Western University Scholarship@Western Electronic Thesis and Dissertation Repository 12-10-2014 12:00 AM HcpE, a potential immuno-modulatory protein from Helicobacter pylori that is dependent on the Disulfide bond protein DsbHP Jeff Lester The University of Western Ontario Supervisor Dr. Carole Creuzenet The University of Western Ontario Graduate Program in Microbiology and Immunology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Jeff Lester 2014 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Bacteriology Commons, Biochemistry Commons, and the Immunology of Infectious Disease Commons Recommended Citation Lester, Jeff, "HcpE, a potential immuno-modulatory protein from Helicobacter pylori that is dependent on the Disulfide bond protein DsbHP" (2014). Electronic Thesis and Dissertation Repository. 2599. https://ir.lib.uwo.ca/etd/2599 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. HcpE, a potential immuno-modulatory protein from Helicobacter pylori that is dependent on the Disulfide bond protein DsbHP (Thesis format: Monograph) By: Jeffrey Lester Graduate program in Microbiology and Immunology A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © Jeffrey Lester 2014 i ABSTRACT H. pylori is a human gastric pathogen that colonizes ~ 50% of the world’s population. It can cause gastritis, gastric or duodenal ulcers and also gastric cancer. H. pylori produces Helicobacter cysteine rich protein HcpE, a secreted protein which may play a role in virulence. In this study we show that HcpE is secreted in the culture supernatant both as a soluble protein and in association with outer membrane vesicles, and may play a role in the modulation of H. pylori inflammatory responses. We identified that DsbHP is necessary for HcpE production and secretion in H. pylori, and demonstrated DsbHP has DiSulfide Bond (Dsb) forming activity on reduced lysozyme. Furthermore, we demonstrated that DsbHP has a DsbA-type of activity when expressed in E. coli, despite its similarity with DsbG, and that DsbHP is involved in maintaining redox homeostasis in H. pylori. Key words: Helicobacter pylori. Disulfide bonds. Dsb proteins. Helicobacter Cysteine- rich proteins. Protein secretion. ii DEDICATION This thesis is dedicated to my entire family, specifically Jeff, Lorrie, Rachelle and Justin, who have supported me along the way iii ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Carole Creuzenet for giving me the opportunity to conduct my research in her laboratory. I would also like to extend my thanks to my advisory committee members Dr. David Heinrichs and Dr. Susan Koval for their advice and support throughout my studies. I would also like to thank all of the members of the Creuzenet lab, past and present for your help throughout the duration of my studies. iv Table of Contents Title Page....................................................................................................................................i ABSTRACT ..................................................................................................................................... ii DEDICATION ................................................................................................................................ iii ACKNOWLEDGEMENTS ............................................................................................................ iv Table of Contents ............................................................................................................................. v List of Figures ................................................................................................................................. ix List of Tables .................................................................................................................................. xi List of Abbreviations: .................................................................................................................... xii CHAPTER 1: INTRODUCTION .................................................................................................... 1 1.1 Helicobacter pylori: a human gastric pathogen ....................................................................... 1 1.2 H. pylori virulence mechanisms .............................................................................................. 2 1.2.1 Urease ............................................................................................................................ 3 1.2.2 Flagella ........................................................................................................................... 4 1.2.3 LPS ................................................................................................................................. 5 1.2.4 Toxins ............................................................................................................................ 6 1.2.5 Adhesins ......................................................................................................................... 8 1.3 Protein secretion systems .................................................................................................. 9 1.3.1 Protein secretion in Helicobacter pylori ......................................................................... 9 1.3.2 Sec secretion for the transport of proteins across the inner membrane ........................ 10 1.3.3 Tat-secretion for the transport of proteins across the inner membrane ........................ 13 1.3.4 Protein folding in the periplasm ................................................................................... 14 1.3.5 General secretory pathway for the transport of proteins from the periplasm to the extracellular matrix ...................................................................................................... 15 1.3.6 Direct transport from the cytoplasm to the extracellular environment ........................ 16 1.3.7 Outer membrane vesicle (OMV) mediated secretion................................................... 17 1.4 Disulfide bond proteins ................................................................................................... 18 1.4.1 Pathways of disulfide bond formation ......................................................................... 19 1.4.2 Importance of Dsb proteins in bacterial virulence ....................................................... 22 1.4.3 Dsb proteins in H. pylori .............................................................................................. 23 1.5 Helicobacter cysteine-rich proteins (Hcps) ..................................................................... 24 1.5.1 Sel1-Like Repeats (SLR), the structural components of Hcp proteins ........................ 27 v 1.5.2 Helicobacter-cysteine rich protein E (HcpE) ............................................................... 29 1.5.3 Putative function of Hcp proteins ................................................................................ 34 1.6 Animal models for studying H. pylori colonization ........................................................ 39 1.7 Hypotheses and objectives .............................................................................................. 41 CHAPTER 2: MATERIALS AND METHODS ........................................................................... 42 2.1 Bacterial strains, growth conditions, and reagents................................................................. 42 2.2 SDS-PAGE and Western blot analysis .................................................................................. 43 2.3 Transformation of E. coli competent cells ............................................................................. 46 2.4 Cloning DsbHP-Flag-His in a pET30a vector for over-expression ......................................... 46 2.5 Cloning an expression vector containing both DsbHP and HcpE ........................................... 47 2.6 Construction of vectors expressing E. coli DsbA-Flag-His and DsbG-Flag-His .................. 47 2.7 Over-expression and purification of proteins via nickel chromatography ............................. 47 2.8 Protein quantification using the Bio-Rad Bradford protein assay ......................................... 50 2.9 Solubility assay for expression of HcpE ................................................................................ 51 2.10 Construction of the H. pylori dsbHP knockout mutant ........................................................... 51 2.11 PCR screening of the dsbHP::kan knockout mutagenesis clones ........................................... 52 2.12 Analyzing H. pylori for detection of HcpE ............................................................................ 53 2.13 OMV preparation and analysis .............................................................................................. 54 2.14 Urease activity assay to assess cellular lysis of H. pylori cultures ........................................ 54 2.15 Cell fractionation of H. pylori...............................................................................................