A pan genome reverse vaccinology approach to prevent Streptococcus agalactiae infection in farmed tilapia Minami Kawasaki BSc Honours A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in {2018} School of Biological Sciences 1 Abstract Streptococcus agalactiae (Group B Streptococcus; GBS) is a Gram-positive pathogenic bacterium that causes disease in a wide range of animals including fish. Outbreaks of GBS in farmed fish have substantial economic impacts on the aquaculture industry, especially in tilapia production, a staple food product in tropical and subtropical low- and middle-income countries and the second most widely farmed fish after carp. Although vaccination can effectively prevent disease caused by GBS in fish it often fails after relatively short periods of use. Capsular polysaccharide (CPS) is the major protective antigen in GBS vaccines and a critical virulence factor. There are currently ten CPS serotypes of GBS and, whilst vaccination against one serotype confers protection in fish, it is not cross-protective against other serotypes. In addition, under the strong selective pressure of vaccination-driven adaptive immunity, evolution of novel serotypes can occur. To improve the serotype cross- protective efficacy of GBS vaccines for aquaculture, I employed a pan-genome reverse vaccinology approach to design candidate serotype-independent protective vaccines based on conserved proteins in fish-pathogenic GBS serotypes. Reverse vaccinology exploits recent advances in genomics to predict antigens in the genome sequence. Reverse vaccinology combined with pan-genome can identify the antigens that are conserved across a diversity of strains of a pathogen. To identify potential vaccine candidate antigens, a pan-genome was constructed for GBS comprising 82 genomes including representative isolates from wild marine fish and stingrays, farm and companion animals and from human clinical cases in Australia, along with isolates from farmed tilapia in Honduras and representative genomes from all current serotypes available via the NCBI Genbank database. Core genome single nucleotide polymorphism 2 (SNP) analysis indicated dissemination of the aquatic host-adapted ST-261 lineage in wild Australian fish from an ancestral tilapia strain, which is congruent with several introductions of tilapia into northern Australia from Israel and Malaysia during the 1970s and 1980s. Aquatic serotype Ib GBS strains have lost many virulence factors during adaptation, but six adhesins, named ST-261 adhesins, were well-conserved across the aquatic isolates and might be critical for virulence in fish and good targets for vaccine development. Three of six ST-261 adhesins were also conserved across all strains including terrestrial isolates and were chosen as vaccine candidates. Expressions of ST-261 adhesin genes were inversely co-regulated with CPS expression dependent upon carbon source and metal ions in the medium. However, gene transcription was not reflective of surface expression as serum antibodies derived in tilapia against recombinant adhesins did not detect similar variation in the level of ST-261 adhesins in whole cell ELISA or Western blot. This may be due to post- transcriptional processing causing a disconnect between gene and protein expression. To evaluate the efficacies of recombinant ST-261 adhesins as vaccines in tilapia, a vaccination and challenge experiment was conducted. All recombinant ST-261 adhesin vaccines resulted in strong antibody response detected by ELISA against the recombinant adhesins. However, no protection from disease was elicited by any of the recombinant vaccines in the challenge model. This may be due to antigenic masking by CPS. It may also be that adhesins are not expressed during the critical stages of proliferation in the host and are required at the mucosal surfaces during pathogen entry via the gastrointestinal tract. Future work should elucidate the role of these ST-261 adhesins in pathogenicity in fish, including where and when they are expressed on GBS during the infection. 3 Prevention of disease in aquaculture caused by highly antigenically diverse pathogens such as GBS might be achieved through deployment of autogenous (custom-made) vaccines supported by careful surveillance to enable incorporation of variants as they arise. Use of rapid, low cost genomics as employed in this thesis will aid in accurate surveillance and inform vaccine modification as required. Genomics may also aid in defining the epidemiological unit over which an autogenous formulation might be employed, increasing the cost-effectiveness of custom-made autogenous vaccines. In the era of “one health”, with global antimicrobial use in animal production far exceeding use in human medicine, it is critical that affordable disease prevention methods are promoted in the world’s fastest growing food production sector. 4 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis. 5 Publication included in this thesis Chapter 2: Kawasaki. M., Delamare-Deboutteville, J., Bowater, R. O., Walker, M. J., Beatson, S., Zakour, N. L. B., Barnes, A. C., 2018. Microevolution of aquatic Streptococcus agalactiae ST-261 from Australia indicates dissemination via imported tilapia and ongoing adaptation to marine hosts or environment. Applied and environmental microbiology, AEM. 00859-00818. Contributor Statement of contribution Minami Kawasaki Conceived experiments 15% (candidate) Designed experiments 70% Conducted the work and analysed the data 80% Wrote and edited paper 70% Jerome Delamare-Deboutteville Conducted the work and analysed the data 10% Wrote and edited the paper 5% Rachel Bowater Conceived experiments 5% Mark Walker Conceived experiments 10% Wrote and edited the paper 5% Andrew C Barnes Conceived experiments 40% Designed experiments 20% Conducted the work and analysed the data 10% 6 Wrote and edited paper 10% Conceived experiments 20% Nouri Ben Zakour Designed experiments 10% Wrote and edited paper 5% Scott Beatson Conceived experiments 10% Wrote and edited paper 10% 7 Submitted manuscripts included in this thesis No manuscripts submitted for publication Other publications during candidature Conference abstracts Kawasaki. M., Labrie, L., Zakour, N. L. B., Delamare-Deboutteville, J., Barnes, A. C., 2017. A conserved adhesin identified in the Streptococcus agalactiae pan-genome is a cross- protective recombinant vaccine against infection by serotype Ia and Ib in Tilapia. 4th FRDC Australian Aquatic Animal Health & Biosecurity Scientific Conference. Kawasaki. M., Labrie, L., Zakour, N. L. B., Delamare-Deboutteville, J., Barnes, A. C., 2017. A conserved adhesin identified in the Streptococcus agalactiae pan-genome is a cross- protective recombinant vaccine against infection by serotype Ia and Ib in Tilapia. 18th International Conference on Disease of Fish and Shellfish. Contributions by others to the thesis Andrew C Barnes (principle advisor) helped to conceive and design the project, supported analyse and interpret the data, and helped with thesis editing. Mark Walker (co-supervisor) helped to design the experiment to produce recombinant proteins which is the part of work in chapter 3. Nouri Ben Zakour helped to conceive and design the experiment which is part of chapter 2. Scott Beatson helped to conceive and design of some parts of experiments in chapter 2. 8 Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis Research Involving Human or Animal Subjects AEC Approval Number: SBS/398/16 Approving committee: Animal Welfare Unit UQ Research and Innovation Title: Effective vaccination of Tilapia (Oreochromis mossambicus) against Group B Streptococcus 9 Acknowledgements I particularly want to thank my main supervisor Andy Barnes who brought this exciting project to me and kept me motivated