Elucidation of enteric virulence of Campylobacter concisus Hoyul Lee A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Supervisor: Dr. Li Zhang School of Biotechnology and Biomolecular Science Faculty of Science University of New South Wales September 2015 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Lee First name: Hoyul Other name/s: Leo Abbreviation for degree as given in the University calendar: PhD School: The School of Biotechnology and Biomolecular Science Faculty: Faculty of Science Title: Elucidation of enteric virulence of Campylobacter concisus Abstract Campylobacter concisus is a Gram-negative mobile oral bacterium that has been shown to be associated with human inflammatory bowel disease. Various experiments were conducted in this PhD project, aiming to elucidate the enteric virulence of C. concisus. The growth of oral C. concisus strains under different atmospheric conditions was examined. It was found that 92 % of strains grew under anaerobic conditions without H2. However, none of the strains grew under microaerobic conditions without H2. The presence H2 greatly increased the growth of C. concisus under anaerobic conditions and enabled C. concisus to grow under microaerobic conditions. The H2 had no effects on the expression of a number of putative virulence factors in C. concisus. The effects of formate and fumarate, on the production of H2S in oral C. concisus were investigated. Supplementation of formate and fumarate significantly increased the positivity of H2S production. In addition, the fumarate significantly increased C. concisus growth. Bioinformatics analysis was conducted to search for potential virulence factors encoded by prophages. Four prophage elements were identified in the genome of C. concisus strain 13826 with putative attachment sites overlapping with tRNA. Each prophage elements contained a novel Xer phage integrase. It was found that CON_phi2 prophage encodes a Zot protein, and CON_phi3 encodes a Zot-like protein. Moreover, the phylogenetic analysis showed the horizontal gene transfer between Campylobacter species. Monoclonal antibody to C. concisus Zot was produced and verified. The impact of bile on the expression of Zot was examined. It was found that a full length and a cleaved fragment of Zot were released from C. concisus in the presence of ox bile. Whether C. concisus strains affect actin in the intestinal epithelial cell line, Caco2 cells, was examined. Some C. concisus strains significantly reduced the levels of β-actin and caused the redistribution of F-actin in Caco2 cells. These findings show that the enteric virulence of C. concisus is determined by bacterial, host and environmental factors. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). 18 March 2016 …………………………… …………………………… …………………………… Signature Witness signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS Originality statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed Date 18 March 2016 i Copyright statement ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied will apply for a partial restriction of the digital copy of my thesis or dissertation.’ Signed Date 18 March 2016 Authenticity statement ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed Date 18 March 2016 ii Acknowledgements The completion of this PhD project consumed a huge amount of effort on the part of not only myself but also my laboratory colleagues. I would like to acknowledge those who have helped me, and show my gratitude towards them. Studying in another country was probably one of the best decisions that I have ever made. This experience has positively influenced my life as I was able to improve my scientific knowledge, communication skills, my grasp of the English language and culture and perhaps most importantly, I became independent. This experience allowed me to meet a great number of people in Australia including; teachers, friends, families, and academics. The accumulation of all these experiences has made these past 10 years very enjoyable and beneficial. First of all, I would like to acknowledge each family member. I thank my mother, Sung- Yun Cho, who continuously encouraged my studies overseas with her heartfelt pray. I thank my father, Jong-Ho Lee, who inspired me to follow my dreams. I also thank my beloved sister, Seon-Woo Lee, who always refreshed my mind with funny jokes. Lastly, I thank my family for the financial support. I would like to express great thanks to my supervisor, Dr. Li Zhang who has offered me a valuable opportunity of doing my PhD research project in her laboratory. I would not have been able to complete my PhD without her supervision, scientific knowledge and experience. Her patience and enthusiasm makes her a fantastic mentor for young scientific researchers and students. I am grateful to her for making this experience possible. I would also like to thank associate Professor Mark Tanaka and Dr. Wallace Bridge for being members of my panel. Their in-depth comments and constructive criticism in regards to my PhD project. I found their annual progress reviews invaluable. I am also grateful to Dr. Sophie Octavia from associate Professor Ruiting Lan's laboratory, Mrs. Iveta Slapetova from BMIF, Dr. Ling Zhong from BMSF, Mr. Chris Brownlee from BRIL, as well as the staff members from the Ramaciotti Centre for their advice and technical support for RT-PCR, confocal microscopy, LC/MS/MS analyses, flow cytometry analyses, and gene sequencing respectively. iii I would also like to give sincere thanks to my colleagues in my laboratory who have become a team for me; Yazan, Viki, Lisa, Yesing, Omar, Mona, Rena, Connie, Sheryl, Nick, Fang, William, Sandra, Leslie, and Ece. They have all worked with me during my PhD studies. I would especially like to give thanks to my two good friends Yazan Ismail and Vikneswari Mahendran, who have helped me since I first joined this team. Lastly, I would like to thank my good friends, especially Hwang and Choi, in Australia who have been a continuous source of encouragement for me during my PhD journey. I would like to extend my gratitude to my family, supervisor, academic staff, colleagues and friends for their kind cooperation and encouragement, which have been a significant source of comfort in my completion of my PhD. iv Publications and conference proceedings Publications during PhD Ma R, Sapwell N, Chung HK, Lee H, Mahendran V, Leong RW, Riordan SM, Grimm MC and Zhang L (2015) Investigation of the effects of pH and bile on the growth of oral Campylobacter concisus strains isolated from patients with inflammatory bowel disease and controls. Journal of medial microbiology. April 64: 438-445. Doi: 10.1099/jmm.0.000013 Lee H, Ma R, Grimm MC, Riordan SM, Lan R, Zhong L, Raftery M, Zhang L (2014) Examination of the anaerobic growth of Campylobacter concisus strains. International Journal of Microbiology. Vol2014. Doi: 10.1155/2014/476047 Zhang L, Lee H, Grimm MC, Riordan SM, Day AS, Lemberg DA (2014) Campylobacter concisus and inflammatory bowel disease.