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The Entner-Doudoroff Pathway and Development of a Promoter-Trapping System

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The Entner-Doudoroff Pathway and Development of a Promoter-Trapping System HELICOBACTER PYLORI COLONIZATION OF THE MOUSE GASTRIC MUCOSA: THE ENTNER-DOUDOROFF PATHWAY AND DEVELOPMENT OF A PROMOTER-TRAPPING SYSTEM DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Amy E. Wanken, B.S., M.S. * * * * * The Ohio State University 2003 Dissertation Committee: Dr. Kathryn Eaton, advisor Approved by Dr. Brian Ahmer Dr. Kathleen Boris-Lawrie _______________________________ Advisor Dr. Charles Brooks Department of Veterinary Biosciences ABSTRACT Helicobacter pylori is a microaerophilic, Gram-negative bacterium that persistently colonizes the mucous layer overlying the gastric epithelium of humans. Colonization with H. pylori results in chronic superficial gastritis, which increases the risk of development of peptic ulcer disease and distal gastric carcinoma. However, the majority of individuals colonized with H. pylori remain asymptomatic. Although several strain-specific factors have been identified and may be markers for the different clinical outcomes of colonization, our present understanding of the role of the bacterial factors that might affect the course of disease is largely incomplete. The work in this dissertation approaches two different aspects of colonization. The first is the analysis of a metabolic pathway, the Entner-Doudoroff (ED) pathway. The enzymes of the ED pathway are present in H. pylori, but the impact of this pathway on colonization has not yet been examined. The objectives of this project were to confirm the presence and activity of 6-phosphogluconate dehydratase (6PGD), the key enzyme in the ED pathway, in H. pylori, to create a mutant strain and confirm loss of enzyme activity, and finally to use the mutant strain in mouse colonization experiments. In the first part of this project, the activity of the ED pathway in H. pylori was evaluated by cloning the genes and assaying their activity. Results indicated that the key ED enzyme, 6-phosphogluconate dehydratase, was active in H. pylori but the ii cloned genes were not active in E. coli, indicating the need for an unknown accessory gene or cofactor. Finally, two ED-negative strains, one in H. pylori M6 and one in H. pylori SS1, were created by insertional mutagenesis into 6-phosphogluconate dehydratase. The second part of the project consisted of testing the mutant strains for colonization ability in a mouse model. Two mouse strains, C57BL/6J and 129, were inoculated with wild-type and/or mutant H. pylori strains M6 or SS1. All mutant and wild-type strains colonized, but colonization was lower for both mutant strains compared to the parental strains. In addition, minimum infectious dose was 100-1000- fold lower for the wild-type than for the ED-negative mutants. Surprisingly, in spite of lower colonization density and higher minimum infectious dose, co-inoculation experiments revealed that wild-type H. pylori did not displace the mutant strain, indicating that competition between wild-type and mutant did not occur in vivo. Results from this study indicate that loss of the key ED enzyme in H. pylori diminishes fitness of the organism in vivo, but that the pathway is non-essential for colonization. However, conservation of this pathway in H. pylori and the fact that colonization was diminished by loss of 6PGD, suggests that the ED pathway has some function in H. pylori metabolism. Although our data show that the pathway is not vital for colonization, it may have some other function in colonization or transmission of the organism among hosts. The second focus of this dissertation was the development of a technique to screen the H. pylori genome for genes which may be important for colonization. This technique, in vivo expression technology (IVET), has been used in other pathogens with iii success and would be a useful tool in H. pylori. The objectives in this project were to develop an IVET plasmid for H. pylori, and to develop methods for testing and screening an H. pylori promoter library. The IVET plasmid was developed so that promoter fragments would be inserted into a StuI site upstream of a promoter-less ureB gene. Expression of this construct in urease-negative H. pylori would be a marker for functional promoters. Various methods for generating a genome library were tested, and it was found that the best method for achieving random fragments of the appropriate size was by sonication of the genomic DNA. Agar plates were also developed for the in vitro urease testing of a large number of colonies by replica plating. This was a necessary step for screening the cells after they had passed through the mouse challenge, as promoters that are up- regulated in vivo but are turned off in vitro would be of most interest for further study. iv Dedicated to my parents and in memory of Robert Kitchen v ACKNOWLEDGMENTS I wish to thank my advisor, Dr. Kathryn Eaton, first and foremost for accepting me as a student when my primary advisor moved to another university and allowing me to continue the work that I had begun. I would also like to thank her for intellectual support, encouragement, and enthusiasm which made the completion of this dissertation possible, as well as personal support through the process. I am also grateful to Tyrrell Conway, my previous advisor, for allowing me to pursue a project that used an organism with which he was not familiar and for encouraging this project from the beginning. I would also like to thank my committee members, Dr. Brian Ahmer, Dr. Kathleen Boris-Lawrie and Dr. Charles Brooks, for input and critical comments on my work. I would like to acknowledge and thank current and past lab members, and especially Elizabeth Murray, Patrick Baker and Richard Peterson, for always answering questions and offering both intellectual and personal support. I am also grateful for the support and friendship that I found throughout my years at Ohio State from Jon Foster, Rob Walczak, Sue Ann Frank, Traci Hatcher, Jean MacDonald, Brian Mark, Tessa Moir, Julie Maybruck, Gloria Sivko, Larry Dearth, Daniel Sanford, Tiffiney Roberts and Stacey Hull. Finally, I would like to thank my family, James and Susan Wanken, Angela and Ian Kitchen, and Jamie and Kristin Wanken for their love, patience and support. I could not have done this without you! vi VITA October 14, 1972............................................Born – Rantoul, IL 1995................................................................B.S. Biology, University of Notre Dame 1995-1999 ......................................................Graduate Teaching and Research Associate, Ohio State University 1999................................................................M.S. Microbiology, Ohio State University 2000-present...................................................Graduate Research Associate, Ohio State University PUBLICATIONS Research Publications 1. A. E. Wanken, T. Conway, and K. Eaton. 2003. The Entner-Doudoroff pathway has little role in Helicobacter pylori colonization of mice. Infect. Immun. 71(5): 2920-2923. 2. K. A. Eaton, J. V. Gilbert, E. A. Joyce, A. E. Wanken, T. Thevenot, P. Baker, A. Plaut, and A. Wright. 2001. Restoration of the ability of Helicobacter pylori to colonize by in vivo complementation of ureB. Infect. Immun. 70(2): 771-778. 3. L. K. Fuhrman, A. E. Wanken, K. W. Nickerson, T. Conway. 1998. Rapid accumulation of intracellular 2-keto-3-deoxy-6-phosphogluconate in an Entner-Doudoroff aldolase mutant results in bacteriostasis. FEMS Microbiol. Lett. 159(2): 261-266. 4. K. Mo, C. O. Lora, A. E. Wanken, M. Javanmardian, X. Yang, and C. F. Kulpa. 1997. Biodegradation of methyl t-butyl ether by pure bacterial cultures. Appl. Microbiol. Biotech. 47(1): 69-72. vii FIELDS OF STUDY Major Field: Veterinary Biosciences viii TABLE OF CONTENTS Page Abstract.............................................................................................................................ii Dedication......................................................................................................................... v Acknowledgments............................................................................................................vi Vita..................................................................................................................................vii List of Tables ...................................................................................................................xi List of Figures.................................................................................................................xii Chapters: 1. Introduction........................................................................................................... 1 2. Analysis and cloning of the Entner-Doudoroff genes in Helicobacter pylori and creation of a 6-phosphogluconate dehydratase mutant ................................ 51 Abstract................................................................................................... 51 Introduction............................................................................................. 52 Materials and Methods............................................................................ 54 Results..................................................................................................... 66 Discussion............................................................................................... 69 3. Colonization of the mouse gastric mucosa by Helicobacter pylori strains deficient in
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