Characterisation of a novel antinematode agent produced by the marine epiphytic bacterium Pseudoalteromonas tunicata and its impact on Caenorhabditis elegans Nor Hawani Salikin A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biological, Earth and Environmental Sciences Faculty of Science August 2020 Thesis/Dissertation Sheet Surname/Family Name : Salikin Given Name/s : Nor Hawani Abbreviation for degree as give in the University : Ph.D. calendar Faculty : UNSW Faculty of Science School : School of Biological, Earth and Environmental Sciences Characterisation of a novel antinematode agent produced Thesis Title : by the marine epiphytic bacterium Pseudoalteromonas tunicata and its impact on Caenorhabditis elegans Abstract 350 words maximum: (PLEASE TYPE) Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of antinematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytic bacteria are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising, yet underexplored source for antinematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce a number of bioactive compounds. Screening genomic libraries of P. tunicata against the nematode Caenorhabditis elegans identified a clone (HG8) showing fast-killing activity. However, the molecular, chemical and biological properties of HG8 remain undetermined. A novel Nematode killing protein-1 (Nkp-1) encoded by an uncharacterised gene of HG8 annotated as hp1 was successfully discovered through this project. The Nkp-1 toxicity appears to be nematode-specific, with the protein being highly toxic to nematode larvae but having no impact on nematode eggs. A putative carbohydrate binding module was identified at the N-terminus of Nkp-1 protein sequence which is suggested to bind to a yet unknown nematode glycoconjugate receptor. This study also provides the first insights into the mode of action of Nkp-1 and the nematode response towards its toxicity. The Nkp-1 expressing clones; HG8 and HP1 (expressing respectively either the original 13.8 kb genomic insert of HG8 or the hp1 gene only) colonised C. elegans intestine, in addition exposure to both strains and protein extracts resulted in multiple physical damages and necrosis. As a defence, C. elegans utilised its innate and associative learned avoidance behaviour to prevent contact with the Nkp-1 strains. Further I found evidence for the involvement of daf-2/daf-16 ILR and sek-1 p38_MAPK immune pathways in response to Nkp-1 exposure and the subsequent expression of genes involved in lysozyme, superoxide dismutase production and dar (deformed anal region) formation. Moreover, this study revealed the impact of different gut microbiota has on nematode survival and the resulting physical damages upon exposure to the Nkp-1. The outcome of these studies not only kickstart the development of Nkp-1 as a future antinematode drug but has re-affirmed the potential of marine epiphytic bacteria as a new source of novel antinematode drugs. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents a non-exclusive licence to archive and to make available (including to members of the public) my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known. I acknowledge that I retain all intellectual property rights which subsist in my thesis or dissertation, such as copyright and patent rights, subject to applicable law. I also retain the right to use all or part of my thesis or dissertation in future works (such as articles or books). 13 August 2020 ………………………………………………… ……….……………………...…….… 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 can be made when submitting the final copies of your thesis to the UNSW Library. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. i 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 …………………………………………….............. 13 August 2020 Date …………………………………………….............. ii INCLUSION OF PUBLICATIONS STATEMENT UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure. Publications can be used in their thesis in lieu of a Chapter if: • The candidate contributed greater than 50% of the content in the publication and is the “primary author”, ie. the candidate was responsible primarily for the planning, execution and preparation of the work for publication • The candidate has approval to include the publication in their thesis in lieu of a Chapter from their supervisor and Postgraduate Coordinator. • The publication is not subject to any obligations or contractual agreements with a third party that would constrain its inclusion in the thesis Please indicate whether this thesis contains published material or not: This thesis contains no publications, either published or submitted for ☒ publication Some of the work described in this thesis has been published and it ☐ has been documented in the relevant Chapters with acknowledgement This thesis has publications (either published or submitted for publication) incorporated into it in lieu of a chapter and the details are ☐ presented below CANDIDATE’S DECLARATION I declare that: • I have complied with the UNSW Thesis Examination Procedure • where I have used a publication in lieu of a Chapter, the listed publication(s) below meet(s) the requirements to be included in the thesis. Candidate’s Name Signature Date (dd/mm/yy) Nor Hawani Salikin 13 August 2020 iii COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents a non-exclusive licence to archive and to make available (including to members of the public) my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known. I acknowledge that I retain all intellectual property rights which subsist in my thesis or dissertation, such as copyright and patent rights, subject to applicable law. I also retain the right to use all or part of my thesis or dissertation in future works (such as articles or books).’ ‘For any substantial portions of copyright material used in this thesis, written permission for use has been obtained, or the copyright material is removed from the final public version of the thesis.’ Signed ……………………………………………........................... 13 August 2020 Date …………………………………………….............................. AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis.’ Signed ……………………………………………........................... 13 August 2020 Date …………………………………………………………………. iv ABSTRACT Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of antinematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytic bacteria are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising, yet underexplored source for antinematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce a number of bioactive compounds. Screening genomic libraries of P. tunicata against the nematode Caenorhabditis elegans identified a clone (HG8) showing fast-killing activity. However, the molecular, chemical and biological properties of HG8 remain undetermined. A novel Nematode killing protein-1 (Nkp-1) encoded by an uncharacterised gene of HG8 annotated as hp1 was successfully discovered through this project. The Nkp-1 toxicity appears to be nematode- specific, with the protein being highly toxic to nematode larvae but having no impact on nematode eggs. A putative carbohydrate binding module was identified at the N-terminus of Nkp-1 protein sequence which is suggested to bind to a yet unknown nematode glycoconjugate receptor. This study also provides the first insights into the mode of action of Nkp-1 and the nematode response towards its toxicity. The Nkp-1 expressing clones; HG8 and HP1 (expressing respectively either the original 13.8 kb genomic insert of HG8 or the hp1 gene only) colonised C. elegans intestine, in addition exposure to both strains and protein extracts