Mechanisms of Predation Resistance and the Effect Ofpredation on the Pathogenicity of Marine Bacteria
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Mechanisms of predation resistance and the effect of predation on the pathogenicity of marine bacteria Parisa Noorian A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales Sydney, Australia July 2018 Thesis/Dissertation Sheet Surname/Family Name :Noorian Given Name/s :Parisa Abbreviation for degree as give in the University calendar :Ph.D. Faculty :Science School :BABs :Mechanisms of predation resistance and the effect of predation on Thesis Title the pathogenicity of marine bacteria Abstract 350 words maximum: (PLEASE TYPE) Vibrio spp. are an autochthonous inhabitant of coastal marine environments where predation by protozoa is a shaping force leading to the evolution of antiprotozoal mechanisms that may also function as virulence factors in animal and human hosts. Vibrio cholerae and Vibrio vulnificus were used in this study in order to further elucidate bacterial adaptations against different model protozoa and the potential role of these antiprotozoal factors in providing fitness in the environment and in a host. The transcriptomic profile of established V. cholerae biofilms to predation by the amoeba, Acanthamoeba castellanii, was investigated. Genes that were significantly differentially expressed between grazed and ungrazed cultures were identified. Tyrosine metabolic genes were among the down-regulated transcripts in the grazed population. Homogentisic acid is the main intermediate of the L-tyrosine catabolic pathway, and is known to auto-oxidize, leading to the formation of the pigment, pyomelanin. Indeed, a pigmented mutant, disrupted in the gene encoding homogentisate 1, 2 - dioxygenase (hmgA) was more resistant to grazing by A. castellanii than the wild type. Grazing resistance of V. vulnificus of different genotypes and places of isolation were evaluated using the protozoan predators, Tetrahymena pyriformis and A. castellanii, but no significant correlation was found in relation to grazing resistance. However, an oyster isolate, V. vulnificus Env1, showed significant grazing resistance and toxicity towards T. pyriformis. The whole genome sequence of Env1 was completed, annotated and compared to grazing sensitive strains to identify Env1 unique features. Further studies revealed one of the antiprotozoal mechanisms of Env1 was secreted and iron-dependent. The transcriptomic profile of V. vulnificus Env1 under iron-replete and -deplete conditions was characterised. A master virulence regulator, arcA, was up-regulated when iron was readily available and an arcA mutant, showed a significant decrease in grazing resistance. Therefore, ArcA is a novel global regulator controlling the grazing resistance of V. vulnificus. In summary, this project revealed new defence systems against protozoan grazing expressed by V. cholerae and V. vulnificus that also play dual roles in environmental survival and pathogenicity, accentuating how protozoan grazing drives the evolution of pathogenicity in bacteria in the environment. 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). ……………………………………………………… ……………………………………..……………… ……………..……………… 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: II | P a g e 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 …………………………………………….............. III | P a g e 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 ……………………………………………........................... 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 ……………………………………………........................... IV | P a g e Acknowledgements I would like to thank to all those who made the completion of this work possible. First and foremost, I wish to express my immense gratitude to my supervisor Associate Professor Diane McDougald; without whom none of this would have been possible. Thank you for everything. I could have not imagine a better mentor. I would like to extend my appreciation to Professor Ruiting Lan for his generous support and accepting me as his student. I would like to express my gratitude to Professor Liz Harry for her continuous and warm support. It was a great experience to work in ithree institute. I would also like to thank Associate Professor Scott Rice, for all the help, advice and generous support. Many people helped me along the way and I want to thank them for all their help, support, interest and valuable hints. Especially, I am indebted to thank Dr. Shuyang (Garfy) Sun. You are one of the first people who trained me back in my Mphil when I had just started real research and continued to support and help me in developing real research to this day. I would like to thank Gustavo Espinoza for all those long talks on science, experiments and life. I would like to express my heart-felt gratitude to Nasim Shah Mohammadi. You have been a great friend and support along the way. I would like to thank Dr. Sean booth for collaboration on extraction of the toxic factor even if it still needs work. I received generous support from Dr. Florentin Constancias and would like say thank you for all your help in RNA-seq. We traded so many emails to make it work. I am happy to acknowledge Dr. Zhilliang Chen, Dr. Tonia Russel and Dr. Nandan Deshpande for all the help in technical support. There are not enough words to describe how much I am indebted to my parents. You have continuously supported me towards my goals and have provided me with everything in life. There is no way for me to thank you enough. I would like to specially thank my brother Farzad. You are my pillar of support and strength, without you, I would have been lost long ago. V | P a g e Abstract Vibrio spp. are an autochthonous inhabitant of coastal marine environments where predation by protozoa is a shaping force leading to the evolution of antiprotozoal mechanisms that may also function as virulence factors in animal and human hosts. Vibrio cholerae and Vibrio vulnificus were used in this study in order to further elucidate bacterial adaptations against different model protozoa and the potential role of these antiprotozoal factors in providing fitness in the environment and in a host. The transcriptomic profile of established V. cholerae biofilms to predation by the amoeba, Acanthamoeba castellanii, was investigated. Genes that were significantly differentially expressed between grazed and ungrazed cultures were identified. Tyrosine metabolic genes were among the down-regulated transcripts in the grazed population. Homogentisic acid is the main intermediate of the L-tyrosine