods: slot blots, Northern ELISA,

and real-time RT-PCR. Slot blots were initially used because they offered a high

throughput and straightforward method without the RNA electrophoresis and time- consuming transferal of RNA onto a nitrocellulose membrane. However, this method was hampered by the detection method, which necessitated densitometric analysis to evaluate the level of urease RNA in the isolates. The software used assigned arbitrary values to the bands. These values correspond to the pixel intensity of the bands.

However, even in samples in which the amount of RNA was kept consistent, the bands were sometimes of differing widths, and a reproducible method to determine the size of the band and the pixel intensity could not be developed.

Northern ELISA appeared to be a better alternative. Hybridization of RNA and the DNA probe takes place in solution. Because molecules of RNA diffuse freely rather

than binding to a membrane, hybridization in solution is more efficient. Also, the 52 amount of urease RNA is directly detected by a spectrophotometer. However, a major

problem with this method is that it was designed for analysis of enriched samples of

eukaryotic mRNA, and non-specific binding to ribosomal RNA may occur. In the

experiments described here the urease probe was often less intense than the r16S probe.

A greater amount of r16S RNA is to be expected over urease RNA since r16S accounts

for about 27% of total RNA (190). However, in order to thoroughly eliminate the

possibility of nonspecific binding, unlabeled probe can be added to the hybridization

reaction. Competition for urease RNA should reduce the absorbance if the nature of the

initial results was the specificity of the probes used. Unfortunately, because of the

sudden unavailability of Northern ELISA kits nor its individual components from their

supplier and the absence of a comparable product from other molecular biology companies, the specificity of this assay could not be thoroughly verified.

The slot blots and the real-time RT-PCR both indicated that urease expression was the same regardless of the number of passages in vitro and the source of the bacteria. However, according to the Northern ELISA data, urease was expressed twice as much in 26695 than in 91-124 indicating that urease would be differentially expressed. However as stated earlier, the specificity of the probes for this assay was not established.

In order to determine whether post-transcriptional regulation of urease occurs, the urease activity of the isolate was also determined. It has already been shown elsewhere that urease activity can be regulated post-transcriptionally by the UreI H+-

activated urea channel (275). Because urease activity was done under the same pH

conditions, UreI regulation is not relevant in this study.

53 Two methods were used to determine urease activity. The first method was

adapted from a commercially available kit for determining the amount of urea. By

substituting the supplied urease enzyme with H. pylori and a defined amount of urea,

the rate of urea conversion to NH3 and CO2 can be determined by light absorbance.

However, in this assay, the NH3 production is reliant on a linked reaction with GlDH.

The phenol-hypochlorite assay was a more direct approach in that the NH3 liberated by

urease converts phenol and NaOCl into indophenol.

Optimization of urease RNA quantification and urease activity has led to the

conclusion that urease expression in the different isolates of 26695 is constitutive

regardless of passage in vivo and in vitro. This is in contrast to motility, which has

been known to be lost after prolonged passage in vitro. Thus, these data indicate that

there is no genetic or phenotype change in the urease enzyme with animal passage.

However, this does not eliminate the possibility thtat urease is regulated and expression

in situ differs from expression in culture. To determine if urease activity differs in in

vivo and in vitro conditions, we have developed a technique for determining urease

activity from stomach homogenate of infected mice (69). Using this technique we able

to analyze the transcriptional activity of promoters of the Cag pathogenicity island fused

to the promoterless urease gene. One Cag promoter (that of cag15) was shown to be

upregulated in vivo (122). Perhaps, the promoter of the urease gene cluster is also

induced in vivo. To demonstrate this would require the determination of urease

expression in vivo. I attempted to use real-time RT-PCR to determine in vivo urease

expression in total RNA in gastric tissue from mice infected with H. pylori. However, I

was not able to detect urease RNA or r16S RNA in the gastric homogenate of infected

54 mice (data not shown). This was probably due to the amount of host RNA

overwhelming the bacterial RNA in the samples. Therefore, an alternative method of

detecting RNA levels of H. pylori on gastric tissue needs to be developed before

determining whether urease expression is upregulated in H. pylori during infection.

Another aspect of urease expression of interest is whether different strains of H.

pylori also express urease at the same level as observed in the strain 26695. An

attractive possibility is that urease activity may underlie differences in colonization

potential and pathogenicity of different H. pylori strains. Thus, this study should be repeated using different H. pylori strains to more fully understand the transcriptional regulation of urease.

CHAPTER 3

Variable Expression of Redundant α-3-Fucosyltransferase Genes futA and futB Lacks

Correlation with Phase Variation of Lewis x and Lewis y Antigens in Helicobacter

pylori

ABSTRACT

The gastric pathogen Helicobacter pylori causes stomach ulcers and gastric

cancer. This bacterium expresses two α-3-fucosyltransferase enzymes, which may play different roles in the selection of the phenotype of Lewis antigens found on H. pylori

lipopolysaccharide (LPS). These enzymes are encoded by futA and futB, which contain a polycytidine (poly(C)) tract vulnerable to slip-strand mispairing during replication that can result in the addition or deletion of cytyidines. Alterations in the length of poly(C) may disrupt the reading frame of genes, which could result in the creation of a premature stop codon prevention the expression of a functional, full-length protein. The purpose of this study was to determine the relationship of futA and futB expression,

Lewis antigen phenotype, and animal colonization. The poly(C) regions of futA and

56 futB were amplified from lab-passed and animal-passed H. pylori strains, and DNA sequencing was used to determine the length of the poly(C) tract. The LPS phenotype of the strains were analayzed by western blot and ELISA using antibodies specific for

Lewis x or Lewis y antigens. In the two animal-virulent strains examined, SS1 and

26695, there was no relationship between futA sequence, Lewis x or y expression, or animal passage. Although the length of the poly(C) region of futA varied between strains and between isolates of the same strain, a relationship between the on/off status of the gene and bacterial phenotype could not be established. Variations were seen in the poly(C) tract of futB, but the predicted amino acid sequence revealed that futB was expressed in all the strains examined. Therefore, no relationship could be determined between futB, Lewis antigen expression, and animal passage.

INTRODUCTION

Helicobacter pylori is a gram-negative, microaerophilic, bacterial pathogen that is adapted to colonize the human glandular stomach. Infection by H. pylori can result in gastroduodenal disease ranging in severity from gastritis to peptic ulcer disease and cancer. H. pylori is the only bacterium classified as a group I carcinogen by the WHO

International Agency for Cancer Research for its direct link to gastric cancer (8).

Infection is common, ranging from 70-90% in developing countries to 25-50% in developed countries (257). Thus, high rates of infection and severity of disease underscore the significance of H. pylori in global health, and there is considerable

57 interest in how H. pylori is able to evade the host’s immune response, establish colonization, and cause disease.

In an effort to elucidate these processes, a number of possible bacterial virulence factors have been identified. One of these that has received intense research interest is lipopolysaccharide (LPS). LPS is a complex glycolipid molecule found at the cell surface of gram-negative bacteria. It consists of a surface-expressed O-antigen polysaccharide, a core oligosaccharide, and lipid A, which is embedded in the bacterial outer membrane. The LPS of H. pylori is distinct from the LPS of enterobacteria in that the lipid A of H. pylori has relatively low endotoxic activity (179) and that its O- antigen has terminal polysaccharide structures identical to human Lewis blood group antigens (17, 177, 178). Human Lewis blood group antigens are fucosylated oligosaccharides found on the surface of gastric epithelial cells (44). NMR analysis(17,

58 Gal1 3GlcNAc- 2

Fuc1

H-type 2

Gal1 4GlcNAc- 3 Gal1 3GlcNAc- 4

Fuc1 Fuc1

Lewis x Lewis a

Gal1 4GlcNAc- 2 3 Gal1 3GlcNAc- 2 4

Fuc1 Fuc1 Fuc1 Fuc1

Lewis y Lewis b

3(Gal1 4GlcNAc)n

i-antigen (polylactosamine)

Figure 3.1: Lewis antigens share a common disaccharide precursor composed of N-acetyl-D-glucosamine (GlcNAc) and D-galactose (Gal). One determinant of the phenotype of the Lewis antigens is the linkage between C1 of Gal and C3 of GlcNAc (Lea and Leb) or C1 of Gal and C4 of GlcNAc (Lex and Ley). Another phenotype determinant is the linkage of the sugar fucose to either GlcNAc (Lea and Lex) or linked to both Gal and GlcNAc (Leb and Ley)) or fucose linked to Gal only (H type 2 ). Also, there is the i-antigen that is not fucosylated and has a 1,4 linkage joining Gal and GlcNAc.

18, 176, 177), immunoelectron microscopy (239), and enzyme-linked immunosorbent

assay (241, 281) of H. pylori has shown that the bacterium expresses a variety of Lewis

antigens including H-type 1, i antigen,Lewis a, Lewis x (Lex) and Lewis y (Ley). Lex

and Ley are the most prevalent Lewis antigen epitopes, occurring in greater than 80% of

H. pylori strains (241).

The mechanism by which this molecular mimicry of human Lewis antigens may

benefit H. pylori and contribute to pathogenesis is not well understood. Some have proposed hat Lewis antigens mediate binding of H. pylori to the surface of gastric epithelial cells (78, 256). Another possibility is that it induces autoimmunity via antigen cross-reactivity and, thus, could cause or exacerbate disease in the host. This hypothesis is supported by detection of anti-Lex and anti-Ley antibodies in sera of

individuals infected with H. pylori (14, 103, 239) and the production of autoantibodies

against antigens of the gastric mucosa of mice immunized with H. pylori (189).

Alternatively, H. pylori Lewis antigens may camouflage the bacterium from the

surveillance of the host immune system, thus protecting it from elimination (15, 31, 57,

59, 166, 180, 193, 201, 211). This hypothesis is consistent with findings that H. pylori

isolates that are recovered from experimentally infected Rhesus monkeys exhibit the

same Lewis antigen phenotype as the host (279). Also, Helicobacter mustelae isolates from naturally infected ferrets express the same blood group A antigen as the host

(198).

These proposed effects of Lewis antigen cross-reactivity suggest that the host

Lewis antigen phenotype may determine the ability of H. pylori to colonize, to cause

60 disease, or both. If this is true, it would be advantageous to the bacterium to be able to

adapt its own Lewis antigen phenotype to that of the host. In fact, H. pylori is able to

vary its Lewis antigen expression in vitro, and the Lewis antigen phenotype of clinical

isolates has been reported to undergo spontaneous switching at a rate of 0.2-0.5% (13).

The mechanism behind this phase variation is believed by some to be slip-strand

mispairing occurring at a polycytidine (poly(C)) tract of two α-3-fucosyltransferase

genes, futA and futB. Alpha-3-fucosyltransferase catalyzes the last step of Lewis antigen synthesis, linking fucose to C-3 of N-acetyl-glucosamine (273).

Although the gene products of futA and futB appear to have redundant functions, there is evidence to suggest that phase variation in their expression determines Lewis antigen phenotype (9). Analysis of Lewis phenotypes of α-3-fucosyltransferase knockouts has shown that loss of futB correlates with the absence of Lex expression (9).

This suggests that futA and futB gene products possess fine specificities in their functions that designate the expression of either Lex or Ley. A later study showed that

the length of the poly(C) of the α-3-fucosyltransferase genes did not coincide with the

determination of Lewis antigen phenotype (226). Because this study used DNA primers

that could amplify the poly(C) region of both futA and futB due to the sequence

homology shared between the two genes, changes in the lengths of the poly(C) and, therefore, the on/off status of the genes could not be attributed to individual α-3-

fucosyltransferase genes. This is paramount to establishing a mechanism of Lewis

antigen specification based on the on/off status of two α-3-fucosyltransferase genes that

possess the same function yet differ in their preferences of fucosylation sites that

determine the Lewis antigen phenotype. The purpose of this study was to determine if 61 there exists a pattern of on/off status of futA and futB that predicts the Lewis antigen phenotype in two animal-virulent strains of H. pylori.

MATERIALS AND METHOD

Bacterial Strains

Several isolates of two H. pylori strains, 26695 and SS1, were used in this

study. Wild-type strain 26695 was originally isolated from a patient with gastritis and is

weakly virulent for gnotobiotic piglets (74). The piglet-adapted isolate, 98-580,

colonizes piglets 103–104-fold more densely than wild-type 26695 (3). Strain SS1 is a

mouse-virulent isolate also originally isolated from a human patient (137). In the

current study, wild-type, mouse virulent isolates, designated as SS1-Eaton and SS1-

Logan, were used. These isolates were passaged separately in the Eaton or Logan

laboratories but retained their mouse virulence. In addition, two SS1 mutants were

used. These mutants had been passaged in the laboratory in the course of introducing

mutations in the putative virulence genes, virB9 (isolate 83-1) and virB10 (isolate 101-

1). The mutations themselves did not affect colonization (70), but one of the mutants,

83-1, lost mobility and the ability to colonize after laboratory passage. Thus, four

separate strains of SS1 were evaluated. SS1-Eaton, SS1-Logan, and 101-1 retained

their mouse-virulence; however, 83-1 did not.

Sequencing of the Poly(C) Region

Bacterial genomic DNA was isolated using QiaAmp DNA mini prep kit (Qiagen

Inc., Valencia, CA). The poly(C) region and surrounding sequence were amplified from

62 genomic DNA of the isolates via polymerase chain reaction (PCR). The forward primers and reverse primers and PCR conditions that were used are shown in Table 3.1.

The Ohio State University Core DNA Sequencing Facility sequenced the resultant PCR product in forward and reverse directions using the same primers. Predicted amino acid sequence was determined using the Clone Manager program (Scientific and Educational

Software) and the San Diego Supercomputer Center (SDSC) Biology Workbench website (http://workbench.sdsc.edu/).

Primer Sequence PCR Conditions FTA-F 5'-GCGTTTTCTAGGCTCTCACAATTGG-3' 94º C, 1 min; 55ºC, 1 min; FTA-R 5' -GGTGTCATTAACAAGCTCGGC-3' 72º C, 2 min – 35 cycles HPFT-15 5' -CCTAAATTAGCTTAAAGGATAACC-3' 94º C, 1 min; 50ºC, 1 min; HPFT-16 5' -GCGATGATAGCGCAAGGGGTTTGA-3' 72º C, 2 min – 35 cycles HPFT-3 5'-TGGCAAACCCTCTTTTCAAAG-3' 94º C, 1 min; 50ºC, 1 min; HPFT-5 5'-TAGCCCTAATCAAGCCTTTG-3' 72º C, 2 min – 35 cycles FTB-R 5'-GCCGAAGTATCACGTTTACC-3' 94º C, 1 min; 50ºC, 1 min; FTB-F 5'-GCGACTATCACCTTTTACTGCG-3' 72º C, 2 min – 35 cycles

Table 3.1: Primer sequences used in this study – DNA oligonucleotide primers and the PCR conditions used to amplify the poly(C) region. Primers were also used in sequencing

Determination of LPS Phenotype Using Western Blot Analysis

Approximately 1 × 107 and 1 × 109 bacteria were washed in sterile deionized distilled water and resuspended in a final volume of 100 µL. Fifteen µL of 20mg/mL

Proteinase K (Sigma-Aldrich, St. Louis, Mo) was added and the samples were

63 incubated for 1 hour at 60º C. The samples were then placed in a boiling, water bath for

10 minutes to inactivate the Proteinase K. Electrophoresis was done on a SDS-PAGE gel at 200 volts for 35-45 minutes followed by electroblotting on a nitrocellulose membrane at 100 volts for 1 hour at 4º C. The membrane was air-dried, blocked in 3%

milk/0.05% Tween in PBS (Gibco, Carlsbad, CA) for 1.5 hours at 37o C, washed 3

times in PBST (PBS + 0.05% Tween), and incubated with either Lex antibody (BG-7,

Signet Pathology Systems, Inc, Dedham, MA) or Ley antibody (BG-8) at 1:1000 in 1:1

PBST:blocking buffer at 37º C overnight. The blot was then washed in PBST and

treated with anti-mouse IgM-alkaline phosphotase conjugate (Sigma-Aldrich, St. Louis,

Mo.) in 3% milk in PBST for 1.5 hours at 37º C. Following washes in PBST, reacting

bands were detected using the BCIP/NBT- phosphate substrate kit (Kirkegaard & Perry

Laboratories, Gaithersburg, Md).

Determination of Lewis antigen phenotype using ELISA

Ninety-six-well microtiter plates were coated with 1 ×106 of whole bacteria

resuspended in PBS and incubated overnight at 4ºC. After washing three times with

PBS, BG-7 and BG-8 were added at a concentration of 1:1000 in PBS/10% fetal calf

serum, and the plates were incubated for 1 hour at room temperature. After washing

three times with PBS, anti-mouse IgM-alkaline phoshphatase was used to probe for the

primary antibodies. Detection was done with the BCIP/NBT-phosphate substrate kit.

Optical density at 450 nm was recorded. Statistical analysis of ELISA data was done

using the Tukey-Kramer multiple comparison test (Instat Graphpad Software).

RESULTS

Comparison of the length of the poly(C) regions and predicted amino acid sequence

Sequencing data revealed that the number of cytidines in the poly(C) tract of futA differed between strains 26695 and SS1 and between laboratory and animal passed isolates. Lab-passed strain 26695 had 13 C’s (Figure 3.2A), which is consistent with the DNA sequence database published for this strain (260). In contrast, the piglet- passaged variant 98-580 contained 14 C’s in the poly(C) region (Figure 3.2B). This addition of an extra cytidine introduced a premature stop codon downstream of poly(C) region (Figure3.1B) and was expected to prevent the expression of futA (9). The futA gene of SS1 also differed between isolates. In the SS1-Eaton and the mouse-passaged variant 101-1, futA had only 7 cytidines (Figure 3.2C), predicting a truncated transcript, while the non-colonizing variant 83-1 had 13 cytidines like lab-passed 26695.

Interestingly, strain SS1-Logan, a wild-type strain which colonizes mice but has been passaged in the laboratory independently of SS1-Eaton also had 13C’s similar to 26695 and unlike SS1-Eaton. Random Amplification of Polymorphic DNA (RAPD) analysis was used to confirm that SS1-Eaton and SS1-Logan were the same strain (data not

65 shown). Thus, poly(C) tract length and predicted futA expression did not correlate with

either bacterial strain or virulence.

A. Poly(C) region of futA of 26695

1 ATGTTCCAACCCCTATTAGACGCCTTTATAGAAAGCGCTTCCATTGAAAAAAT 1 Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Glu Lys Met

54 GGCCTCTAAATCTCCCCCCCCCCCCCTAAAAATCGCTGTGGCGAATTGGTGGGG 19 Ala Ser Lys Ser Pro Pro Pro Pro Leu Lys Ile Ala Val Ala Asn Trp Trp Gly

108 AGATGAAGAAATT 37 Asp Glu Glu Ile

B. Poly(C) region of futA of 98-580

1 ATGTTCCAACCCCTATTAGACGCCTTTATAGAAAGCGCTTCCATTGAAAAAATG 1 Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Glu Lys Met

54 GCCTCTAAATCTCCCCCCCCCCCCCCTAAAAATCGCTGTGGCGAATTGGTGGGG 19 Ala Ser Lys Ser Pro Pro Pro Pro Pro Lys Asn Arg Cys Gly Glu Leu Val Gly

108 AGATGAAGAAATT 37 Arg Stop

C. Poly(C) region of futA SS1-Eaton and 101-1

1 ATGTTCCAACCCTTACTAGACGCCTTTATAGAAAGCGCTCCAATTAAAAAAAAAT 1 Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Pro Ile Lys Lys Lys Leu

62 TACC TCTAAATCTCCCCCCCTAA 20 Pro Leu Asn Leu Pro Pro Stop

Figure 3.2: Nucleotide sequence and the resultant amino acid sequence of the poly(C) region of futA including some flanking sequence within the gene. The predicted amino acid sequence was determined by using the Clone Manager program (Scientific and Educational Software) and SDSC Biology Workbench website. (A) In H. pylori lab- passed strain 26695 futA was determined to have a string of 13 cytidines indicating that the gene was “on” and, thus, it is capable of expression of the complete gene. (B) In pig-passed strain 98-580, 14 cytidines is in the poly(C) region of futA resulting in the

66 creation of a premature stop codon. Because the complete gene cannot be expressed, futA is “off”. (C) futA of mouse-passed strain 101-1 and lab-passed SS1-Eaton both exhibited a truncated poly(C) chain of 7 cytidines. This also resulted in a premature stop codon. The poly(C) chain is underlined and the stop codons are in bold.

The number of cytidines in the poly(C) of futB varied among the strains.

However, unlike the futA sequences, expression of full-length FutB was predicted in all

strains. All SS1 isolates, regardless of source had 5 C’s predicting full-length

expression, and all 26695 isolates had 13 C’s (Figure 3.2 and 3.3). In spite of the

difference in poly(C) region length between the two strains, the predicted sequence of

SS1 futB (5 C’s) has 74% identity with the 26695 futB gene product (13C’s) (Figure

3.4). Thus, differences in length of futB poly(C) correlated with parental strain and did

not predict a specific phenotype.

* - single, fully conserved residue - no consensus

26695 1 ATGTTCCAACCCCTATTAGACG 22 101-1 1 AAAAAATTTTTTTGTAAAATTCCTTTAAAAGGATAATCATGTTTCAGCCCCTATTAGACG 60 ***** ** *************

26695 23 CCTTTATAGAAAGCGCTTCCATTGAAAAAATGGTCTCTAAATCTCCCCCCCCCCCCCTAA 82 101-1 61 CTTATATAGACAGCACCCAAATAGAAGAGACAACCCATAAG------CCCCCATTAA 111 * * ****** *** * ** *** * * * *** ***** ***

26695 83 AAATCGCTGTGGCGAATTGGTGG-----GGAGATGA-AGAAATTAAAGAATTTAAAAAGA 136 101-1 112 ATATAGCCCTAGCCAATTGGTGGCCTTTGGATAAAAGAGAAAGCAAAGGGTTTAGGCGTT 173 * ** ** * ** ********* *** * * ***** **** ****

26695 137 GCGTTCTTTATTTTATCCTAAGCCAACGCTACGCAATCACCCTCCACCAAAACCCCAATG 196 101-1 183 TTATCTTGTATTTCATCCTAAGCCAACGCTATACAATCACTTTACACCAAAACCCTAAAA 233 * * ***** ***************** ******* * *********** **

26695 197 AATCTTCAGATCTAGTTTTTAGCAATCCTCTTGGAGCGGCTAGAAAGATTTTATCTTATC 256 101-1 234 AACATGCAGACATCGTCTTTGGCAGTCCTATTGGATCAGCCAGAAAAATCCTATCCTATC 293 ** * **** * ** *** *** **** ***** * ** ***** ** **** ****

26695 257 AAAACACTAAACGAGTGTTTTACACCGGTGAAAACGAATCACCTAATTTCAACCTCTTTG 316 101-1 294 AAAACACTAAAAGAGTGTTTTACACCGGTGAAAATGAAGTCCCTAATTTCAACCTCTTTG 353 *********** ********************** *** ******** **********

26695 317 ATTACGCCATAGGCTTTGATGAATTGGATTTTAATGATCGTTATTTGAGAATGCCTTTGT 376 101-1 354 ATTACGCCATAGGCTTTGACGAATTGGATTTTAACAATCGTTATTTGAGAATGCCTTTAT 413 ******************* ************** ********************** *

Figure 3.3 - Gene sequence alignment of the poly(C)region of futB from SS1 variant 101-1 and laboratory-passaged 26695. Alignment was performed using the SDSC Biology Workbench website. Asterisk indicates a fully conserved nucleotide. Absence of an asterisk signifies no consensus between the two sequences at that residue. Dash marks indicates a sequence or a single nucleotide not shared by the two sequences. The sequence just 5’ of the start codon of 26695 was not determined

Score = 288 bits (730), Expect = 8e-80 Identities = 137/184 (74%), Positives = 156/184 (84%), Gaps = 5/184 (2%)

101-1: 13 MFQPLLDAYIDSTQIEETTHK---PPLNIALANWWPLDKRESKGFRRFILYFILSQRYTI 69 MFQPLLDA+I+S IE+ K PPL IA+ANWW E K F++ +LYFILSQRY I 26695: 1 MFQPLLDAFIESASIEKMASKSPPPPLKIAVANWW--GDEEIKEFKKSVLYFILSQRYAI 58

101-1: 70 TLHQNPKKHADIVFGSPIGSARKILSYQNTKRVFYTGENEVPNFNLFDYAIGFDELDFNN 129 TLHQNP + +D+VF +P+G+ARKILSYQNTKRVFYTGENE PNFNLFDYAIGFDELDFN+ 26695: 59 TLHQNPNEFSDLVFSNPLGAARKILSYQNTKRVFYTGENESPNFNLFDYAIGFDELDFND 118

101-1: 130 RYLRMPLYYDKLHHKAESVNDTTSPYKIKDNSLYTLKEPSHHFKENHPNLCAVVNNEIDP 189 RYLRMPLYY LH+KAE VNDTT+PYK+KDNSLY LK+PSHHFKENHPNLCAVVN+E D 26695: 119 RYLRMPLYYAHLHYKAELVNDTTAPYKLKDNSLYALKKPSHHFKENHPNLCAVVNDESDL 178

101-1: 190 LKRG 193 LKRG 26695: 179 LKRG 182

Figure 3.4 – Predicted amino acid sequence alignment of futB of SS1-Eaton and its variant, mouse-passaged 101-1. Alignment of the amino acid sequences was performed using the SDSC Biology Workbench website. Blank spacing between the two sequences indicates a non-conserved residue at that position. “+” indicates that the residues at that position are not identical but belong to the same side chain group.

Determination of Lewis antigen phenotype

Appelmelk et al showed that in some strains of H. pylori, the Lewis antigen phenotype is regulated by the on/off status of both the futA and futB genes (2). To determine if such a relationship exists in animal virulent strains 26695 and SS1, the

Lewis antigen phenotypes of SS1-Eaton and its variants and wild-type 26695 and its variant were determined by Western blot and by ELISA.

69 A. n n n n ga ga to to o o 0 0 5 5 Ea Ea 8 1 8 1 - - - - 5 5 1 1 1 1 - - 1 1 69 69 S1-L S1-L S S 10 98 10 98 83- SS 26 83- SS 26

Lewis y

B. n n n n o o ga ga t t 0 0 5 5 Lo Lo Ea Ea 8 8 5 5 1 1 - - 69 69 1-1 1-1 SS1- SS1- SS1- SS1- 26 26 98 98 83- 83- 10 10

Lewis x

Figure 3.5: Western blot analysis of lipopolysaccharide (LPS) of H. pylori strains. Whole bacteria were treated with Proteinase K prior to Western blotting. (A) For Ley determination strain SS1-Eaton (lane 3) and its variants, 101-1 (lane1) and 83-1(lane 2), and strain 26695 (lane 4) and its variant 98-580 (lane 5) were analyzed. In addition, a lab-passed SS1 strain not previously handled in our lab, SS1-Logan (lane 6), was also included to compare with SS1-Eaton in the instance that phase variation occurred to SS1-Eaton while undergoing lab passage. Ley was expressed by wild-type SS1 (Eaton and Logan isolates) and the SS1 variants but was not detectable in 26695 or its variant 98-580. (B) Lex expression was detected in 26695 (lane 5) and 98-580 (lane 6), but not in SS1-Eaton (lane 4), SS1-Logan (lane 1), 83-1 (lane 2), nor 101-1 (lane 3).

70 Western blot revealed that SS1-Eaton and its variants all expressed Ley

(Figure 3.5A). SS1-Logan was identical to the other SS1 isolates. However, all SS1 strains were negative when probed with the BG-7 antibody, indicating that they do not express high enough levels of Lex to be detected by this method. (Figure 3.5B). In contrast to SS1, wild-type 26695 and 98-580 were positive for Lex but negative for Ley.

Unlike the Western blot data, ELISA data indicated that 26695 and 98-580 do express

y x Le epitopes and that Le is detectable in 83-1 and 101-1 although at a low level (Figure

3.6). The difference between Western blot and ELISA results is likely attributable to the difference in sensitivity of these two assays.

1.600

1.400

1.200

1.000

Lewis x 0.800 Lewis y

Abs. (450 nm) 0.600

0.400

0.200

0.000 SS1-Logan SS1-Eaton 831 101-1 580 26695 Strains

Figure 3.6 –Expression of Lex and Ley by H. pylori as measured by ELISA. BG-7 and BG-8 monoclonal antibodies are use to detect Lex and Ley, respectively. Lewis antigens were quantified spectrophotometrically at a wavelength of 450 nm. All isolates expressed Ley, but 26695 and its piglet-passaged variant, 98-580 had significantly lower expression than did SS1 and its variants (p<0.05). Strain 26695 and its variant had the highest expression of Lex. Lex expression varied among SS1 isolates.

Phenotype of Strains ELISA Variant Strain FutA FutB Lewis x Lewis y

Eaton SS1 7C (Off) 5C(On) - +

a 101-1 SS1 7C (Off) 5C(On) +/- +

83-1 SS1 13C (On) 5C(On) +/- +

Logan SS1 13C (On) 5C(On) - +

98-580 26695 15C (Off) 13C(On) + +

WT b 26695 13C (On) 13C(ON) + +

a Weak interaction with BG-7 antibody b Wild-type

Table 3.2 – Correlation between genotype and phenotype of H. pylori strains and variants

DISCUSSION

Previous studies have suggested that alterations of the length of the poly(C) chain of the fucosyltransferase genes futA, futB, and futC due to slip-strand mispairing during replication can prevent the expression of a functional, full length protein (9, 230,

72 271). Based on data from the comparison of the on/off status of the two α-3-

fucosyltransferase genes, futA and futB, and the resultant Lex/y phenotype in H. pylori strains, some authors have suggested that the futA and futB gene products possess distinct preferences for Lex and Ley synthesis and that the on/off status of these two genes underlies the phase variation of Lex and Ley epitopes (9). A later study concluded

that the poly(C) lengths of the α-3-fucosyltransferase genes could not be correlated with a specific Lex/y phenotype (226); however, this study failed to differentiate the poly(C) lengths of futA and futB. Such a distinction is necessary to establish whether the α-3- fucosyltransferase genes have divergent specificities and whether the presence or absence of one of these genes influences the Lex/y phenotype of the LPS. In the present

study we determined whether the on/off regulation of futA and futB could be directly

correlated with the Lewis antigen phenotype of the LPS of H. pylori. Our results

indicate that in the strains used in this study there was no correlation between futA or futB genotype and Lex or Ley expression (Results summarized in Table 3.2).

The length of the poly(C) of futA of SS1-Eaton and 101-1 were different from

that of SS1-Logan and 83-1, but the four strains had virtually identical Lewis antigen

expression. Furthermore, the lengths of the poly(C) of futA in 98-580 and 26695 WT were different and predicted different patterns of gene expression, but there were no

observable differences in Lewis x/y expression. In addition, comparison of futA/B

status between 26695 strains and SS1 strains did not yield a pattern for Lewis x/y

expression. For instance, in 83-1 and SS1-Logan that expressed both α3-

fucosyltransferase genes Lex was either weakly detected or not detected all, while 26695

73 WT that likewise expressed both genes possessed both Lex and Ley. This suggests that futA is not critical in synthesis of Lex or Ley antigens.

The full-length gene product of futB has been proposed to specifically

synthesize terminal monomeric and oligomeric Lex epitopes due to its preference for

internal lactosamine substrates for fucosylation (9). However, in our study Lex was not expressed in SS1-Eaton and SS1-Logan and only weakly expressed in 101-1 and 83-1 despite futB being intact. This suggests that intact futB gene product is not sufficient to

allow Lex expression. We cannot rule out the possibility that futB is necessary for Lex

expression since in all of the strains tested in this study futB was “on”.

The poly(C) of futA appears to be more hypervariable than the poly(C) of futB.

Although the poly(C) region of futB does vary between 26695 and SS1 strains, the

poly(C) of futA even varies within isolates of the same strain while the number of

cytidines in futB is stable within the same strain. This variability does not appear to

impact the outcome of the Lewis antigen phenotype, and its significance remains to be

determined.

Taken together the results of this study strongly suggest that phase variation of

Lex/y phenotype cannot be solely attributed to slip-strand mispairing of the poly(C) region of the futA and futB genes. Also, functional differences of the two α-3- fucosyltransferases accounting for Lex versus Ley expression could not be determined based on the resultant Lex/y phenotype. Future work should focus on enzymatic assays

to separate out these functional specificities of futA and futB if they do indeed exist. It

may be that these genes are strictly redundant in function. It has been proposed that the

on/off switching caused by slip-strand mispairing contribute to the ratio of the α-3-

74 fucoslytransferase enzyme to α-2-fucosyltransferase enzyme (270). Alpha-2- fucosyltransferase functions downstream of α-3-fucoslytransferase in the synthesis of

Ley. The consequence of reduction of the amount of α-3-fucosyltransferase enzyme as a result of one two copies of its gene being switched “off” may in turn diminish Lex

expression and favour the Ley phenotype. In addition sequence analysis of the futC

gene that encodes for α-2-fucosyltransferase reveals that it also has a poly(C) region

vulnerable to slip-strand mispairing (28, 260). The consequence of the regulation of

futC in relation to the on/off status of futA and futB on the Lewis antigen phenotype has

not yet been investigated. Regulation at the replication level of futA, futB, and futC may

be important for establishing the balance of Lex and Ley expression. Therefore,

disruption of this balance via “on/off” switching of any of these genes would alter the

Lewis antigen phenotype of H. pylori causing phase variation.

CHAPTER 4

The Role of the Lewis Histo-blood group Antigens Expressed on the

Lipopolysaccharide of H. pylori in Modulating Host Immunity

ABSTRACT

The Helicobacter pylori SS1 mutant strain SS1:0826 was unable to synthesize

O-antigen and induced lower levels of gastritis when infected in mice compared to the parental SS1 strain. This suggests that the O-antigen may modulate the host’s immune response. Infection with H. pylori stimulates a cell-mediated, TH1, immune response via CD4+T-cells. The hypothesis of this chapter is that O-antigen modulate the host immune response by interaction with CD4+T-cells. This was evaluated by stimulating splenocytes derived from mice inoculated with SS1 H. pylori strain with sonicate preparations of the H. pylori M6 strain and M6:0826 and LPS samples from SS1 and

SS1:0826. In additon, splenocytes from T-bet+/- and T-bet-\- mice, which are deficient in the T-bet transcription factor required for interferon gamma (IFNγ) were evaluated for their responsiveness to these antigens. Stimulation of the splenocytes was determined by enzyme-linked immunosorbent assay (ELISA) quantitation of IFNγ secreted into the supernatant after five days in cell culture.

76 The data generated from this study suggested that IFNγ induction of splenocytes from infected mice was not significantly reduced by lack of O-antigen in M6:0826 sonicate compared to the M6 sonicate. Likewise, IFNγ induction by both the SS1 and the SS1:0826 LPS samples at 0.5 µg/mL were indistinguishable and comparable to that of the sonicate samples. However, at 50 µg/mL the LPS samples stimulated IFNγ secretion from splenocytes of both uninfected and infected mice to levels approximately

7 times that of M6 sonicate stimulation of splenocytes from infected mice. SS1:0826

LPS induced IFNγ synthesis at a higher level than SS1 LPS but not significantly so

(p>0.05). Interestingly, IFNγ induction of by SS1:0826 LPS at the higher concentration was reduced when treated with proteinase K prior to incubation with naive splenocytes although induction by SS1 appears to be unaffected by proteinase K.

This indicates that at the higher concentration cellular protein in SS1:0826 LPS may in part account for stimulation of splenocytes. Also, because splenocytes from uninfected mice stimulated with SS1 LPS pre-treated with proteinase K also induced IFNγ production, the response of the splenocytes not treated with proteinase K is probably due to non-specific to the cellular protein in the LPS. Also, IFNγ induction of the splenocytes from infected mice by the LPS samples is not significantly reduced by proteinase K suggesting that this response, especially in the case of SS1:0826, is probably specific to antigens in the cellular protein fraction of the LPS samples.

IFNγ induction in the splenocytes from infected, T-bet+/- mice was decreased compared to the splenocytes from infected, wild type mice for all the antigens except for M6:0826 sonicate which was actually elevated. For the splenocytes from infected,

T-bet-/- mice stimulation was undetectable for all the antigens except for the 50 µg/mL

77 LPS samples which were at levels comparable to uninfected T-bet-/- splenocytes but lower than infected wild type splenocytes treated with 50 µg/mL LPS . This indicates that CD4+ cells are essential to splenocyte stimulation in H. pylori sonicate preparations and 0.5 µg/mL LPS samples but may not be necessary for stimulation at 50 µg/mL of

LPS.

The data presented here suggest that the presence of O-antigen does not play a major role in stimulating the host immune system and may even downregulate the inflammatory response to H. pylori based on the higher stimulation of IFNγ in SS1:082

LPS. Also, the response to LPS is at least in part independent of infection and CD4+ with H. pylori since the infection status and the responsiveness of CD4+ cells are not required for IFNγ induction. However, further studies must be done to determine which cell populations in the splenocytes cultures are stimulated and whether LPS levels used in this assay are biologically significant.

INTRODUCTION

Lipopolysaccharide (LPS) is a complex macromolecule found on the outer cell membrane of gram-negative bacterium. LPS has three components: O-antigen, the core oligosaccharide, and lipid A. The O-antigen is the outermost moiety of the LPS and is composed of repeating sequences of either linear trisaccharides or branched tetra- or pentasaccharides that link to the heptose sugars of the core oligosaccharide. The variability of the O-antigen greatly contributes to the antigenic differences of the LPS between bacterial species and strains (142). The core oligosaccharide and lipid A of

78 LPS are relatively constant in structure and composition compared to the O-antigen.

The core oligosaccharide is the central region of the LPS and consists of a short chain of

heptose attached to keto-3-deoxyoctulonic acid (KDO), which is the point of attachment

for lipid A. Lipid A is a glycolipid and anchors the LPS to the outer cell membrane.

Lipid A is responsible for the endotoxic effect of the LPS of a number of

enterobacteria, such as S. minnesota and E. coli (34). LPS of these bacteria can elicit a potent inflammatory response from an infected host which in extreme cases can result in shock (a severe and sudden drop in blood pressure and tissue perfusion), end-organ failure, and death (167). However, the endotoxicity of the LPS of the gastric pathogen

H. pylori is greatly reduced compared to E. coli and S. minnesota. The LPS of S. minnesota has been calculated to have a 1000-fold more potency than H. pylori when

using a monocyte activation assay (179) . Despite this low potency, H. pylori LPS is

still able, to some degree, to induce the secretion of Il-6, IL-8, IL-1, tumor necrosis

factor (TNF), monocyte chemotactic protein 1, epithelial neutrophil-activating peptide

78, and reactive oxygen species from human monocytes in culture (31, 34).

A unique feature of the LPS of H. pylori is the expression of Lewis histo-blood group antigens on the O-antigen (17, 177, 178). Lewis x (Lex) and/or Lewis y (Ley)

antigens are found in 80% of H. pylori strains and Lewis antigens Lewis a, Lewis b, i antigen, and H type 1 have been identified on H. pylori strains. The Lewis antigens were originally found in human body secretions, such as saliva and gastric fluids, and adsorbed to the surface of red blood cells and the surface of tissues, including the gastric epithelial cells (44). Lewis antigens are closely related to the ABO histo-blood group antigens. They all share a common disaccharide precursor composed of N-

79 acetyl-D-glucosamine (GlcNAc) and D-galactose (Gal). The phenotypes of the Lewis antigens are determined by either the linkage between GlcNAc and Gal (Linkage between C1 of Gal and C3 of GlcNAc or C1 of Gal and C4 of GlcNAc) or the fucosylation pattern (Fucose linked to GlcNAc or fucose linked to both Gal and

GlcNAc).

Molecular mimicry of human Lewis antigens by the LPS of H. pylori has been the impetus of recent endeavors to determine the role of H. pylori LPS in the immune response to the pathogen. The expression by H. pylori of Lewis antigens that are identical in phenotype to that of the host could camouflage H. pylori from the host’s immune system preventing clearance of the bacterium and, in effect, promoting persistence of infection. Host mimicry has been shown experimentally in Rhesus monkeys infected with H. pylori strains of mixed Lewis antigen phenotype. The Lewis antigen phenotype of H. pylori recovered from these monkeys tended to match that of the host (279). Also, H. mustelae has been isolated from ferrets, its natural host, with the same Lewis antigen phenotype of its host (56). It may be that H. pylori is able to alter its Lewis antigen phenotype, presumably, to match that of the host during colonization. This proposal was based on the observation that H. pylori clinical isolates have been shown to be able to undergo spontaneous Lewis antigen phenotype switching at a frequency of 0.2 –0.5 % (12).

However, a more recent study indicated that H. pylori may influence the host

Lewis antigen phenotype. This study showed that H. pylori infection induced expression of sialyl-Lewis x antigens on the gastric epithelium of Rhesus monkeys

(156). The sialyl-Lewis x antigens were shown to be receptors for the H. pylori adhesin

80 SabA (156). SabA is believed to enable H. pylori adherence to the gastric epithelium

preventing the bacterium from being swept into the lower gastrointestinal tract.

Another H. pylori adhesin, BabA, has been theorized to function in the same role.

BabA binds Lewis b of gastric epithelial cells, and the presence of babA2 allele has

been correlated with increased virulence (94).

These observations of H. pylori adhesin/host Lewis antigen interactions have led

some to speculate that the reverse could also be true: that H. pylori Lewis antigens

interact with an epithelial cell adhesin. Consistent with this hypothesis, fluorescent

latex beads carrying synthetic Lewis x antigens mapped to the luminal surface of

mucosal cells and to the cells lining the gastric pits where H. pylori is normally

localized (78). However, an epithelial cell adhesin that binds Lewis antigens has not

yet been identified, and a recent study was unable to determine a correlation between

Lewis antigen expression, H. pylori in situ adherence to gastric sections, and gastric

histopathology (155).

Alternatively, the sharing of Lewis antigen phenotypes between the host and H.

pylori may lead to an autoimmune response. There are indications that this could take place in humans. Anti-Lex and anti-Ley antibodies have been detected in the sera of

individuals infected with H. pylori (14, 104, 239). There is also evidence of H. pylori-

induced autoimmunity in the mouse animal model (189).

Insertional inactivation of the β-1,4-galactostransferase gene (HP0826) in H.

pylori results in truncated O-antigen (150). The β-1,4-galactostransferase enzyme

directs the addition of Gal to GlcNAc of the Lewis antigen precursor on the lactosamine

chain of the O-antigen(150); therefore, the mutant does not express Lewis antigens.

81 Infection of severe combined immune-deficient (SCID) mice that were adoptively

transferred with splenocytes from wild type mice with the SS1 knockout mutant

(SS1:0826) results in reduced gastritis compared to transferred SCID mice infected with

wild-type SS1 strain (Infection and Immunity, in review). As in humans, infection of

adoptively transferred SCID mice with H. pylori results in a cell-mediated, or a TH1, immune response which is characterized by IFNγ production in tissue, splenic helper T

(TH) cells in response to H. pylori antigen, and the development of a delayed-type

hypersensitivity to H. pylori antigens, and gastritis (76, 173). Therefore, the infection

study with SS1:0826 strongly suggests that O-antigen have a significant role in

modulating the immune response in the mouse animal model.

Therefore, the purpose of this study is to test the hypothesis that through a novel

mechanism LPS modulates the host’s primarily TH1 response to H. pylori infection via

the O-antigen. Specifically, this study will seek to 1) determine whether H. pylori LPS

alone can stimulate mouse splenocyte cell cultures in a similar manner as H. pylori

sonicate, 2) to determine if loss of O-antigen in H. pylori sonicate abrogates stimulation

of mouse splenocytes, and 3) to evaluate whether the effect of O-antigen on the immune

system includes CD4+ T-cell involvement.

MATERIALS AND METHODS

Mice

C57BL/6J and C57BL/6J-Prkdcscid (Severe combined immunodeficient, SCID)

mice were purchased from Helicobacter-free colonies at the Jackson Laboratory (Bay

Harbor, Maine). C57BL/6Tbet-/- mice were obtained from Harvard University. Mice

82 were kept in sterile microisolator cages in a barrier facility where they were provided

with sterile water and fed Teklad lab chow ad libitum (Teklad, Madison, WI). Mice

were inoculated orally with 0.2 mL of approximately 108 cfu of live broth-cultured H.

pylori strains SS1 or M6 in Brucella broth. At the end of the infection phase, mice are

killed with phenobarbital and exsanguinated. The stomach and spleen are removed

aseptically. The stomach is bisected along the lesser and greater curvature. The

squamous is removed and half the stomach is homogenized for determination of

bacterial colonization by plate dilution, and the other half is sectioned in 1 mm sections

and stored in formalin for histologic analysis. All procedures involving animals were

approved by the Ohio State University institutional laboratory animal care and use

committee.

Bacteria

H. pylori strains SS1:0826, M6:0826, SS1, M6 were used in this study. M6:0826

and SS1:0826 are isogenic nonpolar mutant strains of M6 and SS1, respectively, in

which the β-1,4-galctosyltransferase gene (HP0826) is inactivated by insertional

mutagenesis of a kanamycin cassette. Strain SS1 and M6 are mouse-adapted strains

isolated from human patients (137).

Adoptive Transfer

Spleens for adoptive transfer were excised from the donor C57BL/6 mice and

disaggregated in cold (4oC) Hanks buffered salt solution. After clumps were allowed to settle out, cells were sedimented at 1000 rpm for 10 min at 4oC for 10 minutes. For

transfer, erythrocytes were removed by hypotonic lysis. After examination for cell

viability by trypan blue exclusion, splenocytes were counted, and adjusted to 1x107/mL

83 in Dulbecco’s PBS, pH 7.5. For adoptive transfer recipient SCID mice were given 0.1

mL by intraperitoneal injection.

Splenocyte cultures

Spleens were excised from the mice and disaggregated in cold (4oC) Hanks

buffered salt solution. After clumps were allowed to settle out, cells were sedimented at

1000 rpm for 10 min at 4oC for 10 minutes. For transfer, erythrocytes were removed by

hypotonic lysis. After examination for cell viability by trypan blue exclusion, splenocytes were counted, and adjusted to 1x107/mL in RPMI 1640 (Gibco BRL,

Rockville, MD) supplemented with 10% fetal calf serum (FCS) (Gibco BRL, Rockville,

MD), HEPES (Gibco BRL, Rockville, MD), 2-mercaptoethanol (Sigma, St.Louis, MO),

and antibiotics. 0.2 mL of the cells was pipeted into 96-well plates. Cells were treated with either 10 µg/mL of concanavalin A (conA) (Sigma), 10 µg/mL of sonicate of M6 or M6:0826 bacteria, 0.5 or 50 µg/mL of SS1 or SS1:0826 LPS preparations (courtesy of Susan Logan).

ELISA

Enzyme-linked immunosorbent assay was done using the OptEIA Mouse IFN-γ

Set (BD PharMingen, San Diego, CA) according to the included protocols. 100 µL of anti-mouse IFN-γ antibody in 0.1M carbonate, pH 9.5 (1:2000) was added per well to

96-well plates. The plates were covered and incubated overnight at 4oC. Afterwards,

the plates were washed 5 times in phosphate buffered saline (PBS) (Gibco) with 0.05%

Tween-20 (Biorad, Hercules, Ca) (PBST) and blocked with 200 µL of 10 % FCS in

PBS for 1 hour at room temperature. Plates were washed 5 times in PBST, and 100 µL

of supernatant from the splenocyte cultures were added to the wells along with IFN-γ

84 standards provided in the kit. After an incubation of 2 hours at room temperature, the plates were washed 5 times in PBST, and 100 µL of biotinylated anti-mouse IFN-γ

(1:250) /avidin-horseradish peroxidase conjugate (1:250) in 10% FCS in PBS was pipeted into each well and allowed to incubate for 1 hour. Plates were washed 10 times with PBST, and 100 µL of tetramethlybenzidine (TMB) and hydrogen peroxide (1:1) was added to each well. After 30 minutes, the reaction is stopped with the addition of

50 µL of 1 M H3PO4 and measured with a spectrophotometer at an optical density of

450 nm.

Purpald Assay

For this assay 10 µg (protein) of SS1:0826 sonicate preparation and 2-keto-3- deoxyoctonate (Kdo) standards (Sigma) of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mM concentrations were used. This assay was performed based on a method previously published (138). Briefly, the unsubstituted terminal vicinal glycol groups of Kdo and heptose in the sonicate preparation and the standards are oxidized by addition of 50 µL of NaIO4 (Sigma) in a microtiter plate. This was performed at room temperature for 25 minutes. Periodate oxidation of these groups produced formaldehyde. This was followed by the addition of 50 µL of 136 mM of purpald (Sigma) in 2N NaOH and incubation at room temperature for 20 minutes. The formaldhyde formed an adduct with purpald. Upon further oxidation with periodate, the adduct was converted into a purple substrate. Twenty µL of 2-propanol is added to remove bubbles and the substrate was measured with a spectrophotometer at a wavelength of 550 nm. Using the

Kdo standards, the amount of LPS was determined once the calculations were corrected for the amount of heptose molecules in the LPS macromolecule.

85 Delayed-type hypersensitivity

Delayed-type hypersensitivity (DTH) to H. pylori antigens were determined by

giving adoptively transferred SCID mice infected with M6 H. pylori strain 10 µg of M6

or M6:0826 sonicate or 1 µg of SS1 or SS1:0826 LPS by injection into a rear hind

footpad. Before and after 24 hours of the injection, footpad thickness was measure with

a dial thickness gauge, and the results expressed as the change in thickness (in mm)

RESULTS

Effect of LPS and O-antigen on mice splenocyte cells

An earlier study demonstrated that transferred SCID mice infected with the

SS1:0826 (Lex-/Ley-) mutant developed dramatically reduced gastritis compared to

infection with the SS1 (Lex+/Ley+) parental strain (Eaton et al, submitted). Therefore, I

first sought to determine the effect of SS1:0826 sonicate on in vitro cultures of

unfractionated splenocytes from uninfected mice and mice infected with H. pylori strain

SS1. Splenocyte activation was determined in the cell cultures derived from uninfected mice. Neither the wild type M6 (Lex+/Ley+) sonicate nor M6:0826 (Lex-/Ley-) mutant

produced levels of IFNγ significantly higher than the untreated splenocytes (p>0.05), as

expected (Fig. 4.1). However, in the splenocytes cultures from infected mice, IFNγ was

elevated, although not statistically significant, when stimulated with M6 sonicate

compared to M6 sonicate-stimulated uninfeceted splenocytes (p= 0.971) and M6:0826

sonicate-stimulated, infected splenocyte cultures (p=1.000) (Figure

4.2).

86 4000

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0 Untreated ConA M6 Sonicate M6:0826 SS1 LPS (0.5 SS1:0826 LPS SS1 LPS (50 SS1:0826 LPS Sonicate ug/mL) (0.5 ug/mL) ug/mL) (50ug/mL)

Figure 4.1: ELISA analysis of IFNγ secretion in uninfected C57BL/6 mouse splenocytes after stimulation with either conA, M6 sonicate, M6:0826 sonicate, SS1 LPS, or SS1:0826 LPS. 10µg/ml of bacterial sonicate was used, and either 0.5 µg/ml or 50 µg/ml of LPS was administered for 5 days. (n=3)

4000

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gamma (pg/mL) 1500 - IFN 1000

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0 Untreated ConA M6 Sonicate M6:0826 SS1 LPS (0.5 SS1:0826 LPS SS1 LPS (50 SS1:0826 LPS Sonicate ug/mL) (0.5 ug/mL) ug/mL) (50ug/mL)

Figure 4.2: ELISA analysis of IFNγ secretion by splenocytes derived from C57Bl/6 mice infected with SS1 H. pylori strain. Splenocytes were stimulated with M6 and M6:0826 sonicate (10 µg/mL ) , SS1 and SS1:0826 LPS at 0.5 µg/mL and 50 µg/mL for 5 days. (n=3)

87 In order to determine whether the stimulatory effects of Helicobacter sonicate on splenocytes could be localized to the LPS, LPS isolated from the SS1 H. pylori strain and the SS1:0826 mutant strain were also evaluated. To ensure that the amount of LPS used for this assay was equivalent to what is present in the sonicate preparations previously used to stimulate unfractionated splenocyte cultures (10 µg/mL)(73), LPS in

10 µg/mL of SS1 sonicate preparation was quantified by using the purpald assay (138).

Using the purpald method 0.5 µg/mL of LPS was determined to be sufficient for the assay. In addition to the 0.5 µg/mL dose of LPS, a 100-fold dosage of 50 µg/mL was also used to stimulate cultures. The SS1 and SS1:0826 LPS samples are not pure

LPS samples. They have a protein concentration of 7% and 7.5%, respectively. The lower 0.5 µg/mL dosage of both SS1 and SS1:0826 LPS stimulated IFNγ secretion at approximately the same levels regardless of the infection status of the mice from which the splenocytes were derived (p>0.05) (Fig 4.1 and 4.2). At the higher dosage levels,

SS1:0826 LPS induced IFNγ secretion at a higher level than SS1 LPS from uninfected mice (p<0.05) (Figure 4.1). Also, at the lower dosage levels in splenocyte cultures from infected mice, SS1:0826 LPS appears to stimulate more IFNγ production than SS1 LPS; however, the levels were not statistically significant (p= 1.0) (Figure 4.2).

The lower dose SS1 LPS appears to induce IFNγ from the splenocytes from infected mice at as similar levels as M6 sonicate (p>0.05) (Figure 4.2). Because, the untreated, infected splenocytes produced IFNγ at a level indistinguishable from the M6 sonicate-stimulated, infected splenocytes (p>0.05), it is difficult to determine whether the results are specific to the lower SS1 LPS dose and to the M6 sonicate. SS1:0826

88 LPS at the lower dose stimulated higher levels of IFNγ than M6:0826 sonicate, but the

difference was not statistically significant (p>0.05).

In contrast, stimulation of infected splenocytes with the higher dose of SS1 LPS

resulted in a greater production than the M6 sonicate (p<0.05) (Figure 4.2). Likewise,

SS1:0826 LPS at the higher dose was able to induce significantly more IFNγ than

M6:0826 sonicate (p<0.001) (Figure 4.2).

Role of O-antigen in TH1 response

Gastritis and gastric epithelial lesions both in humans (62) and in the mouse

+ animal (73) model appear to be mediated by CD4 T lymphocytes via a TH1 type immune response to H. pylori antigens. Because infection of adoptively transferred

SCID mice with the SS1:0826 mutant results in mild gastric lesions compared to infection with the parental SS1 strain, T-bet heterozygous and homozygous mice were infected with SS1 and used to assess the involvement of CD4+ T cells in O antigen

modulation of host immunity. The T-bet protein is a member of the T-box family of

transcription factors (254). T-bet is important in TH1 differentiation and IFNγ

production in CD4+ T cells and natural killer T cells (186, 255). As a result mice

deficient in T-bet have greatly reduced IFNγ induction and reduced inflammation

89 4000

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0 Untreated ConA M6 Sonicate M6:0826 SS1 LPS (0.5 SS1:0826 LPS SS1 LPS (50 SS1:0826 LPS Sonicate ug/mL) (0.5 ug/mL) ug/mL) (50ug/mL)

Figure 4.3: ELISA analysis on INFγ production by splenocytes derived from uninfected T-bet (+/-) mice. Splenocytes were stimulated with either conA, M6 or M6:0826 sonicate (10µg/mL), or SS1 or SS1:0826 LPS (0.5 µg/mL and 50 µg/ml). (n=2)

in response to antigen. Therefore, splenocytes from the T-Bet deficient mice and wild type C57Bl/6 mice were stimulated during in vitro cell culture to determine whether H. pylori sonicate and LPS could stimulate splenocytes in the absence of fully functional

CD4+ cells. In the splenocyte culture from the uninfected, T-bet (+/-) mice, M6 sonicate and M6:0826 sonicate induced IFNγ secretion, however, not significantly above untreated (p<0.05) (Figure 4.3). The M6 sonicate induced secretion

90 4000

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0 Untreated ConA M6 Sonicate M6:0826 SS1 LPS (0.5 SS1:0826 SS1 LPS (50 SS1:0826 Sonicate ug/mL) LPS (0.5 ug/mL) LPS ug/mL) (50ug/mL)

Figure 4.4: ELISA analysis of IFNsecretion by T-bet (-/-) splenocytes from stimulated with either conA M6 or M6:0826 sonicate (10µg/mL), or SS1 or SS1:0826 LPS (0.5 µg/mL and 50 µg/ml). (n=2)

of IFNγ in the T-bet (+/-) splenocytes (Fig. 4.3), but was detectable in the T-bet (-/-) mice but not significantly more than untreated (Fig. 4.3). LPS at the higher doses induced high levels of IFNγ in cultures of the heterozygous and homozygous T-Bet uninfected splenocytes; however, stimulation was lower in the homozygous mutant splenocyte cultures than the heterozygous (SS1:0826 LPS, p=0.605; SS1 LPS, p=0.999)

(Fig. 4.3 and 4.4). Also, SS1:0826 LPS stimulation was higher than in the SS1 LPS in both the homozygous and heterozygous splenocyte cultures (Fig. 4.3 and 4.4) but not significantly (Heterozygous,p= 0.761; homozygous, p=0.994).

91 4000

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L 2500 (pg/m a 2000 m

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0 Untreated ConA M6 Sonicate M6:0826 SS1 LPS (0.5 SS1:0826 LPS SS1 LPS (50 SS1:0826 LPS Sonicate ug/mL) (0.5 ug/mL) ug/mL) (50ug/mL)

Figure 4.5: Stimulation of splenocytes derived from T-bet (+/-) mice infected with SS1 strain of H. pylori. Cells were treated as before with conA, SS1 and SS1:0826 sonicate (10µg/mL), or SS1 or SS1:0826 LPS (0.5 µg/mL and 50 µg/ml). (n=3)

4000

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0 Untreated ConA M6 Sonicate M6:0826 SS1 LPS (0.5 SS1:0826 SS1 LPS (50 SS1:0826 Sonicate ug/mL) LPS (0.5 ug/mL) LPS ug/mL) (50ug/mL)

Figure 4.6: ELISA detection of INFγ levels in cell cultures of splenocytes from SS1-infected T-bet (-/-) mice. Cultures were stimulated with conA, M6 and M6:0826 sonicate (10µg/mL), and SS1 and SS1:0826 LPS (0.5 µg/mL and 50 µg/ml). (n=3)

Stimulation of splenocytes derived from infected T-bet mutant mice (Fig. 4.5 and 4.6) did not differ significantly from uninfected T-bet mice. Sonicate stimulation of the homozygous splenocytes from both infected and uninfected mice seemed suppressed compared to the heterozygous. However, M6:0826 induced higher IFNγ but not significantly more (p=0.849) than M6 sonicate in the heterozygous. Some stimulation was observed for the lower dose of SS1 LPS for the infected heterozygous

(Fig. 4.5) compared to the uninfected heterozygous (Fig. 4.3). As in all the groups tested the higher doses of LPS generated high levels of IFNγ with the highest being observed with the SS1:0826 LPS.

The LPS samples were not absolutely protein-free. The SS1 and SS1:0826 samples had a protein concentration 7.5% and 7.0%, respectively. Because of the strong response of the splenocyte cultures from all groups to LPS of SS1 and SS1:0826,

I attempted to degrade the residual protein in the LPS using Proteinase K. After treatment with Proteinase K the LPS samples were used in the stimulation of the splenocyte cultures.

93 4500

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0 SS1 LPS SS1:0826 SS1 LPS SS1:0826 SS1 LPS SS1:0826 SS1 LPS SS1:0826 SS1 LPS SS1:0826 SS1 LPS SS1:0826 (50 ug) LPS (50 ug) LPS (50 ug) LPS (50 ug) LPS (50 ug) LPS (50 ug) LPS (50ug) (50ug) (50ug) (50ug) (50ug) (50ug) B6 T-Bet (+/-) T-Bet (-/-) B6 T-Bet (+/-) T-Bet (-/-) Uninfected Infected

without Proteinase K with Proteinase K

Figure 4.7: LPS samples were treated with 20 µg/ml of Proteinase K prior to addition to splenocyte culture. ELISA analysis of 5-day cell cultures of splenocytes derived from infectected and uninfected wild type (n=3), and uninfected (n=2) or infected (n=3) T-bet (-/+) and T-bet (-/-) mice that were either. Mice were infected with SS1 H. pylori strain. Splenocyte cultures were stimulated with 50 µg/mL of SS1 LPS and SS1:0826 LPS. (n=2).

In the cultures with splenocytes from the uninfected mice IFNγ secretion was dramatically reduced in all but the wild-type mouse splenocytes stimulated with SS1

LPS. In cultures derived from infected mice, only T-bet (-/-) splenocytes showed a significant reduction in IFNγ production.

94 45.00

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Change in footpad thickness after 24 hrs 0.00 SS1 LPS M6 Sonicate SS1:0826 M6:0826 SS1 LPS M6 Sonicate SS1:0826 M6:0826 (n=5) (n=5) LPS (n=5) Sonicate (n=2) (n=2) LPS (n=3) Sonicate (n=5) (n=3)

Uninfeced M6 infected

Figure 4.8: Delayed-type hypersensitivity was determined for adoptively transferred SCID mice that were either uninfected or infected with M6 H. pylori strain for 8 weeks. SS1 or SS1:0826 LPS (1µg) or M6 or M6:0826 sonicate (10 µg) was injected into the footpads of the mice. After 24 hrs. the thickness of the footpads are measured and compared to the initial footpad thickness.

The development of a DTH response to H. pylori antigen within adoptively transferred SCID mice that have been infected with H. pylori is a indicator of cell- mediated immune response. Therefore, to fully understand the immunomodulation of

O-antigen in the context of a CD4+-mediated immune response, the ability of M6:0826

mutant to induce DTH was evaluated. Sonicate preparation s and LPS cell-free samples

were injected into the footpads of C57Bl/6 mice infected with M6 H. pylori strain of uninfected C57Bl/6 mice. After 24 hours the footpads of the mice were measured to determine the degree of swelling.

SS1 LPS, SS1:0826 LPS, and SS1:0826 sonicate were not able to induce swelling comparable to M6 sonicate in infected nor uninfected mice (SS1 LPS,

95 SS1:0826 LPS, and SS1:0826 sonicate vs. M6 sonicate; p<0.01) (Figure 4.8). Virtually,

no swelling was observed in infected mice given SS1 LPS even though SS1 LPS caused

swelling in uninfected mice; however, it was not statistically significant (Infected/SS1

LPS vs. Uninfected/SS1 LPS; p>0.05). Although M6 sonicate induced higher swelling

than M6 sonicate in uninfected mice it was not statistically significant (p>0.05).

DISCUSSION

There has been considerable interest in assigning significance to the Lewis

antigens expressed on the LPS of H. pylori. Our lab has recently observed that the

SS1:0826 mutant strain, which is unable to synthesize Lewis antigens, induces less

severe gastritis during infection in adoptively transferred SCID mice compared to the

parental SS1 strain (Eaton et al, in submission). This suggests that the O-antigen and,

therefore, the Lewis antigens may be a major contributor to the immune response

elicited by H. pylori. Therefore, in this chapter I sought to determine whether the lack of O-antigen would result in a diminished TH1 response, which would be even more

severe in the T-bet knockout mice that express the inflammatory cytokine IFNγ at a

reduced level. This hypothesis was evaluated by treatment of splenocytes isolated from

infected and uninfected wild type mice and transgenic mice deficient in the T-bet

transcription factor with LPS preparations and sonicate preparations from H. pylori

96 strains with or without O-antigen. The data presented here suggest that any role that

LPS and its O-antigen play in immunity requires other factors.

Treatment of splenocyte cultures with 0.5 µg/mL of LPS from SS1 and

SS1:0826 induced little or no IFNγ production regardless of the source of the splenocytes. What complicates the interpretation of this data is that in the groups of splenocyte cultures from wild type C57Bl/6 mice the untreated cultures have detectable levels of IFNγ (Fig. 4.1 and 4.2). This clouds the meaningfulness of the IFNγ induction by the LPS at the lower concentration and stimulation by the positive control conA, especially in the splenocyte cultures from the infected mice.

A common feature of all of the groups of splenocyte cultures was the high level of IFNγ induction by 50µg/ml of both SS1 and SS1:0826 LPS. However, in most cases the mutant SS1:0826 LPS produced higher levels despite lacking O-antigen. There are several possible explanations for this observation. Rasko et al proposed that long O side chain repeats of some H. pylori strains could possibly prevent access of the bacterium to the host epithelial cells and, thus, diminish virulence of these strains (217).

This hypothesis was based on previous studies that have shown that O side chain repeats of less than 3 repeats are common on type I H. pylori strains while the less virulent type II strains tend to have more than 10 repeats (176, 178). Therefore,

SS1:0826 LPS may allow some cell surface interaction with the splenocytes in culture that elicits an enhanced T cell stimulation that is normally blocked by Lewis antigen repeats in SS1 via stearic hindrance or by masking epitopes. For instance, it could be possible that the absence of Lewis antigens provides better access to the lipid A

97 structure of LPS to the LPS binding protein (LBP) that transports LPS to the CD14 receptor of macrophages.

However, application of this model to in vivo situations is undermined by observations in this lab that the SS1:0826 causes reduced gastritis in the transferred

SCID mouse model. This could suggest that the effect of the SS1:0826 cell-free, LPS sample on splenocytes in cell culture does not adequately recreate the immunomodulation of the whole organism especially during colonization in the host.

The LPS samples were purified by gel-permeation-chromatography on a column of Bio-Gel P-2. However, the SS1 LPS and SS1:0826 LPS samples still consist of 7% and 7.5% of cellular protein, respectively. This would mean that SS1:0826 LPS when added to splenocyte cultures would have 250 ng more protein than SS1 LPS (3.5 vs.

3.75 µg). This amount of proteinis 35% and 37.5% , respectively, of the quantity of protein used to determine the dose of sonicate used in the splenocyte stimulation assays.

Whether the differing amount of protein in the LPS samples contributes to the differing level of IFNγ induction is speculative without further repetition of this experiment accounting for these differences.

Because of the protein detected in the LPS samples, they were pre-treated with

Proteinase K before being added to the splenocyte cultures. This procedure resulted in reduction of the IFNγ production in all the uninfected groups except for the wild type splenocytes treated with SS1 LPS. This indicates that IFNγ in most of the splenocyte groups from uninfected mice were mostly induced in part by the protein fraction in the

LPS samples. This also suggests that LPS is only partially involved with the stimulation of naïve splenocytes upon treatment with the LPS samples. Unlike SS1

98 LPS induction, SS1:0826 LPS induction of IFNγ is decreased, although down to a similar level as SS1 LPS, when pre-treated with proteinase K. However, in both the T- bet homozygous and heterozygous splenocytes, proteinase K-treatment diminished

IFNγ production to nearly undetectable levels. Therefore, the response of the

+ uninfected splenocytes to untreated LPS is probably dependent on CD4 TH cells. The infected groups were not as affected by Proteinase K treatment except for the T-bet homozygous splenocytes treated with SS1:0826 LPS. Because, for the most part,

Proteinase K treatment of LPS was unable to diminish IFNγ induction in the infected groups, these results seem to emphasize the importance of LPS in IFNγ induction.

The lack of O-antigen in the M6:0826 sonicate resulted in suppression of DTH response compared to M6 sonicate. This suggests that O-antigen recognition by CD4+ cells is involved in DTH response. Because the SS1 LPS samples failed to induce

DTH, other antigenic factors may also contribute. The LPS samples may exclude carrier protein that is present in the sonicate preparations and is associated with the LPS enabling recognition of the LPS by memory CD4+ T-cells. In addition antigenic variation between inoculum strain M6 and the test strain SS1 strains cannot be ruled out as a possible explanation for the lack of DTH from the SS1 LPS.

Also of note was the substantial production of IFNγ by both T-bet (+/-) and T- bet (-/-) splenocytes. Although loss of the T-bet transcription factor diminishes IFNγ production by CD4+ T cells, CD8+ T cells are unaffected by the loss of T-bet and are capable of IFNγ synthesis (255). Therefore, CD8+ T cells could account for the strong

IFNγ production in the T-bet deficient splenocytes. Previous studies have reported that

LPS can induce CD4+ and CD8+ T cell to proliferate and to secrete IFNγ and IL-2

99 secretion (164, 165, 181, 262). In a study with T cells isolated from DO11.10

d d (OVA323-339/H2-A ) mice and OT-1 (OVA257-264/H2-K ) mice it was not only shown

that both CD4+ and CD8+ cells are activated by LPS in the presence of OVA antigen but also that at 100 ng/mL (454.5 times less than my study) LPS was able to induce the cells independent of the OVA antigens (181). Also, this study as well as others has shown that CD14-expressing antigen presenting cells (APC’s) account for most of the stimulatory effects of LPS (164, 165, 181, 262).

Establishing a correlation between a cell-mediated immune response to H. pylori and O-antigen and LPS will require determining the mechanism of the activation of the splenocytes by LPS. Macrophages and monoctyes are among the splenic population and, therefore, would be likely candidates. The LPS-stimulation of CD4+

and CD8+ T cells is dependent on CD14+ monocytes and macrophages (181). CD14 is

a surface glycoprotein that binds LPS complexed with the LPS binding protein (LBP), a

liver-secreted transport protein. This triggers a signal transduction pathway that

activates cytokine expression via the nuclear factor κβ transcription factor (127, 287).

Several of these cytokines including IFNγ, tumor necrosis factor α, IL-12 direct CD4+

cells into a TH1 fate (224).

The data here suggest that perhaps CD8+ T cells are likely mediators since IFNγ

induction still occurs in the absence of CD4+ T cell-production of IFNγ. Therefore,

flow cytometry of stimulated splenocyte cells double stained for proliferation, such as

with fluorescent-labled antibodies for BrdU incorporation, and for several cell types,

such as CD8+, CD4+, and CD14 cells. These experiments could yield novel interactions

100 of LPS with immune cells that could uncover new ways of addressing the disease caused by infection with H. pylori and other gram-negative bacterium.

101

CHAPTER 5

Perspectives

The body of work presented in this dissertation seeks to identify molecular mechanisms that control virulence. The chapters two and three are an attempt to define regulation of virulence at the DNA level. The second chapter does this by analyzing the expression level of urease in the different points of adaptation to their environment.

The 91-124 and 99-2310 isolates of the 26695 strain had been recovered from the pig and afterwards had undergone only a few passages in vitro, while the 96-1106 pig isolate had undergone 14 passages in vitro and should be well-adapted to the growth in laboratory conditions. The parental 26695 strain in this study had not been previously used for animal infection studies. However repeated assays using slot blot analysis, real-time RT-PCR, and the hypochlorite-indophenol assay indicate that all of these isolates urease expression and activity are indistinguishable and, therefore, urease is constitutively expressed in the 26695 strain.

The third chapter focused more on a specific mechanism for the regulation of virulence. This chapter investigated the significance of tracts of cytidine repeats

(poly(C)) in two genes encoding for enzymes that exhibit α-3-fucosyltransferase

102 activity. These enzymes have been shown via biochemical analysis and insertional

inactivation and spontaneous mutation of their respective genes to catalyze the addition

of the sugar fucose to the lactosamine chain of the O-antigen of the LPS of H. pylori

(106, 163, 273). Depending on the fucosylation pattern either Lex or Ley are

synthesized.

The poly(C) tract of the α-3-fucosyltransferase genes, futA and futB, have been shown to undergo slip-strand mispairing. This could result in the addition or loss of the cytidines. These events could alter the reading frame of these two genes resulting in the production of truncated, inactive gene product. Appelmelk et al have have hypothesized that genes are not simply redundant, but possess preferential sites for fucosylation that effects whether the Lex or Ley are synthesized (10). FutA is

envisioned to preferentially fucosylate the N-acetyl-D-glucosamine (GlcNAc) moiety of

the dissacharide precursor creating Lex (10). Conversely, FutB is believed to prefer to

add fucose to D-galactose (Gal) of the precursor forming the Ley structure (10).

Therefore, inactivation of one or either both of these genes could alter the Lewis antigen phenotype of H. pylori.

However, the data here could not be used to find a correlation between the length of the poly(C) of futA and futB with the Lewis antigen phenotype of variants of two strains of H. pylori. This in agreement with one study that compared the poly(C) regions of the α-3-fucosyltransferase genes, Lewis antigen phenotype and the disease outcome from clinical isolates (226). Another study indicated that poly(C) sequences previously identified in the α-2-fucosyltransferase gene, futC, (260) also undergoes slip- strand mispairing and demonstrated that the inactivion of this gene prevents Ley

103 synthesis (230). The on/off status of futA, futB, and futC as determined by the length of the poly(C) tracts of these genes together may determine the Lewis antigen phenotype. To this date, there has not been published a study that examines the poly(C) of all three genes together and their consequence on Lewis antigen expression.

The fourth chapter of this dissertation is an extension of the third chapter in that this chapter pertains to the determination of the effect of the expression of the Lewis antigen has on the host immune response. However, the phenotype of the Lewis antigen is not taken into consideration, however, it is the absence or presence of Lewis

antigens that is the focus of this study. In this study we utilize the SS1:0826 and

M6:8026 mutant strains in which the gene encoding galactotransferase is inactivated by

insertional mutagenesis. Therefore, these strains are incapable of producing Lewis

antigens on their O-antigen. Preliminary studies within this lab have shown that

gastritis is reduced in mice infected with SS1:0826 compared to the wild-type strain

SS1 (In submission for publication). This indicates that Lewis antigens may modulate

the host immune response.

To explore this further, the galactotransferase knockout mutants were used in

splenocyte stimulation assay and delayed-type hypersensitivity (DTH) assays. The

induction of interferon-γ (IFN-γ) secretion by splenocytes and the triggering of DTH in

infected mice are two events characteristic of a CD4+-dependent Th1 immune response

to H. pylori antigens. This study utilized sonicates from the M6:0826 strain and the M6 wild-type strain and LPS samples from SS1:0826 and SS1 strains.

This study showed that LPS has a stimulatory effect on splenocyte cultures.

Elevated IFN-γ production of splenocytes from infected mice were observed with 50µg/

104 mL of both LPS samples despite treatment of the LPS samples with Proteinase K. Of particular interest, the LPS of the mutant strain still had a stimulatory effect on the splenocytes comparable to the wild-type strain despite not being able to express Lewis antigens. Therefore, Lewis antigens may not play a substantial role in the Th1 immune response to H. pylori. Although initially stimulation of the splenocytes from uninfected mice was observed with LPS samples, IFN-γ was greatly reduced upon pre-treatment of the LPS with Proteinase K. This indicates that the protein fraction of the LPS samples not treated with Proteinase K was largely responsible for the high level of IFN-γ secretion. Conversely, in the DTH study, the LPS samples did not significantly induce swelling of the mice footpads as expected. This suggests that T cells do not mediate the immune response seen in splenocyte studies.

Although the work presented in this dissertation was unable to definitively describe the molecular mechanisms that were proposed to attribute to variations in virulence, this work should serve as a springboard for future work in elucidating these mechanisms. Sequence analysis of the genome of H. pylori has revealed a smaller than expected number gene regulatory mechanisms compared to other bacterium (260).

Therefore, it is quite possible that H. pylori employs some novel gene regulation strategies.

An extension of the work presented on urease expression could be the determination whether urease transcription activity of H. pylori during colonization in the host is elevated compared to H. pylori grown in in vitro culture. This would require a technique sensitive enough to detect urease RNA from total RNA isolated from the gastric tissue of an infected host. Also, it would be interesting to determine whether H.

105 pylori strains of differing degrees of virulence and colonization potential express urease at the same level.

While the data in Chapter Three was unable to demonstrate that random, slip- strand mispairing in the poly(C) regions of futA and futB regulates Lewis antigen phase variation, this study did emphasize the need to broaden this study to include other factors such as α-2-fucosyltransferase gene, futC. FutC is required for Lewis y synthesis and, like futA and futB, it also has a hypervariable region subject to slip-strand mispairing. It could be that phase variation of Lewis antigen phenotypes is the result of the aleration of the relative levels of α-3-fucosyltransferase and α-2-fucosyltransferase due to the on/off switching of these three genes via slip-strand mispairing.

The study in Chapter Four calls for a closer examination of which population of the splenocytes are stimulated by the LPS samples. This would mean the use of flow cytometry and double labeling for cell-type specific surface markers, such as for CD14,

CD4, and CD8, and for proliferation, such as with anti-BrDU for BrDu incorportation.

Fractionation of splenocytes by the removal or the enrichment of a cell population may not be so informative as it seems that LPS stimulation is a corporative event in light of stimulation by LPS of T-bet -/- mice splenocytes in which CD4+ cells are unable to produce IFN-γ.

A better understanding of how H. pylori is able to regulate virulence factors will facilitate the identification of H. pylori strains that have a greater tendency to cause disease and of new means of combating H. pylori infection that are both cheaper and more efficient than current treatments.

106

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