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Investigation of Prophage in Clinical Isolates of H Pylori

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W&M ScholarWorks Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects 2013 Investigation of Prophage in Clinical Isolates of H pylori Kevin Alexander Leslie College of William & Mary - Arts & Sciences Follow this and additional works at: https://scholarworks.wm.edu/etd Part of the Microbiology Commons Recommended Citation Leslie, Kevin Alexander, "Investigation of Prophage in Clinical Isolates of H pylori" (2013). Dissertations, Theses, and Masters Projects. Paper 1539626941. https://dx.doi.org/doi:10.21220/s2-be0k-kz58 This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. Investigation of Prophage in Clinical IsolatesH. pylori of Kevin Alexander Leslie Great Falls, VA B.S., College of William & Mary, 2009 A Thesis presented to the Graduate Faculty of the College of William and Mary in Candidacy for the Degree of Master of Science Department of Biology The College of William and Mary January, 2013 APPROVAL PAGE This Thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science Approved b >12 uummuifcffcf unaii Associate Professor Mark Forsyth, Biology The College of William and Mary Associate Professor Oliver Kerscher, Biology The College of William and Mary A iology \R I s. S( II \< ! s Institutional Compliance Committee Form Every A&S graduate student must answer all questions below by checking either the “yes” or the “no” box for each question. If you answer YES to any question, you must provide the appropriate committee protocol number and the date when written approval was received. Every A&S graduate student must provide this form, signed by both the student and the faculty research advisor, to the Office of Graduate Studies and Research as a separate page (not inserted into the thesis/dissertation) when the final original thesis or dissertation is submitted. If you answer YES to any question, you must also include the completed as a required page in your thesis or dissertation. Yes No Questions Name of Provide committee protocol committee from number and date of written which approval approval needed if answer to question is YES □ □ Did this project involve surveys, Protection of testing of human subjects, or Human Subjects collection of information from Committee living human beings? □ □ Were live vertebrate animals Institutional Animal used in this project? Care and Use Committee s ^ □ Did this project use any (a) Institutional recombinant DNA molecules Biosafety (including transgenic animals or Committee the transfection of cell lines), (b) infectious agents, (c) human 1 1 ! ' tissue or body fluids (including saliva, urine, blood, semen, or primary human cell cultures), or (d) wild-caught or random source animals or animal tissue (for anyone employing animals that may carry zoonotic disease)? □ □ Did this project involve research Institutional with radioactive material? Radiation Safety Committee I - i , Student Name (printed) Faculty Research Advisor (printed) Signature OQ/1 Q /O S COMPLIANCE PAGE Research approved by Institutional Biosafety Committee Protocol number(s): IBC-2012-4-11-7920-mhfors Date(s) of approval: 5/13/2012 ABSTRACT Helicobacter pylori colonizes the human stomach and causes gastroduodenal complications ranging from ulcer disease to cancer. A major virulence determinant of this pathogen iscag the Pathogenicity Island (cag PAI), and isolates possessing this element are more frequently associated with severe disease outcomes. In many bacterial species, prophage are also frequently associated with bacterial virulence. The work presented in this thesis aims to characterize the completeness, location, and effects of prophage DNA across different clinical isolates H.of pylori. In addition, identification of potential antivirulence loci and the potential for strains to produce viral particles following induction are investigated. PCR data indicate a significant correlation between the absence of prophage sequence and the presence cagof the PAI in our collection of clinical isolates. Additionally, amongcag the PAI(-) strains possessing prophage elements, there appears to be preferential insertion of these elements into H.the pylori chromosomal geneHP0453. In sequencedcag PAI(+) strains that harbor prophage, three different intergenic chromosomal insertion sites were identified. The most prevalent insertion site, located between chromosomal genesIpxd and metK, was analyzed using NCBI database tools. This analysis revealed potential bacteriophage att recombination site sequences and novel insertion site characteristics. Supporting recently published studies (mBio, 2011 Nov 15;2[6] and J Virol., 2012 Jun 13 [Epub ahead of print]), TEM and epifluorescence microscopy have indicated the presence of viral­ sized particles from clinical isolates possessing complete prophage sequence that were induced with UV irradiation. Despite the successful generation of a vector for use in the creation of a prophage knockout mutant, attempts at deletion of this locus have been unsuccessful. Continued investigation will enable a better understanding of important virulence mechanisms and the role prophage play in this clinically-relevant species. TABLE OF CONTENTS Acknowledgements ii Helicobacter pylori: A Brief Overview 1 Major H. pylori Virulence Factors: VacA,DupA, OipA, HtrA, CagA 2 Type-IV Secretion Systems and thecag Pathogenicity Island 5 Antivirulence Loci 9 Bacteriophage and Their Effects on Host Cells 10 Prophage inH. pylori 20 Materials and Methods 21 Results and Discussion 37 Future Directions 51 Concluding Remarks 55 Appendices 57 References 93 i ACKNOWLEDGEMENTS This writer wishes to express his or her appreciation to Dr. Mark Forsyth, under whose guidance this investigation was conducted, for his patience, guidance and criticism throughout the investigation. The author is also indebted to Dr. Oliver Kerscher and Dr. Kurt Williamson for their training and careful reading and criticism of the manuscript. Helicobacter pylori: A Brief Overview Discovered in 1982 by Barry Marshall and Robin Warren, H. pylori is a widespread, gram-negative human pathogen infecting over half of the world's population. Infection rates are generally higher in developing countries, and transmission is thought to occur via a fecal- oral route (1-3). Specifically, colonization of new human hosts is posited to most often be a result of transmission from parent to child in the early years of life. Genetically, H. pylori is highly heterogeneous and is divided into sub-species groupings known colloquially as “strains” (4). The bacterium is microaerophilic, approximately 3 pM in length, spiral-shaped, and possesses between four to six flagella (5). It is unique in both its ability to colonize the human gastric mucosa and its characterization as the only known bacterial carcinogen. Though 80% of infected individuals are asymptomatic, infection with H. pylori is associated with the development of gastritis, as well as gastric and duodenal ulcers and cancers. In addition, several studies have linked infection to the development of autoimmune diseases such as Lupus and Sjogren's syndrome (6). Infection is commonly diagnosed via urea breath or stool antigen tests, while the efficacy of real-time PCR for diagnosis is currently being tested (7). In locations with adequate access to medical services, H. pylori infection can generally be treated with antibiotic therapy. Specifically, first-line therapy regimens include the seven-day use of a proton pump inhibitor, combined with amoxicillin, clarithromycin and/or metronidazole. Unfortunately, a rise in antibiotic resistance has decreased the efficacy of triple-antibiotic therapy in recent years. Clarithromycin, prescribed heavily in many countries for the treatment of bacterial respiratory infections, has caused localized increases in resistance. In these localities, quadruple-drug treatment as first line therapy (metronidazole, omeprazole, 1 bismuth subcitrate, and tetracycline) outperforms standard triple-therapy. Given the rise in resistance and rates of reinfection, alternatives to standard antibiotic treatments are being investigated (8). Disease outcomes vary from patient to patient; diet, infection duration, age at infection, and other environmental factors have been implicated. Recently, human host genetic variation has also been suggested as a cause of at least a portion of this variation, with polymorphisms in certain cytokine genes altering gastric cancer risk in infected patients. Regarding H. pylori virulence, more severe disease outcomes are linked to infection by strains possessing a contiguous suite of genes known collectively as the cag Pathogenicity Island {cag PAI). In addition to the cag island, H. pylori possesses several notable virulence factors that affect disease severity, including proteins responsible for vacuolation and induction of inflammation (9,10). Major H. pylori Virulence Factors: VacA, DupA, OipA, HtrA, CagA VacA VacA is both a secreted and membrane-bound protein encoded for by the vacA gene in H. pylori. Alleles of vacA (vacuolating cytotoxin) vary by geographic region, with certain types being associated with increased risk of developing gastric cancer and peptic ulcer disease. To date, several roles have been described for the VacA protein.
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