THE ROLES OF OXYGEN AND DISULFIDE REDUCTASES IN THE PHYSIOLOGY OF CAMPYLOBACTERALES Nadeem O Kaakoush A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Medical Sciences Faculty of Medicine The University of New South Wales Sydney, Australia 2008 “Science is a wonderful thing if one does not have to earn one's living at it.” - Albert Einstein ACKNOWLEDGEMENTS This thesis would not have been possible without the contributions of many people who have helped and supported me throughout the various stages of this candidature. I must thank my supervisor, Prof. George L Mendz, for giving me the opportunity to carry out my PhD degree in his laboratory at UNSW, and for his support throughout the years. I hope this quest was as rewarding for you as it was for me. You have taught me many valuable “tricks of the trade”; I hope we can continue to pursue not only a scientific collaboration together but also a good friendship. I’d like to thank Prof. Hazel Mitchell for providing support, especially the necessary lab space, during the final stages of my candidature. To Edward, Megan, Arinze, Phil, Justin, Bernice, Zsuzsanna, Johnny, Alfred and Sophie fellow warriors of science, thank you for your friendship and support throughout the years, and best of luck in the future. To A/Prof. Hilde De Reuse at the Institut Pasteur, thank you for allowing me to join your team and work in your lab for four months during my candidacy. To Damien Leduc, for your help and support in the lab and your countless translations of French to English; and to the rest of the group, especially Kristine and Marie for making me feel like I was part of the team. On a more personal note, thank you to Ali in Orlando, Adeline in Sydney, Amer and Ziad in Beirut, and Hussein and Jana in Dubai for staying in touch all this time and for their concern about my well-being. Finally, a special thank you to my sisters Nina and Nada, my Mum and Dad– you know how much you mean to me. ABSTRACT This work has studied several aspects of the physiology of the animal-colonizing species Campylobacter jejuni, Helicobacter pylori, Wolinella succinogenes and Arcobacter butzleri. C. jejuni and H. pylori were found to be obligate microaerophiles and W. succinogenes an anaerobe. A. butzleri was found to be an aerobe able to grow anaerobically. Comparative analyses of the responses of C. jejuni, H. pylori and W. succinogenes to various oxygen concentrations were investigated using transcriptomics and genes differentially expressed at higher oxygen concentrations were identified. At the time of this study no microarrays were available for A. butzleri. These comparative studies provided a better understanding of bacterial adaptation to and interaction with their environment. Several enzymes involved in oxireduction processes, including disulfide reductases, were upregulated under oxidative stress. Disulfide reductases of host-colonizing bacteria are involved in the expression of virulence factors, resistance to drugs, and elimination of toxic compounds. CXXC and CXXC-derived motifs are present in the active sites of disulfide reductases and are essential for the catalysis of these redox reactions. Large-scale genome analyses of 281 prokaryotes identified CXXC and CXXC-derived motifs in each microorganism. The total number of these motifs showed correlations with genome size and oxygen tolerance of the prokaryotes. Specific bioinformatic analyses served to identify putative disulfide reductases in the four Campylobacterales species. The project investigated the involvement of these enzymes in resistance to the antibiotic metronidazole, cadmium detoxication and pathogenesis. The activities of disulfide reductases were modulated by the presence of metronidazole, and its reduction was inhibited by the presence of disulfide reductase substrates. In addition, proteins involved in oxireduction of the low redox potential ferredoxin were downregulated in metronidazole resistant strains, suggesting that ferredoxin is involved in the resistant phenotype. Cellular processes and pathways regulated under cadmium stress included fatty acid biosynthesis, protein biosynthesis, chemotaxis and mobility, the tricarboxylic acid cycle, protein modification, redox processes and heat shock response. Notably, the data provided evidence for a role of oxireduction processes in the development of metronidazole resistance and the detoxication of cadmium. Furthermore, a method was developed to identify thiol disulfide oxidoreductases in the four Campylobacterales. The results suggested that H. pylori contained a novel disulfide bond formation system. Investigation of their potential involvement in virulence or colonization indicated that the putative thiol disulfide oxidoreductases HP0231 and HP0595 are related to the colonization efficiency of H. pylori. Finally, the only known disulfide reduction system in Campylobacterales, the thioredoxin system, was investigated in more detail. Phylogenetic analyses of the thioredoxin reductases TrxB1 and TrxB2 of the four bacteria were performed. The phylogenetic features of the TrxB2 suggested a special role for this enzyme in the physiology of these bacteria; thus, the enzyme was investigated further in H. pylori. TrxB2 was found to be an NADPH reductase, possibly involved in important oxireduction processes within the cell. CERTIFICATE OF ORIGINALITY I hereby declare that this submission is my own work, and to the best of my knowledge materials previously published or written by another person have been properly acknowledged. 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 to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged. Nadeem O Kaakoush PUBLICATIONS JOURNAL ARTICLES N. O. Kaakoush and G. L. Mendz (2005) Helicobacter pylori Disulphide Reductases: Role in Metronidazole Reduction. FEMS Immunology and Medical Microbiology 44: 137-142. N. O. Kaakoush and G. L. Mendz (2006) Involvement of disulphide reductases in the response of Campylobacter jejuni to cadmium stress. Metal Ions in Biology and Medicine (Eds. Collery P., Morais P. V., Alpoim M. C.) Vol. 9 John Libbey & Co., Montrouge, France. S. Bury-Moné, N. O. Kaakoush, C. Asencio, F. Mégraud, A. Labigne, H. De Reuse and G. L. Mendz (2006) Is Helicobacter pylori a true microaerophile? Helicobacter 11: 296- 303. N. O. Kaakoush, Z. Kovach and G. L. Mendz (2007) Potential Role of Thiol:disulfide Oxidoreductases in the Pathogenesis of Helicobacter pylori. FEMS Immunology and Medical Microbiology 50: 177-183. N. O. Kaakoush, T. Sterzenbach, W. G. Miller, S. Suerbaum and G. L. Mendz (2007) Identification of Disulphide Reductases in Campylobacterales: a bioinformatics investigation. Antonie van Leeuwenhoek 92: 429-441. N. O. Kaakoush, W. G. Miller, H. De Reuse, G. L. Mendz (2007) The oxygen requirement and tolerance of Campylobacter jejuni. Research in Microbiology 158: 644- 650. N. O. Kaakoush and G. L. Mendz (2008) Responses of four Campylobacterales to cadmium stress. Metal Ions in Biology and Medicine (Eds. Collery P., Morais P. V., Alpoim M. C.) Vol. 10 John Libbey & Co., Montrouge, France. N. O. Kaakoush, M. Raftery and G. L. Mendz (2008) Molecular responses of Campylobacter jejuni to cadmium stress. FEBS Journal. (Accepted). JOURNAL ARTICLES IN PREPARATION N. O. Kaakoush, C. Baar, E. M. Fox, J. MacKichan, S. C. Schuster and G. L. Mendz (2008) Comparative analysis of the response of three Campylobacterales species to different oxygen tensions. International Journal of Biochemistry and Cell Biology. N. O. Kaakoush, C. Asencio, M. Raftery, F. Mégraud and G. L. Mendz (2008) A redox basis for metronidazole resistance in Helicobacter pylori. Antimicrobial Agents and Chemotherapy. N. O. Kaakoush and G. L. Mendz (2008) Inhibition of disulfide reductases as a therapeutic strategy. Current Enzyme Inhibition. CONFERENCE PROCEEDINGS N. O. Kaakoush and G. L. Mendz (2008) Responses of four Campylobacterales to cadmium stress. Metal Ions in Biology and Medicine 10th International Symposium. Corsica, France. Oral Presentation. N. O. Kaakoush (2007) Molecular responses of Campylobacter jejuni to cadmium stress. UMR INRA/ENV SECALIM, l'Ecole Nationale Veterinaire de NANTES. Nantes, France. Invited talk. N. O. Kaakoush, C. Asencio, F. Mégraud and G. L. Mendz (2007) A redox basis for metronidazole resistance in Helicobacter pylori. European Helicobacter Study Group XX International Workshop. Istanbul, Turkey. Oral Presentation. N. O. Kaakoush, M. Raftery and G. L. Mendz (2007) Molecular responses of Campylobacter jejuni to cadmium stress. 14th International Workshop on Campylobacter, Helicobacter and Related Organisms. Rotterdam, The Netherlands. (ESCMID grant winner). N. O. Kaakoush, E. M. Fox, J. MacKichan and G. L. Mendz (2006) Molecular responses of Helicobacter pylori to changes in oxygen tension. European Helicobacter Study Group XIX International Workshop. Wroclaw, Poland. Oral presentation. N. O. Kaakoush and G. L. Mendz (2006) Involvement of disulphide reductases in the response of Campylobacter jejuni to cadmium stress. Metal Ions in Biology and Medicine 9th International Symposium. Lisbon, Portugal. Oral presentation. N. O. Kaakoush and G. L. Mendz (2005) Disulphide reductases of Campylobacterales: Are they involved