Helicobacter Pylori Strains and Human Cytokines (Manuscript)

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 112

Identification and Characterization of Biomarkers in Bacterial Infections

MARTIN STORM

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 UPPSALA ISBN 91-554-6475-0 2006 urn:nbn:se:uu:diva-6509 !" # $ % & " '( '))* $+,$- . " . . /" " 0% . & 1# 2" 3 4 "#

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I. Martin Storm, Ingegerd Gustafsson, Björn Herrmann and Lars Engstrand: Real-time PCR for pharmacodynamic studies of Chlamydia trachomatis Journal of Microbiological Methods 61 (2005) 361– 367

II. Martin Storm, Abdolreza Advani, Monica Pettersson, Hans O. Hallander and Kåre Bondeson: Comparison of real- time PCR and pyrosequencing for typing Bordetella pertussis toxin subunit 1 variants Journal of Microbiological Methods (2005)

III. Martin Storm, Christina Persson, Lovisa Lovmar, Anders Andersson, Lars Engstrand and Ann-Christine Syvänen: A Microarray Method for the Simultaneous Genotyping of Helicobacter pylori Strains and Human Cytokines (Manuscript)

IV. Andreas Meinke, Martin Storm, Tamás Henics, Dieter Gelbmann, Sonja Prustomersky, Zoltán Kovács, Duc Bui Minh, Birgit Winkler, Ulrike Stierschneider, Manfred Berger, Alexander von Gabain, Lars Engstrand and Eszter Nagy: Composition of the “antigenome” of Helicobacter pylori defined by human serum antibodies (Manuscrip)

Reprints of paper I and II were printed with permission of the publishers

Contents

Introduction...... 7 Bacterial Genetics ...... 8 The Molecular revolution ...... 10 Methods ...... 11 Fluorescence...... 11 PCR / real-time PCR ...... 12 Taq-Man Real-Time PCR ...... 14 FRET-probe based Real-time PCR ...... 15 Pyrosequencing ...... 16 Mini sequencing Tag array...... 18 The antigenome approach ...... 19 Specific Aims...... 22 Chlamydia trachomatis, Paper I ...... 23 Bordetella pertussis, Paper II...... 26 Helicobacter pylori, Paper III and IV ...... 29 Paper III...... 35 Paper IV ...... 40 Concluding remarks...... 44 Acknowledgements...... 45 References:...... 47 Abbreviations

ACV Acellular vaccine ATP Adenosine tri-phosphate BLAST Basic local alignment tool Bp Base pair CCD Charge coupled device Ct Cycle threshold C-tag Capture tag DNA Deoxyribonucleic acid dNTP Deoxynucleotide-triphosphate ELISA enzyme-linked immunosorbant assay FRET Fluorescence Resonance Energy Transfer- IF Immuno-Fluorescence IL Interleukin LB Luria broth MACS Magnetic affinity cell sorting mbp Mega base pair MIC Minimal inhibitory concentration PCR Polymerase chain reaction PAI Pathogenicity island PPi Pyrophosphate Ptx Pertussis toxin RNA Ribonucleic acis SNP Single-nucleotide polymorphism ssDNA Single stranded DNA WCV Whole cell vaccine Introduction

In 1674 a Dutch biologist named Anton van Leeuwenhoek, looked through a homemade microscope at a drop of water. He discovered millions of tiny, living “animicule”, this discovery of bacteria (and protozoa) would eventually revolutionize the medical field. In 1840 the German pathologist, Freidrich Henle put forth the “germ theory of disease” (the notion that bacteria could cause human diseases). This theory was confirmed in the 1870s and 80s by the work of Koch and Pasteur. Koch, who is called the founder of diagnostic microbiology, designed a series of experiments that resulted in the famous Koch´s postulate. Pasteur is most famous for inventing a process to kill microorganisms in milk, pasteurization, but has contributed im- mensely to the field of bacteriology. Ever since the discovery that microorganisms cause disease and the discovery of the first antibacterial agent, an anti syphilis agent discovered by Paul Ehrlich in 1910, there has been a grate deal of effort directed towards diag- nosing the microorganisms causing disease. As we keep discovering new disease causing agents this task is getting ever more complex. Much of the work conducted today in the clinical microbiology laboratory would be new to Koch and Pasteur; however they probably would recognize many of the basic techniques. Much of the work conducted in the laboratory is still based on Koch’s pure-culture concept. This is however changing. The molecular biology revolution that started in the 1950s is rapidly changing the way the clinical microbiologist is working. It has led to methods and an amount of knowledge that Koch and Pasteur could not have dreamt of.

7 Bacterial Genetics

The bacterial genome is circular (with very few exceptions), haploid and is made up of DNA. The size of the genome varies greatly between species from the smallest of only 0.58 million base pairs (mbp) (Mycoplasma geni- talium) to 9.13 mbp for the largest (Myxococcus xanthus) (www.cmr.tigr.org). The size of the genome determines the amount of genetic information that the bacterium carries, and thus many of the bacteria with very small genomes are obligate parasites (i.e. they need something from a host that they themselves cannot produce in order to live and propa- gate).

The two main functions of the bacterial genome are replication and expression. Replication is a tightly regulated process by which the cell creates an accurate copy of its own DNA, the DNA and thus the genotype is then passed along to the daughter cell via cell division. Like all living things bacteria uses DNA as the template for RNA production in a process called transcription, RNA in turn is then processed into protein by a process called translation. This flow of information from DNA to RNA to protein and the fact that protein is in turn necessary for all these steps to take place is called the central dogma of molecular biology an idea that was presented by Fran- cis Crick in 1970 (Crick 1970).

DNA

Protein RNA

Figure 1. The central dogma of molecular biology, as described by Francis Crick. The solid arrows represent information flow, while dotted arrows represents probable information flow. Information flow from protein to either DNA or RNA is considered impossible

8 The expression of specific genetic material under specific conditions determines the bacterium’s phenotype.

Many bacteria contain extra chromosomal material called plasmids that rep- licates independently of the chromosome. These elements usually carry non- essential but beneficial genes, such as genes that encode toxins or antibiotic resistance. The size of the plasmids can range from very small, only about 5000bp to extremely large, more than 2mbp. One important aspect of plasmids is that they can be transferred between two different bacteria, either by the bacterial form of sexual reproduction, called conjugation or simply by taking the plasmid up from the surrounding environment (transformation). This genetic promiscuity (a bacterium can pick up a plasmid from a different species) is an important survival strategy.

Mutations are substitutions, insertions, deletions or rearrangements of genetic material. If the mutation results in a phenotypic change for the bacteria the mutation is said to be non-silent, if the mutation occurs without a resulting phenotypic change the mutation is called silent. Spontaneous mutations occur at random and the frequency by which it occurs is determined by the accuracy of the DNA replication and the bacterium’s DNA repair system (Miller 1996). The mutation rate varies a great deal both between different bacterial species and between different strains of the same species. Most mutations have either no effect on the organism or are detrimental; however a few mutations are beneficial, such as antibiotic resistance. The penetrance of the mutation will depend on its ability to provide an advantage to the bacterium under certain conditions and the metabolic cost associated with the mutation (Bjorkholm et al. 2001). Mutations do not only occur at random they can be induced by environmental factors such as chemicals and UV- light. An environmental factor that is mutagenic for bacteria is often also a carcinogen for animals.

9 The Molecular revolution

In essence molecular microbiology is based on the detection and characterization of nucleic acids and was born in 1944 when, in a famous experiment, Avery et.al. identified DNA as the transforming factor responsible for turn- ing avirulent strains of pneumococcus into virulent mutants (Avery et al. 1944). The same year Barbara McClintock discovered that genes can move between chromosomes by studying pigment inheritance in corn. Another milestone in molecular biology came in 1953 when Watson, Crick and Wil- kins solved the structure of DNA (Watson et al. 1953). Although the composition of DNA was known, how the information stored in DNA composed of a sequence of four nucleotides was translated into proteins containing any number of up to 20 possible amino acids still remained a mystery. This changed in 1966 when Nirenberg, Mathai and Ochoa “cracked the genetic code” and determined that a combination of three nucleotides (a codon) spe- cifically coded for each of the amino acids (Nirenberg et al. 1966; Stanley et al. 1966). Recombinant DNA technology was developed in the 1970’s and the possibility of combining DNA from two different organisms was first demonstrated in 1972 by Paul Berg who fused DNA from two different vi- ruses (Berg et al. 1974). The next major breakthrough in molecular biology was the development of DNA sequencing in 1975 by Fredrick Sanger and the invention of the polymerase chain reaction in 1984 by Kary B Mullis (Sanger et al. 1977; Mullis et al. 1987). These discoveries are the corner- stones of molecular biology. In later years the field has expanded enor- mously, recently the possibility to sequence a complete bacterial genome in matter of days has become reality and there are an increasing number of new applications being presented each year (Margulies et al. 2005).

I will in this thesis describe how we have applied some of these new methods to develop instruments for solving some of the questions facing us in the field of clinical bacteriology today.

10 Methods

Fluorescence

For many of the methods described in this thesis the interpretation of results are dependent on interpreting signals from fluorescent reporter molecules called fluorophores. A fluorophore is a molecule that can emit energy in the form of light at a certain and specific wavelength after having been “excited” by absorbing energy from light of a different and equally specific wavelength. All fluorophores have in common that they are excited, i.e. the electrons in the molecule accept energy and are transferred into a state of higher energy, best at a certain wavelength of energy. This is called the excitation spectrum. When the electrons fall back into their ground state they emit energy of a certain wavelength, this is called the emission spectrum. The difference in wavelength between the absorption and emission maxima is called Stokes shift. (Figure 2) Fluorophores are used in a variety of biological applications such as protein detection, PCR and microarrays.

Figure 2. The absorption and emission spectra overlaps at lower intensities. For a fluorophore a large stokes shift is an advantage since the background resulting from the overlapping of the two spectra will be lower.

11 PCR / real-time PCR

The polymerase chain reaction (PCR) was invented by Kary B Mullis in 1985. At first the process was labor intensive primarily since the enzyme driving the reaction, DNA polymerase, was degenerated by the heat needed to degenerate the DNA in between each cycle of PCR (Mullis et al. 1986; Mullis and Faloona 1987). In 1988 the process was significantly simplified by the discovery of heat stable DNA polymerase isolated from the bacteria Thermus aquaticus eliminating the need to replace the polymerase between cycles (Saiki et al. 1988). The same year the first thermal cycler for com- mercial use was introduced by the Perkin Elmer Corporation. The PCR process revolutionized molecular biology and Mullis received the Nobel Prize in chemistry in 1993 for his discovery. In the beginning of the 1990s the first commercially available diagnostic tests utilizing this new technology became available and in the years to come PCR gained wide spread use in diagnostic microbiology. The PCR process had its limitations, one of which was poor quantification capabilities. Attempts were made to quantify PCR product in agarose gel by measuring the intensity of the ethidium bromide stained gel using UV sensitive cameras. Ethidium bromide binds to double stranded DNA and fluoresces under UV light, but the process was cumbersome and not very accurate. Another drawback was the fact that the reaction could not be monitored and thus the efficiency of the reaction could not be calculated. This led Higuchi et al to develop a system where a video camera coupled to an adapted thermal cycler could detect the accumulation of PCR product during PCR by including ethidium bromide in the reaction. By plotting the resulting increase in fluorescence versus the cycle number a much more complete picture of the reaction could be produced than was possible by measuring the end product alone. They called this new process Real-Time PCR (Higuchi et al. 1993).

The concept of cycle threshold value (Ct) allows for accurate and reproduci- ble quantification. The Ct value is defined as the least number of cycles it takes to reach a point in which the fluorescent signal is first recorded as statistically significant above background. This means that; the more template DNA is present at the beginning of PCR the fewer cycles are needed to reach the Ct value. By comparing the Ct value obtained from an unknown sample

12 to a standard curve generated by a set of known samples a very accurate measurement of the initial concentration of template DNA can be calculated. The ability to monitor the PCR process in real time has completely revolutionized the way PCR is used in the clinical field. Since the introduction of real-time PCR; the idea of measuring fluorescence during the PCR reaction has led to the development of a many new methods and applications. One of the more significant developments was the introduction of different fluorophores, other than ethidium bromide.

The use of fluorophores coupled to DNA allowed the design of probes specific to a target sequence of the DNA amplified, which greatly increases the specificity of the PCR reaction.

Real-Time PCR is based on the ability of one molecule to absorb the energy released from another molecule. This is a concept known as fluorescence resonance energy transfer or FRET (Fig 3). All fluorophores has in common that they are excited, i.e. the electrons in the molecule accept energy and are transferred into a state of higher energy, best at a certain wavelength of energy. When the electrons fall back into their ground state they emit energy of a certain wavelength. FRET occurs when the energy emitted from one molecule is absorbed by a second molecule. In order for this to happen, two conditions have to be met: 1. the emission spectrum of the first molecule has to roughly match the excitation maximum for the second molecule. 2. the molecules has to be in close proximity of each other for the energy to transfer usually between 10-100 Å.

S1’ 2

3 Energy

S 0 Figure 3 The FRET concept: 1. The electrons of molecule 1 are excited (S1’) When the electrons return to the ground state (S0) energy is released. If this energy is absorbed by molecule 2 FRET has occurred (S1), when the electrons of molecule 2 return to S0 energy is released at a different wavelength than when it was absorbed.

13 Taq-Man Real-Time PCR

A Taq-man probe is based on the concept of FRET. A ssDNA probe (~18- 25 bases) is labeled in the 5’ end with a reporter fluorophore and in the 3’ end (or some times internally) with a quencher. A quencher is a molecule that can absorb the energy released by another molecule either emitting, or more commonly, without emitting light as a result. Quenchers commonly used in Taq-man assays do not emit light but dissipate the absorbed energy as heat. The Taq-man probe is designed so that it is complementary to a sequence within the amplicon generated by the PCR. If the probe binds to the amplicon during the PCR process the probe will be degraded by the 5 to 3’ nuclease activity of the Taq DNA polymerase. During each cycle in the PCR process a laser will illuminate the sample with light at a wavelength corresponding to the excitation maximum of the reporter molecule and a CCD camera will measure the emission of light from the sample at a wavelength corresponding to the emission maximum of the same molecule. As long as the probe remains intact and the reporter and quencher molecules are in proximity to each other the light energy emitted by the reporter molecule will be absorbed by the quenching molecule. However if the probe anneals to a sequence within the amplicon and is degraded the reporter and quencher molecules will separate and the emission light from the reporter molecule will be detected by the camera (Fig 4).

Ȝ In

Out Ȝ In Ȝ

Taq

Figure 4 The nuclease activity of the Taq-polymerase degenerates the Taq- man probe, separating the fluorophore from the quencher.

FRET-probe based Real-time PCR

Although the Taq-man based real-time PCR is versatile there are applications where other methods based on real-time technology are more suited. The FRET-probe assay is an example of this. The FRET probe assay is based on the use of two probes, an anchor and a reporter probe. The probes are designed to anneal next to each other on the template DNA, with the reporter probe annealing over the region of interest. Further they are designed to have different melting temperatures with the anchor probe having the higher melting temperature. The two probes are labeled with fluorophores either in the 5’ or in the 3’ end of the probe so that when both probes are annealed at the same time the fluorophores are in direct proximity to each other. The two fluorophores have an overlapping emission /excitation spectra so that when the two probes are in proximity FRET can occur. At the conclusion of each PCR cycle the sample is illuminated with a wavelength corresponding to the excitation maximum of the anchor fluorophore and emission is measured at a wavelength corresponding to that of the fluorophore on the reporter probe. If the second fluorophore emits light FRET has occurred and thus the two probes have to be in proximity to each other (Fig 5).

Ȝ In Ȝ Out 1

Ȝ In Ȝ Out 2

Figure 5 When the anchor and the reporter probes both are annealed to the template DNA, FRET will occur.

15 One of the main applications of FRET probes is end-point melt analysis. At the completion of the PCR the sample is slowly heated while fluorescence is measured in the same manner as describes above. At a certain temperature the reporter probe will disassociate from its template DNA with a loss of FRET as a result. This will give an accurate indication of the melting temperature of the probe/ template complex. This allows this type of assay to identify one or more single nucleic base polymorphisms based on the melting temperature, as long as they are covered by the reporter probe, since the melting temperature will differ depending on the number of mismatched bases.

Pyrosequencing

Pyrosequencing is a “sequencing by synthesis” technology, first presented in 1998 by Rhogani et al, based on sequential addition of nucleotides to a reaction solution and real-time monitoring of DNA synthesis (Ronaghi et al. 1998). Like other sequencing methods pyrosequencing is dependent on the addition of complementary nucleotides to a single stranded DNA template by the specific action of DNA polymerase primed by a sequencing primer. What sets pyrosequencing apart is that the release of pyrophosphate (PPi) from the reaction is used to generate light, allowing for real-time monitoring of the reaction.

The pyrosequencing reaction can be seen in figure 6. After PCR amplification of the target sequence of interest, a single stranded template of the amplified cDNA is immobilized in a 96 well plate. The reaction then takes place in a solution, containing the enzymes; DNA polymerase, ATP sulfyrase, Lucifirace and Apyrase and the substrates Adenosine 5’ phospho- sulfate (APS) and Luceferin.

16 Figure 6 A schematic of the Pyrosequencing enzymatic cascade, and a representation of a Pyrogram (right)

A specific sequencing primer that is designed to anneal 5’ upstream of the target sequence is added to the reaction. Nucleotides are sequentially added to the reaction and the reaction is driven by heat, like a regular PCR reaction. If the nucleotide is incorporated PPi is released in a molar amount that is directly dependent on the amount of nucleotide incorporated. ATP sulfyrase and APS converts the released PPi to ATP which in turn drives the conver- sion of luceferin to oxyluceferin, which will generate visible light in direct proportion to the amount of ATP available. The light is detected using a charged coupled device (CCD) camera and the intensity of the light will be registered as a peak in a graph called a pyrogram. In the pyrogram the order of added nucleotides are on the X axis while the intensity of the light is on the Y axis, the height of the peak over a nucleotide in the pyrogram is determined by the amount of light detected and thus is in direct proportion to the amount of nucleotide incorporated. After each addition of nucleotide to the reaction and before a new nucleotide is added, the apyrase (a nucleotide degrading enzyme) degrades un incorporated dNTP’s and residual ATP. The sequential addition of up to 300 nucleotides is registered in the pyrogram and at the end of the process the DNA sequence can be determined.

17 Mini sequencing Tag array

A microarray is a collection of ssDNA spots attached to a solid surface, most commonly glass, forming an array. They were first developed to study rela- tive gene expression (Schena et al. 1995; Lockhart et al. 1996). Tag-array minisequencing was mainly developed to analyze SNP’s but it is a versatile technology that takes advantage of the specificity and accuracy of DNA polymerase by the cyclic incorporation of a terminating, fluorescently labeled di-deoxy nucleotide directly next to a minisequencing primer designed to anneal complementary to a specific position of the target DNA. By using four different fluorophores, one for each possible nucleotide the specific base that was incorporated can be determined (Syvanen et al. 1990; Pastinen et al. 2000).

Figure 7 A. A single array in an “array of arrays” can contain well over 100 c-tags that each identifies a specific target. B. A single, fluorescently labeled ddNTP is incorporated by DNA polymerase in direct proximity to the mini sequencing primer. C. Each “spot” in the array contains a specific c-tag complementary to the tag-sequence in the 3’ end of the mini sequencing primer. Figure adapted with authors permission (Lovmar et al. 2005)

The target sequences of interests are PCR amplified in highly multiplex reactions and the resulting PCR products are pooled so that all targets are repre- sented in a single solution. A second round of PCR is performed using the pooled samples as template and the minisequencing primers are added to the reaction. The minisequencing primer carries a 20 bp tag-sequence in the 5’ end that allows it to hybridize to a complementary sequence, called a capture tag (c-tag) printed on a glass slide. At the conclusion of the second PCR the sample is hybridized to the tag-array. The c-tags are constructed with similar thermodynamic properties so that they can hybridize under the same con-

18 ditions. Further the c-tag sequences are designed to be unique both among them selves and in respect to the target DNA, a 20bp DNA sequence has 1012 (420) possible combinations of the four (A, T, G and C) bases. After hybridization the microarray slide is analyzed in a microarray scanner, an instrument that detects fluorescence.

By using a collection of unique c-tags printed in an array on the slide (figure 7) a large number of genetic targets can be investigated using a single array. By printing up to 80 identical arrays on a single slide an “array of arrays” is created. By using a custom made silicone grid to separate samples, up to 80 different samples can be investigated in the same hybridization reaction (Lindroos et al. 2002).

The antigenome approach At present there are 295 complete bacterial genome sequences available in the public domain (TIGR, 2006). This wealth of information is fast becoming an invaluable tool for vaccine design. The possibility to develop acellular vaccines (ACV) based on a few highly antigenic epitopes rather than vaccines based on attenuated or killed organisms (WCV) is revolutionizing the way novel vaccines are designed. The use of bioinformatics and bacterial genome sequences in order to predict candidate antigens by sequence homology to known antigens, cell surface location and secreted proteins is now relatively easy (Zagursky et al. 2003). The process of conducting a genome wide in silico search for vaccine candidates has been named reverse vaccinology and was first demonstrated by the identification of antigens protecting against Meningococcus serotype B and Pneumococcus (Adu- Bobie et al. 2003). Due to the inaccuracy of available prediction algorithms a genome based prediction usually identifies antigens representing 10-25% of all genome encoded proteins. This makes the validation process of the antigens very cumbersome and the inclusion of other selection criteria is necessary. Since evaluation of the immune response against a candidate antigen is a crucial validation task in ACV design, the use of human immunogenicity as the primary screening tool is desirable; this also has the added advantage of ensuring that antigens selected are expressed in vivo. Recently a novel approach called antigenome technology combining the advantages of reverse vaccinology with the selection criteria mentioned above has been used to identify novel antigens in Staphylococcus aureus (Etz et al. 2002).

19 Genomic DNA from the pathogen is mechanically shredded by sonication or chemically degraded by the use of Dnase I, these methods produce fragments of approximately 100-300 bp or 50-100 bp, respectively. After frag- mentation is confirmed by agarose gel electrophoresis the fragments are blunt ended by T4-DNA polymerase. The fragments are cloned into pMAL 4.1 vector with an antibiotic selection cassette and transferred into E. coli (Fig. 8).

Figure 8 The creation of a clone library by fusion into E. coli surface proteins

Two clone libraries, one of small fragments and one of lager fragments, cov- ering the entire genome is thus created. Once positive clones are selected DNA fragments are excised using restriction enzymes FseI and NotI, the fragments are cloned into vectors coding for E. coli surface proteins LamB and FhuA. The resulting clones will then express these fusion proteins and present them on their surface. Clones expressing the fusion proteins are again selected by antibiotic resistance and washed followed by incubation over night either with antibodies from pooled human sera from patients with confirmed infection or with negative sera (to serve as the control). At the end of the incubation the cells are again washed and then incubated with biotinylated goat-anti-human IgG antibodies. After the wash with LB, MACS micro beads coupled to streptavidin are added and the streptavidin coated beads bind the biotinylated antibodies. After a washing cycle the cells are transferred to a MS column and a magnet is used to separate cells that have bound patient antibodies.

20 The region containing the inserted fragment in the LamB or FhuA genes are sequenced and are aligned against the whole genome sequence for the pathogen using the BLAST algorithm (Altschul SF 1990) (http://www.ncbi.nlm.nih.gov/BLAST/) and an antigenome is created (Fig 9).

Figure 9 The sequences obtained from the MACS screening is aligned against the genomic sequence of H. pylori resulting in the creation of an antigenome. In this fashion open reading frames that code for novel antigens can be identified. Here the immunogenic region of HP0485 (catalase-like protein) can be seen.

Results from the MACS assay is considered together with results from a peptide ELISA assay with partial antigens using either patient or normal sera and finally a gene distribution study. Thus potential antigens can be selected (Fig 10).

Figure 10 Final selection process in the antigenome approach to vaccinology.

21 Specific Aims

I. The aim of this study was to develop a simple method that would reliably measure the viability of Chlamydia trachomatis in cell culture in order to determine the pharmacodynamic properties of C. trachomatis and to create a suitable platform for multicenter studies of resistant / persistent C. trachomatis infection. II. This study’s main purpose was to determine which of two novel methods was best suited for surveillance of pertussis toxin variants circulating in vaccinated populations, and further to validate the methods against the currently used Sanger sequencing standard. III. In this study we wanted to develop a platform technology that would allow us to determine if there are biomarkers in Helicobacter pylori and in humans that could be used in combination to predict the outcome of infection. Fur- ther we wanted to prove that it was feasible to use this platform to investigate archived material, such as formalin preserved, paraffin embedded gastric biopsies. IV. The aim of this investigation was to determine the comprehensive antigenic profile of H. pylori, in order to identify novel antigens that could be used in a future H. pylori vaccine; we also wanted to identify potential antigenic mak- ers that could be related to disease and the development of cancer.

22 Chlamydia trachomatis, Paper I

Chlamydiae are divided into two lineages, the Chlamydia genus comprising C. trachomatic, Chlamydia muridarum and Chlamydia suis and the Chlamy- dophila genus that is constituted of Chlamydophila pneumoniae, Chlamydo- phila pecorum, Chlamydophila psittaci, Chlamydophila abortus, Chlamydo- phila caviae and Chlamydophila felis. Of these nine species only C. trachomatis and C. pneumoniae have humans as natural hosts, while C psittaci occasionally causes zoonotic infections transmitted by birds.

Chlamyidae are obligate intracellular bacteria that infect eukaryotic cells, a fact that when the organism was first discovered led researchers to believe that they had found a virus. It is now known that like all bacteria they process their own DNA, RNA and synthesizes their own proteins, they are however facultative ATP parasites (Gerard et al. 2002). They possess both inner and outer membranes as well as lipopolysaccharides similar to those found in the gram negative group of bacteria but unlike the gram negative bacteria they lack the peptidoglycan layer that separates the inner and outer membranes. Chlamydiae also have a unique life cycle (Fig 11) including two morphologically distinct forms.

The infective but metabolically inactive elementary bodies (EB) attaches to the epithelial cells on mucosal surfaces (A.) after which they enter the host cell (B). After 6-8 hours the EB starts to rearrange into the metabolically active reticulate bodies (RB). 8-24 hours later the RBs start to divide by bi- nary fission (C). 18-24 hours after the cell has been infected the RB reor- ganize back to the EB state and eventually the host cell will rapture and the RBs are released (C).

Figure 11 Life cycle of Chlamydia

23 Chlamydia trachomatis is the worlds most common sexually transmitted disease, with a world wide prevalence around 2-10% in reproductive age groups and genital chlamydial infection is the most common notifiable infectious disease in both Unites States and several European countries (CDC 1996; Burstein et al. 1998; Bunnell et al. 1999). The prevalence among males seems to be lower than among women (Shafer et al. 2002; van den Brule et al. 2002). The fact that many more men than women remain asymptomatic and that women develop more severe symptoms might be responsible for a misrepresentation of the reported prevalence among men. In fact, in a recent study by Sylvan et.al., on the prevalence of C. trachomatis among asymptomatic or mildly symptomatic attendants of a Swedish youth- health clinic the prevalence was almost 30% higher in men than in women (6.0% as to 9.8%) (Sylvan et al. 2002).

The C. trachomatis species consists of at least 15 main serovars with a clear distinction between the three serotypes belonging to the lymphogranuloma venereum biovar, causing invasive genital tract infection, and those belonging to the TRIC biovar causing trachoma or genital infections. Genital chlamydia infections may lead to severe complications such as: salpingitis, ectopic pregnancy, infertility and epididymitis and trachoma is the leading cause of preventable blindness in the world. Uncomplicated infection with C. trachomatis is usually treated with doxycycline or azithromycin. Treat- ment with antibiotics is usually effective in eradicating the infection but therapeutic failure does occur and is a recognized problem. Strains resistant to tetracyclines as well as the appearance of multi-drug resistant C. trachomatis has also been reported (Lefevre et al. 1997; Somani et al. 2000). Although antibiotic resistance could explain why treatment may fail, the few incidences of resistant C. trachomatis reported makes it unlikely. An alter- nate explanation for treatment failure might be altered replication levels due to antibiotic treatment; resulting in a small but persistent population of C. trachomatis surviving the treatment regiment (Beatty et al. 1994; Dreses- Werringloer et al. 2000). Persistent C. trachomatis has been reported by several investigators both in vivo and in vitro (Coles et al. 1993; Nanagara et al. 1995; Raulston 1997). There is circumstantial evidence suggesting that antibiotic treatment induces slow growth and persistent chlamydia infection however no studies has of yet shown that to be the case. In order to better understand how C. trachomatis is effected by antibiotic treatment and to enable multi-center studies there is a need for a rapid and standardized method that can produce comparable results

24 Using Taq-man based real-time PCR we have devised a method to measure concentrations of viable C. trachomatis in cell culture. The results show the method to be comparable with immunofluorescent (IF) staining of cultured cells, the current golden standard. The method was used to determine the minimal antibiotic concentration (MIC) needed for a 95% reduction of viable Chlamydia for two isolates.

Table 1. In the study we investigated two strains, C2028 from a patient that responded well to treatment and C6206 from a patient with reoccurring symptoms. MIC was defined as a 95% decrease of viable C. trachomatis

The method we developed is believed to be a clear improvement over exist- ing methods primarily because it removes the need for IF staining which is both cumbersome and subjective. Further our method has the added advantage of being suitable for determining the viability and thus the replication rate of C. trachomatis more precisely than does traditional IF-staining. This is a necessity if you want to better understand the persistence of chlamydial infections.

25 Bordetella pertussis, Paper II

Bordetella pertussis is a very small (0.2-0.5 ȝM), strictly aerobic, Gram- negative, coccobacillus and the etiological agent responsible for whooping cough. It is a highly contagious respiratory disease that is most severe in infants and young children (Greenberg et al. 2005; Mattoo et al. 2005). There is also evidence that adolescents and adults, which serve as an important reservoir for the disease, are developing symptomatic disease (von Konig et al. 2002). There are between 20-40 million cases each year with approximately 200 000- 400 000 fatalities per year (Crowcroft et al. 2003; Celentano et al. 2005). The vast majority (90%) of these cases occur in developing countries (Preziosi et al. 2002). In the pre vaccination era nearly every child contracted this disease and pertussis was a leading cause of in- fant deaths (Willems et al. 1996). Whooping cough is characterized by par- oxysm of numerous rapid coughs followed by a characteristic whooping sound. The most common complication of pertussis infection is secondary pneumonia. In many cases infants that contract pertussis will require hospi- talization (Tanaka et al. 2003). The high morbidity and mortality rates associated with pertussis led to the introduction of whole cell vaccines composed of killed bacteria in the 1950s and 60s. Once the vaccination programs were in place the incidence of pertussis was drastically reduced (Hardwick et al. 2002; Olin et al. 2003). In the 1970 the interest in pertussis was rekindled due to the many side effects of the whole cell pertussis vaccine (Cherry 1996). This led to the development of acellular pertussis vaccines based on pertussis toxoids, pertactin etc. (Trollfors et al. 1995; Gustafsson et al. 1996). In recent years the interest in the whole cell pertussis vaccine has once again increased. A number of countries such as Canada, the United States, Australia and the Netherlands have reported an increase in the incidence of pertussis, despite a continuously high vaccine coverage (Bass et al. 1987; Bass et al. 1994; DeSerres et al. 1995; Andrews et al. 1997). It is thought that polymorphisms in key antigens in circulating strains are responsible for reducing the effect of currently used vaccines (Mooi et al. 1998; Mooi et al. 1999; van Amersfoorth et al. 2005). Studies conducted in the Netherlands suggest that the structure of the B. pertussis population has changed over time and that these changes have been driven by vaccination (van der Zee et al. 1996; Mooi et al. 2001). One of these key antigens in pertussis vaccine formulations is the pertussis toxin sub unit 1 (ptxS1). The

26 S1 subunit is part of the 6 subunit pertussis toxin. The S1 subunit has ribo- sylating activity on membrane G proteins and causes the unregulation of cyclic AMP levels in epithelial cells, which increases respiratory secretions and mucus production (Katada et al. 1983; Moss et al. 1983). There are four variants of this antigen named ptxS1A,B,D and E. Each variant is separated by a single nucleotide polymorphism (SNP) within a short region of the gene (10 bp) the two variants with the highest degree of variability found between the A and D variants (three A-G substitutions) (Fig 12). All four mutations in the ptxs1 gene are non-silent and can be found in a region identified as a T-cell epitope (Peppoloni et al. 1995). Investigations comparing the ptxS1 variants circulating before the vaccination era to those found after vaccination suggest that B. pertussis has undergone an antigenic shift driven by immune selection. Studies of the ptxS1 gene showed that variants D and B common before the onset of vaccination has all but been replaced by ptxS1A (Mooi, van Oirschot et al. 1998; Cassiday et al. 2000).

Fig 12. The polymorphic region of the ptxS1 gene, the complete sequence for ptxS1D is given, * denotes identical bases. The indicator and reporter probes used in the assay described are underlined.

It is hypothesized that the introduction of vaccines initially reduced the circulation of B. pertussis and that the bacterium adapted and managed to re- store its circulation in vaccinated populations. In order to determine if there is a connection between theses polymorphisms and the reoccurrence of whooping cough in vaccinated populations there is a need to investigate B. pertussis strains currently in circulation in these populations. Such investigations have been conducted on smaller scales (Bass and Stephenson 1987; Bass and Wittler 1994; DeSerres, Boulianne et al. 1995; Andrews, Herceg et al. 1997). However there is a need to expand these investigations in order to gain a better understanding of the reasons behind the resurgence of B. pertussis. Sanger sequencing is currently the only effective method available for investigating the ptxS1 allele in circulating strains. The time and cost associated with this method has hampered large scale investigations.

27 Two novel methods for typing the four ptxS1 genotypes were developed and evaluated. The first based on hybridization probe and real-time PCR technology and the second on Pyrosequencing technology. We estimated the specificity, sensitivity; cost and time spent handling the samples between the two assays, all factors that are important to consider before implementing a novel method. 143 clinical isolates collected in Sweden were typed and the results were compared to Sanger Sequencing.

Both systems were comparable in reliability and cost, with a specificity and sensitivity of 100% as compared to Sanger-sequencing. When looking at sample handling and analysis of the results the real-time PCR assay was simpler and faster to perform. However, pyrosequencing results, with a reading length of up to 300 bases, will offer a much higher resolution of the ptxS1 allele. This might prove important for the discovery of novel, previously unknown sub-types.

Both systems handled the task with the same sensitivity and specificity as Sanger Sequencing. The simplicity and speed of real-time PCR might be better suited for large scale investigation of circulating ptxS1 types, however if one is interested in finding and identifying novel ptxS1 types pyrosequencing might be better suited for the task. Both pyrosequencing and real-time PCR offers advantages over the currently used Sanger sequencing.

28 Helicobacter pylori, Paper III and IV

The species Helicobacter was probably first observed by Bizzozero, an Ital- ian physiologist at the end of the 19th century. Bizzozero observed gram negative, spiral shaped bacteria in the stomach of dogs (Bizzozero 1892). At around the same time other medical professionals also reported presence of a spiral shaped bacteria in upper gastrointestinal disorders (Pel 1899). How- ever these discoveries went largely unnoticed. In 1938 an American pathologist discovered a spiral shaped bacteria in 43 % of 242 subjects in an autopsy study, further it was known that peptic ulcer disease responded well to treatment with bismuth salts (Doenges 1938). Any further research into the findings was hindered by the lack of fresh specimens of human gastric tissue as well as the fact that the newly discovered bacteria could not be cultured, Thus, the findings soon fell into oblivion.

In 1983, almost a century after it had first been discovered, two Australians, Barry Marshal and Robin Warren noticed a gram negative, flagellated, spiral shaped bacteria growing on agar plates containing human gastric biopsies, that were accidentally left in an incubator over the Easter holidays. The close physical resemblance of this new bacterium to that of the Campylobac- ter species and the fact that peptic ulcers frequently occur in the pyloric gland region of the stomach, led them to name it Campylobacter pyloridis (Warren 1983). The bacteria has undergone two name changes since, first to C. pylori and then in 1989 Goodwin et al. changed the name to the currently used Helicobacter pylori (Goodwin CS 1989). Early observations established a link between H. pylori and gastro-duodenal disease, such as peptic ulceration and gastritis. But it was not until Dr. Marshall took it upon him self to fulfill Koch’s postulate by ingesting a liquid culture of the bacteria that the link was proven (Marshall et al. 1985). After ingesting the culture Dr. Marshall developed mild illness for 14 days; by day ten, gastritis had developed which lasted for four days. The discovery of H. pylori and the realization that it caused gastric ulcers earned the two Australians the 2005 Nobel Prize in physiology or medicine.

Helicobacter species belong to the epsilon sub-group of proteo-, or purple bacteria. The group contains over thirty members of the Helicobacter species colonizing a wide range of hosts (Gasbarrini et al. 2000; Gueneau et al. 2002).The species is diverse in morphology but some features are shared

29 among almost all members of the genus Helicobacter, such as low (35-44 mol %) chromosomal guanine/ cytosine (G/C) content, strong urease activity, the presence of sheathed flagella etc (Owen 1998). The fact that there is such a wide degree of morphological diversity and host specificity suggest that the Helicobacter spp. is an old bacterium and has co- evolved with its hosts for a long period of time (Fox 2002).

Helicobacter pylori is a microaerophilic, gram negative, spiral shaped rod, between 2.5 and 4 uM in size, and under certain circumstances it can be U- shaped or coccoid (Enroth et al. 1999). H. pylori is actively motile using 4- 6 unipolar, sheathed flagella (Dunn et al. 1997; Owen 1998). It resides natu- rally in the gastrointestinal tract of Humans and non-human primates there is also evidence that it can infect pigs, cats, sheep and pups (Dubois et al. 1994; Dubois et al. 1995; Fox et al. 1997; Dore et al. 1999; Fox 2002; Solnick et al. 2003). In the stomach the majority of H. pylori can be found in the gastric mucosa, however a few are found adhered to the gastric mucosal epithelium. The bacterium is highly adapted to survive in the hostile environment of the stomach where few other organisms can survive. All though H. pylori is considered to be a extra cellular bacteria there is evidence suggesting that the bacteria has a mechanism for intracellular invasion (Bjorkholm et al. 2000).

Figure 12 Scanning electron micrograph of H. pylori, kindly provided by Christina Nilsson, Swedish Institute of Infectious Disease Control, 2005.

30 The adherence to the gastric mucosa is an important colonization strategy for H. pylori (Evans et al. 2000; Mahdavi et al. 2002; Odenbreit 2005). Like other bacteria that colonize a turbulent environment, for example uropatho- genic E. coli, H. pylori must adhere strongly to the epithelial cells in order to counteract the movement of the stomach and the fast turn around time of gastric epithelial cells. Attachment to the gastric epithelial cells is accomplished primarily by binding to the frucosylated blood group antigen Lewis B (Aspholm-Hurtig et al. 2004; Nilsson et al. 2006).

Helicobacter pylori is the causative agent in the development of most gastric and duodenal ulcers (Graham et al. 1992; Hosking et al. 1994; Kuipers et al. 1995). Since 1975 it has been known that when the normal gastric mucosa is disturbed by active gastritis; atrophy, intestinal metaplasia, dysplasia and finally adenocarcinoma can ensue. This was first described by Correa et al who also hypothesized that the triggering mechanism for these was niroso- compounds or gastric irritants such as salts (Correa et al. 1975; Correa 1988). Later it was realized that H. pylori can initiate all these events and was a more likely trigger; a realization the led the WHO to conclude that the bacteria was a class 1 carcinogen (Correa, Haenszel et al. 1975; Fox et al. 1992; "Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon, 7-14 June 1994" 1994; IARC 1994). An attempt to prove the relationship between H. pylori and gastric cancer was performed in Mongolian gerbils. 37% of the H. pylori infected gerbils had at the end of the study developed gastric adenocarcinoma while none of the uninfected had, further strengthening the association between infection and the development of cancer (Watanabe et al. 1998).

At present two H. pylori strains have been completely sequenced, J99 isolated from a British patient with gastritis and 26695 isolated from an Ameri- can patient with duodenal ulcer (Tomb et al. 1997; Alm et al. 1999). A comparison between the two strains showed that approximately 7% of the genes were unique for each stain and that there was an abundance of substitutions in the third base of the codons (Alm, Ling et al. 1999). The similarity of the Cag pathogenicity island from four Swedish H. pylori isolates was investigated by Blomstergren et.al. Their study also indicated that there was a high degree of diversity between H. pylori strains (Blomstergren et al. 2004). Although almost all gastric and duodenal ulcers are related to infection with H. pylori only a small number of those infected ever develop any of these diseases (Bjorkholm et al. 2003). This has led to the conclusion that there must be other factors either associated with the bacteria, the host, the environment; or more likely, a combination of these factors that lead to the development of disease.

31 One of the important predictors of disease is the Cag pathogenicity island (Cag PAI), a large horizontally acquired region containing about 30 genes (Covacci et al. 1993; Censini et al. 1996; Akopyants et al. 1998; Nilsson et al. 2003). The Cag PAI can be found in most but not all H. pylori strains and among other things it encodes for a type IV secretion system, the cytotoxin associated gene A (CagA), related to an increased inflammatory response (Peek et al. 1995; Tummuru et al. 1995; Odenbreit et al. 2000; Back- ert et al. 2002) and CagE related to increased production of IL-8 also a recognized predictor of the severity of disease (Tummuru, Sharma et al. 1995). Other important virulence factors are the vacoulating cytotoxin (VacA) which was initially recognized because it induced vacuoles in epithelial cells (Figura et al. 1989; Cover et al. 1993; Bereswill et al. 2002). The VacA gene has further been related to increased inflammation, apoptotic activity and inhibition of T-cell proliferation (Boncristiano et al. 2003; Boquet et al. 2003; Gebert et al. 2003), IceA (induced by contact with the epithelium) and the neutrophil activating protein (napA) to mention a few (Evans et al. 1995; Cooksley et al. 2003; Wu et al. 2005). There are also some genes that should be defined as essential and not as virulence factors since their presence seem to be essential for colonization. Examples of such are the Urease, that enables the bacteria to regulate the pH and the flagella, that enables motility (Eaton et al. 1991; Eaton et al. 1992). As examples of environmental factors that has been associated with increased risk of gastric disease high intake of salt as well as cigarette smoking can be mentioned (Kono et al. 1996; Tredaniel et al. 1997).

Since there is no specific mutagen or carcinogen produced by H. pylori it is currently thought that the chronic inflammatory process triggered by the infection is responsible for the progression to gastric carcinoma. The combinations of host and bacterial genetic polymorphisms are thought to con- tribute to the severity of the inflammation and the progression to gastric neoplasia and in the end cancer (Correa 2005; Correa et al. 2005). Host genetic susceptibility has been investigated by several groups and most of the interest has been geared towards single nucleotide polymorphisms in cytokines. The key role that these types of molecules have in regulating the immune response to infection makes them likely candidates for contributing to the progression through Correa’s model to gastric cancer. Single nucleotide polymorphisms in many of the key antigens have been investigated in order to determine if they have any relation to the development of gastric cancer:

IL-1E is a potent cytokine that has been implicated in mediating both acute and chronic inflammation and is responsible for a wide variety of biological effects. Among other things IL-1 beta is responsible for inducing the expression of the proinflamatory cytokines, IL-2, IL-6, IL-8, IL-12, TNF-alpha; the

32 oncogenes c-myc, c-jun, c-fos; proinflamatory mediators such as cyclooxogenase 2 and the inducible form of nitric oxide synthase (iNOS) (El-Omar 2001). IL-1 beta is also one of the most potent inhibitors of gastric acid secretions known (Robert et al. 1991; Wallace et al. 1991; Beales et al. 1998). Several single nucleotide polymorphisms (SNP) has been identified in the IL-1E gene, two of which have been associated with an increased risk of gastric cancer; IL-1E -511 (rs16944) and IL-1E -31 (rs1143627). Both SNP’s are implicated in increasing the transcription of IL-1 beta and in European populations the alleles are in almost complete linkage disequilib- rium a finding that does not seem to be true in Asian populations A third SNP, Il-1B +3954 (rs1143634) was reported by El-Omar et al to confer protection against gastric cancer in homozygote’s for the T allele although these findings were not statistically significant (El-Omar et al. 2000; Furuta et al. 2002).

Several SNP’s have been reported in the promoter region of the TNF-D gene (a proinflamatory cytokine) but their association to gastric cancer remains unclear. Zambon et al reported associations between the TNF-D -308 SNP (rs1800629) and H. pylori infection, the -857 SNP (rs1799724) and infection outcome; further they found a relation between the -1031 SNP (rs1799964) and the degree of antral inflammation (Zambon et al. 2005). The TNF-D - 308 A allele has been associated with an increased risk of CagA positive infections and with non-cardia gastric cancer where as the G allele has been associated with peptic ulcer (Kunstmann et al. 1999; Lanas et al. 2001; El- Omar et al. 2003; Machado et al. 2003). Further the TNF-D -238 SNP (rs361525) A allele has been suggested to confer protection against gastric cancer (Jang et al. 2001).

There are three SNP’s in the IL-10 gene promoter region that has been implicated in the regulation of IL-10 production; IL-10 -1082 G/A (rs1800896), -819 C/T (rs1800871) and -592 C/A (rs1800872). These alleles are present in three main haplotypes GCC (high IL-10 producer), ACC and ATA (low Il-10 producer (Crawley et al. 1999; Rad et al. 2004). The relevance of IL- 10 in the development of gastric inflammation was demonstrated by Berg et al who showed that Helicobacter felis infected IL-10 knockout mice developed more severe gastritis than did controls (Berg et al. 1998). Further El- Omar et al showed that homozygote’s for the low producing haplotype had a significantly higher risk of developing non-cardia gastric cancer (El-Omar, Rabkin et al. 2003).

Expression levels of IL-8 have been associated with tumour angiogenesis and with an increased invasive activity of gastric cancer cells ((Kido et al. 2001). The gene encoding Il-8 contain three SNP’s, -251 (rs4073); +396 (rs2227207) and +781 (rs2227306) that has been investigated in association

33 with the development of gastric cancer. The -251 allele has been determined to alter the expression of IL-8 and the -251 A; +396 G and +781 T haplotype have been associated with a higher risk of developing gastric cancer in a high risk population (Hull et al. 2000; Sharon et al. 2004). Two polymorphisms in the IL-4 promoter are known to effect the expression; -590 C/T (rs2243259) and -33 C/T (rs2070874)(Rosenwasser et al. 1995; Nakashima et al. 2002). IL-4 is thought to reduce gastric atrophy (pre-cancerous stage) by down regulating interferon gamma which in the end could promote H. pylori adherence, thus a low production of IL-4 would increase the risk of gastric cancer (Vercelli et al. 1990).

Disease resulting from the infection with Helicobacter pylori is a major threat to the general health both in the developed world, where infection rates are comparatively low as well as in the developing world where the infection rates are much higher. Therefore it is important to either identify factors that are important for progression of the disease or to prevent infection altogether. Since eradication of Helicobacter from the human population by antibiotics is neither desirable nor feasible, there has to be another way; either by identifying specific bacterial and/ or host traits that increases the risk of developing disease or by preventing initial colonization all together. Currently there is no way of accomplishing either.

34 Paper III

There is an urgent need to identify risk factors for developing gastric cancer. Since so few of those infected with H. pylori actually become symptomatic and fewer still develop cancer, studies that would allow for identifications of such factors have to be very large. Since no suitable method currently exists for undertaking these studies we designed a tag-array minisequencing array that would give information about the Human SNP genotypes related to the risk of developing cancer, and also at the same time give genotypic information about the H. pylori that infected the individual. Further the tag-array format is well suited for high throughput studies. Since there is a good possibility that H pylori may have adapted to the changes in gastric environment that typically accompany the carcinogenic process and it is difficult to isolate the bacteria from patients with confirmed gastric cancer we wanted to develop a method that would be useful for genotyping H pylori using archived, paraffin-embedded biopsy or resection material

In order to design such an assay it was necessary to find homologous regions in the H. pylori genes of interest. Sequences of less than 150bp that were more than 90% homologous were determined to be suitable for the assay. The minisequencing primers were designed so that the 3’ end of the primer would anneal 1 base upstream from the SNP. No special design constrains were used when designing the mini-sequencing primers for Human SNP’s. However, in order to assure that the primers annealed in regions with the highest possible sequence conservation the annealing site of the MS-primers for H. pylori genes was determined by hand. A 20 bp nucleotide sequence tag, obtained from the Affymetrix GeneChip Tag Collection (Affymetrix, St Clara CA) was attached to the 5’ end of each of the MS-primers in order to facilitate binding to the tag-minisequencing array. At the conclusion of the assay the slide was scanned and fluorescence for each of the fluorophores in each spot was measured.

A total of 12 strains isolated in a study performed in Sweden, for which the Cag-PAI status was known was included (Enroth et al. 2000; Nilsson, Sillen et al. 2003). For four of the isolates the matching formalin preserved paraffin embedded gastric biopsies was obtained, also a gastric biopsy for which

35 Figure 13. Analysis of 12 H. pylori isolates and 5 gastric biopsies (indicated by an asterix) using the minisequencing array. Dark field indicates the presence of the gene, white absence and gray indicates that the result was inde- terminant. the presence of H. pylori had been confirmed by histology but no genotypic information was known was included. We compared the results the Cag-PAI genes from the minisequencing array to the previously reported results from Nilsson et al. and found them to be in over all good agreement, with exception of 12:3 which had previously been reported to lack all Cag genes except CagA. In our assay we found an addi- tional 8 genes of the Cag-PAI. A closer comparison of 2 of the strains with matching gastric biopsies can be seen in figure 14. An interesting fact is that the MS-array detected large numbers of Cag genes in the gastric biopsies that was not detected in the isolated strain, although the strains were isolated from the same biopsies we investigated. This is most likely due to infection with several different clones of H. pylori, which is a recognized phenomena, a fact that supports the argument that it is necessary to look at the population of the bacteria infecting a person rather than separate isolates in order to determine a “risk genotype” of H. pylori (Figura et al. 1998; Enroth 1999; Israel et al. 2001). The biopsy obtained from patient Ca 9 (a cancer patient)

36 is actually the only one where fewer genes in the biopsy than in the in the isolate were found, most likely a result of low bacterial presence in the biopsy.

Cag20 Cag6 Cag14 Cag4 Cag-Į Cag1 Cag10 Cag18 Cag25 Cag2 Cag5 Cag8 Cag9 Cag12 Cag16 Cag19 Cag22 Cag7 Cag23 CagA 26695

Cag15 Cag17 cag21 Cag24 9:1 (Nilsson et al.) Cag11 Cag13 A.

9:1 B.

C. 9:1*

D. 18:1 (Nilsson et al.)

E. 18:1

18:1* F.

Figure 14. A detailed view of the Cag-PAI typing results and the expected results of three isolates and two gastric biopsies. Solid arrows indicate genes that are present, dotted arrow indicate that the result was indeterminate and striped arrows indicate genes that were not included on the MS-array. 26695 contain the complete Cag-PAI. A and D is a representation of previously reported results, B, C, E and F are results from the array typing of isolates (B and E) and gastric biopsies (C and F).

DNA obtained from 52 Swedish donors was investigated in order to validate the SNP genotyping. We also investigated the five biopsies previously mentioned. The success rate for assigning genotype varied between 76% and 95% depending on the SNP investigated. We further compared the minor allele frequencies in order to determine if the assay gave results consistent with what has previously been reported for each SNP to dbSNP, a central repository for SNP information (www.ncbi.nlm.nih.gov/SNP/). Our results were over all in good agreement with what had previously been reported to the database. Since the data in the dbSNP database is calculated for all sam- pled populations some variations of allele frequencies was to be expected due to the small geographical spread of our sample population (blood donors in Uppsala, Sweden). IL-1 beta -511 was the SNP that deviated most from

37 Table 2. Genotyping results for the SNPs in human cytokine genes SNP dbSNP Major / Minor allele Genotyping Minor allele Genotype frequency7 1 3 4 6 Gene name and referenceposition ID minor allele freqdbSNP success (%) freqMS-array 2 5 1 1 1 2 2 2

Interleukin-1 beta -511 rs16944 A/G 0.47 92 0.05 0,94 0,02 0,04 El Omar et al 2000 -31 rs1143627 T/C 0.48 77 0.59 0,43 0,33 0,25

3954 rs1143634 C/T 0.17 81 0.23 0,73 0,10 0,18 Tumor necrosis factor alpha -1031 rs1799964 T/C 0.23 95 0.19 0,65 0,33 0,02 Zambon 2005 -857 rs1799724 C/T 0.1 76 0.01 0,86 0,09 0,05

-376 rs1800750 G/A 0.02 94 0.04 0,92 0,08 0,00

-308 rs1800629 G/A 0.1 90 0.14 0,71 0,29 0,00

-238 rs361525 G/A 0.08 81 0.08 0,89 0,06 0,05 Interleukin-10 -1082 rs1800896 A/G 0.3 83 0.4 0,33 0,56 0,12 CrawleyCrowley 1999 & Berg 1998

-819 rs1800871 C/T 0.38 92 0.24 0,56 0,40 0,04

-592 rs1800872 C/A 0.39 89 0.32 0,41 0,55 0,04

Interleukin-8 -251 rs4073 A/T 0.45 88 0.45 0,39 0,33 0,29 Hull 2000 & Sharon2004

396 rs2227307 T/G 0.46 86 0.38 0,51 0,23 0,27

781 rs2227306 C/T 0.25 88 0.48 0,24 0,55 0,21 Interleukin-4 -590 rs2243259 C/T 0.03 86 0.26 0,63 0,27 0,10 Nakashima2002 &Vercelli 1990 -33 rs2070874 C/T 0.41 92 0.15 0,76 0,18 0,06

1990 Interferon-Gamma 874 rs2430561 T/A N.A 84 0.45 0,31 0,48 0,21 Pravicia2000 and Zambon 2005

Interleukin-2 -330 rs2069762 T/G 0.24 93 0.25 0,65 0,21 0,14 Hoffman 2001 &Togawa2005 Interleukin-6 -174 rs1800795 G/C 0.21 81 0.42 0,38 0,39 0,23 Basso 1996 & Lobo Gatti 1 See reference list for details 2 The SNP positions are given in relation to the translation initiation site for each gene 3 Reference numbers from the dbSNP database at NCBI: www.ncbi.nlm.nih.gov/SNP 4 According to dbSNP 5 Percentage of samples for which a genotype was obtained 6 Determined in the present study 7 Determined in the present study, 1 denotes minor allele, 2 denotes major allele (Vercelli, Jabara et al. 1990; Basso et al. 1996; Berg, Lynch et al. 1998; Crawley, Kay et al. 1999; El- Omar, Carrington et al. 2000; Hull, Thomson et al. 2000; Pravica et al. 2000; Hoffmann et al. 2001; Nakashima, Miyake et al. 2002; Sharon, C. et al. 2004; Lobo Gatti et al. 2005; Togawa et al. 2005; Zam- bon, Basso et al. 2005) what had previously been reported with a minor allele frequency of 5% instead of almost 50% (Table 2).

Since the final aim of this assay is to identify the bacterial and host biomarkers in paraffin embedded, formalin preserved gastric biopsies we also determined the SNP genotype of the 5 gastric biopsies previously mentioned. The limited number of biopsies we had access to made it impossible to determine SNP genotypes with any accuracy using SNPsnapper software. We

38 did however look at the raw data from the biopsies and were able to determine that the results would be of sufficient quality to be typed if we had access to a larger material.

The results from this study suggest that this assay would be suitable for identifying bacterial genes that are present in gastric biopsies. Further the assay is also suitable for typing human SNP’s from the same type of material, although further studies are warranted to determine optimal analysis conditions.

We believe that more information is required to understand the interaction between H. pylori and the host in gastric cancer development. Presumably, host and bacterial genetic factors underlie the H. pylori associated changes in the infected hosts. These genetic factors may answer to the disease progression in gastric cancer development. The study of bacterial virulence markers in archived paraffin-embedded biopsies can provide us with unprecedented opportunities to investigate H. pylori strain characteristics before and during early stages of cancer development. The identification of host and microbial genomic markers in archived in paraffin-embedded biopsy or resection material can be done simultaneously using slices of the material. This in situ method makes it possible to travel in time to compare present H. pylori strains with strains prior to the onset of gastric cancer diagnosis, taking advantage of both historic materials with frozen bacterial strains and old patient materials with archived paraffin-embedded tissue blocks. The future plan is to identify endoscopic biopsies years before gastric cancer diagnosis. Further performing a nested case-control study, accomplished with record- linkages between files with non-cancer patients undergoing gastric biopsies ascertained in computerized local pathology registers and national registers of cancer, death and migration.

39 Paper IV

Helicobacter pylori’s emerging resistance to antibiotics is rapidly becoming a problem, at present a combinational treatment consisting of two antibiotics and a proton pump inhibitor is the recommended treatment, still the eradication rate is only 80-90% (van der Hulst et al. 1997; Feydt-Schmidt et al. 2002; Leal-Herrera et al. 2003; Parsonnet 2003). Before long a triple antibiotic regiment will be required, the addition of a third antimicrobial substance, although certainly effective, is only a temporary solution. The genetic variability of H. pylori and the propensity for mutations all but assures that multi resistant strains will develop (Bjorkholm, Sjolund et al. 2001). It is becoming increasingly clear that a novel way of combating H. pylori infection is urgently needed.

It has long been realized that vaccinations would be the most effective way of dealing with H. pylori infection. It has been estimated that a vaccination program with an effectiveness of only 50% would all but eliminate H. pylori from the industrialized world (Rupnow MF 2001). Two main uses for a vaccine have been suggested, a prophylactic vaccine that would protect against the initial infection by the bacteria and a therapeutic vaccine that could be used instead of (or in combination with) antibiotics to treat the infection.

Multiple strategies have been employed for identifying suitable antigens to be part of a acellular H. pylori vaccine and so far a few good candidates, mainly CagA, VacA, IceA, urease and NapA, have been identified. The immunogenicity of these antigens has been extensively studied (Manetti et al. 1997; Guy et al. 1998; Satin et al. 2000). However, these known antigens have not proven sufficiently effective for human use. There is a need to identify novel antigens if a H. pylori vaccine is to become reality. Since many of the previously described antigens are also known predictors of disease outcome, new antigens could also prove useful as biomarkers.

We have therefore employed a novel technique in order to generate such a comprehensive library of antigens. We have used the antigenome technology, described in detail in the materials and methods section of the thesis, to identify 179 possible antigens from two H. pylori strains KTH Du and KTH Ca1, isolated from a patient with duodenal ulcer and a patient with gastric cancer, in 13 screens with five IgG pools and 2 IgA pools. Of these possible antigens, 124 corresponded to previously annotated open reading frames (ORFs) as determined by the blastn algorithm against the genbank archive (www.ncbi.nlm.nih.gov/BLAST), we were unable to assign 55 genomic

40 regions to any previously annotated ORF (results not shown). As could be expected some of the most frequently isolated genomic regions belonged to previously known antigens such as CagA (794 hits in 11 screens) and CagY (801 hits in 8 screens) this also served as a confirmation that our selection process had worked. We also identified several novel antigens some that scored very high in the assay, for example a putative iron transporter (Hp1341) scored 170 hits in 7 screens, which can be compared to VacA, a known antigen, which scored 47 hits in 6 screens). We also identified a hypothetical protein (Hp0563) which scored 134 hits in a single screen. By aligning sequences retrieved from the genomic fragment libraries to the specific genes that were selected for further analysis we were able to determine the epitopic regions of the genes (figure 15).

CagA (3558 bp) Hpy1341 (855 bp)

Figure 15. In the CagA gene two immunogenic regions are clearly distin- guished when aligning the fragments from the “small fragment library” (lower arrows) and the “large fragment library” (upper arrows). A single immunogenic region is identified in Hp1341.

Using this information we synthesized peptides that corresponded to these regions. The peptides, in turn, were used in a peptide ELISA where we investigated the individual sera that had been used to create the initial sera pools. The results from the peptide ELISA (not shown) was used together with several other criteria, such as cellular location, conservation, predicted secretion, we narrowed the list of antigens to 14 promising candidates (table 3).

41 Table 3. Ranking of candidate antigens No. of No. of Gene distribu- Rank ORF Common name Hits* screens tion** 1 HP1341 siderophore-mediated iron transport 170 7 28 protein 2 HP0563 hypothetical protein 134 1 28 3 HP0115 flagellin B 56 4 28 4 HP0706 Outer membrane protein 39 3 28 5 HP1116 Hypothetical protein 34 3 28 6 HP0192 fumarate reductase 25 3 28 7 HP0542 cag21 / cagG 22 3 23 8 HP1453 hypothetical protein 22 6 28 9 HP0266 dihydroorotase 21 1 25 10 HP0752 flagellar hook-associated protein 2 21 8 28 11 HP0175 cell binding factor 2 20 3 Nd 12 HP0229 outer membrane protein 16 3 Nd 13 HP1342 outer membrane protein 16 7 Nd 14 HP0289 toxin-like outer membrane protein 15 5 28

Nd, not determined; *, number of screens in which antigen was selected; **, number of strains in which gene was present of a total of 28 strains tested.

The results from the peptide ELISA indicated that the most immunodominant protein was the putative iron-dependent siderophore transporter protein (TonB) Hp1341. In order to detect specific antibodies, towards this protein induced by H. pylori during infection we cloned and expressed the full length (31 kDa) protein and examined the reactivity to patient sera in ELISA. The results from the ELISA with the full length protein were similar to the results from the peptide ELISA. We also compared the reactivity of Hp1341 to that of CagA, and the over all IgG antibody levels were similar. Interestingly, as can be seen in figure 16 there was no correlation between CagA and Hp1341 antibody levels. 1000

800

600 CagA

units HP1341 400

ELISA ELISA 200

1 9 4 6 6 64 65 67 73 77 81 08 19 41 50 57 47 53 56 23 23 23 23 23 23 240 24 24 244 24 25 25 25 P P P P P2375P P P2396P P24 P2416P P2438P243P P2442P P24 P245P P2462P246P P P Figure 16. Antibody levels against recombinant Hp1341 and CagA determined by ELISA against individual sera.

42 The finding of specific antibodies against the putative iron-dependent siderophore transporter Hp1341 is especially interesting since iron acquisition has been suggested as a novel therapeutic target. Iron is an essential element for bacteria (as for most organisms) and sidophore proteins function as irons scavengers. It has been shown in other Gram-negative bacteria that sidophore transport proteins are required for active iron transport (Braun et al. 2002). The findings of antibodies against Hp1341 suggest the possibility of counteracting the bacteria not only by enhancing the host immune response but also by interfering with the conditions under which the bacteria grow by disrupting iron transport.

Many of the previously known antigens, such as CagA and VacA, identified in this assay are also well known predictors of disease. Since the host immune response to H. pylori infection is an important part of the disease progression, identification of the H. pylori antigenome will also prove useful for the identification of novel bio-markers that could in the end, help us to better understand why some of those infected with the bacteria will develop disease while the vast majority will not.

43 Concluding remarks

The studies included in this thesis is a collection of methods and applications that we have developed and established in order to lay the ground work for future studies and discoveries.

B. pertussis is still a problem in the developed world this despite good vaccine coverage. We do not yet understand why, but we do know that changes in antigens have occurred in the bacterial population and that the new variants are different from those used for vaccine production. In order to understand what has happened it is necessary to investigate the circulating population at a large scale and over time. It is our hope that the methods we have established for characterizing pertussis toxin variants will prove to be a significant improvement to the methods currently in use.

We established a platform technology for MIC and viability testing of C. trachomatis, something we hope will result in better understanding why infections that seem to respond well to antibiotic treatment sometimes reoc- cur

We have identified a large collection of novel immunodominant proteins from H. pylori. We hope to use this knowledge not only to find possible components for a vaccine; and also hope that the information gained from generating the H. pylori antigenome will be helpful in identifying biomarkers and even future drug targets.

Finally we also developed a novel application for tag-minisequencing arrays that will allow us to “travel back in time” in order to determine the genotypic makeup of the H. pylori clones that initially was responsible for triggering the events that in the end led to cancer and at the same time we will be able to gain useful information about the genetic variation of cytokines from the same individual. We hope that in the end this information will allow us to establish risk profiles so that prophylactic treatment can be given to patients that are at an increased risk for developing complications from the H. pylori infection.

44 Acknowledgements

I would like to express my sincere gratitude and appreciation to the many people that have in different ways contributed to this thesis. Particularly I would like to thank the following:

My excellent supervisor, Lars Engstrand for taking me on as a PhD student, for always being supportive and encouraging through out these years and for being an unending source of inspiration and ideas.

Also, to my equally excellent co-supervisor Ann-Christine Syvänen for allowing me to become an “exchange student” in the molecular medicine department, for introducing me to microarrays and for all the valuable input.

All my co-authors and contributors to the studies, especially to: Kåre Bon- deson for introducing me to many of the techniques in this thesis. To Hans Hallander for teaching me all I know about whooping cough. Björn Herrmann for sharing his extensive knowledge of Chlamydia. To Ingerd Gustafsson for showing me how to grow it. Lovisa Lovmar for all the help with everything from microarrays to Microsoft Word. Christina Persson for being the rock in the lab. Eszter Nagy for all the input on vaccines and im- munology.

All my co workers at the department of clinical bacteriology here in Upp- sala: My fellow PhD students Sara Olofsson, Marie Edvinsson, Cristian Ehernborg, Elisabeth Nielsen and Patric Jern. The rest of the Uppsala crew: Markus Klint, Åsa Innings and Ylva Molin, for your positive attitude, all the good times we have had and for moving all my stuff while I was in Houston. You guys make this place grate. To honorary bacteriologists Johanna Hen- riksnäs for among other things explaining how the stomach works and Carl- Johan Rubin for being a good buddy both on and off the field. Also to Eva Haxton for helping with all the red-tape and to Astrid Asklin and Eva Hjelm for teaching me the basics of bacteriology and the rest of the people in the “Korridoren”, Marius Domineika, Anita Perols, Juliana Larsson and Otto Cars.

45 All my co-workers at my “second home” at the Swedish Institute of Infec- tious Disease Control. Especially to Mathilda Lindberg, thank you so much for all your help with everything. Also to Sandra Hjalmarsson (tank you for the pottery), Hedvig Jakobsson, Anna Skoglund, Anders Andersson, Helene Kling, Sönke Andres, , Marianne Ljungström, Kristina Schönmeyr and Lena Ericsson. Thank you for always making me feel at home and for making it such a grate place to work at.

The Molecular Medicine Crew: My adoptive office mates: Lovisa Lovmar, Johanna Sandling and Annika Ahlford thank you for letting me set up shop and for all your assistance. And I would also like to thank the rest of the group for helping me with the arrays and all that it entails.

All the helpful people at Intercell AG. Thank you all so much for helping me feel at home in Vienna and for all the good times both inside and outside of work. Especially to Manfred Beger, Andy Mienke, Dieter Gelbmann.

Past co-PhD students: Maria Held, Maria Sjölund, Christina Nilsson, Farzad Olfat, Britta Björkholm, Mårten Kivi, Annelie Lundin you are all sorely missed.

The clinical staff here at the department of microbiology that are always supportive and helpful, especially to past co-worker Caroline Fock, to Eric Torell for the tennis matches and to Åke Gustavsson for getting me involved in this in the first place.

There are many others that deserves to be thanked especially the students and project workers, too numerous to be mentioned here, but you have my gratitude and appreciation. Thank you so much.

Lastly but not least I would like to thank my friends and family for all the help and support and for keeping me aware of the fact that there is life outside of work.

Uppsala, February 2006

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