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Dale Liebenberg INVESTIGATING A BIR-CONTAINING PLASMODIUM FALCIPARUM PROTEIN AND IDENTIFYING ITS BINDING PARTNERS Dale Liebenberg The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at are those of the author and are not necessarily to be attributed to the NRF. A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2018 DECLARATION I, Dale Liebenberg, declare that this Thesis is my own, unaided work. It is being submitted for the degree of Doctor of Philosophy at the University of the Witwatersrand, Johannesburg. It has not been submitted for any other degree or examination at any other University. Dale Liebenberg 1st day of March 2018 ii Declaration In memory of my ever faithful shadow Patches 2012 – 2017 iii RESEARCH OUTPUTS Submitted Manuscript Building a Protein Interaction Network for a P. falciparum BIR-Containing Protein using Phage Display Technology Dale Liebenberg and Thérèsa L. Coetzer - submitted to Parasitology (IF 2.713) International Conferences Identification of Binding Partners for a Putative P. falciparum Inhibitor of Apoptosis (IAP) Protein Dale Liebenberg and Thérèsa L. Coetzer Oral presentation at: • The 6th Multilateral Initiative on Malaria (MIM) Pan-African Malaria Conference, Durban, KwaZulu-Natal, South Africa, October 2013 Study of a Putative P. falciparum Inhibitor of Apoptosis (IAP) Protein Dale Liebenberg, Pierre M. Durand and Thérèsa L. Coetzer Poster presentation at: • The 23rd South African Society of Biochemistry and Molecular Biology (SASBMB) / Federation of African Societies of Biochemistry and Molecular Biology (FASBMB) Congress, Drakensburg, KwaZulu-Natal, South Africa, January 2012 Local Conferences Using CRISPR-Cas9 to Manipulate the Genome of P. falciparum Dale Liebenberg and Thérèsa L. Coetzer Oral presentation at: • The 25th South African Society of Biochemistry and Molecular Biology (SASBMB) Congress, East London, Eastern Cape, South Africa, July 2016 iv Research Outputs Poster presentations at: • The Faculty of Health Sciences Research Day and Postgrad Expo, University of the Witwatersrand, Johannesburg, Gauteng, South Africa, September 2016 - awarded Best Student Poster Presentation in the Molecular and Comparative Biosciences category • The South African Medical Research Council’s Office of Malaria Research (MOMR) 2016 Malaria Research Meeting, University of Pretoria, Pretoria, Gauteng, South Africa, July 2016 Can Protein-Protein Interactions Involved In Apoptosis Be Potential Drug Targets? Dale Liebenberg and Thérèsa L. Coetzer Oral presentation at: • The South African Medical Research Council’s Office of Malaria Research (MOMR) 2015 Malaria Research Meeting, Durban, KwaZulu-Natal, South Africa, August 2015 Novel Binding Partners for a P. falciparum Inhibitor of Apoptosis (IAP) Protein Dale Liebenberg and Thérèsa L. Coetzer Oral presentations at: • The Faculty of Health Sciences Research Day and Postgrad Expo, University of the Witwatersrand, Johannesburg, Gauteng, South Africa, September 2014 • The Molecular Biosciences Research Thrust (MBRT) Research Day, University of the Witwatersrand, Johannesburg, Gauteng, South Africa, December 2014 - awarded 3rd prize in the Oral Category Poster presentation at: • The 24th South African Society of Biochemistry and Molecular Biology (SASBMB) Congress, Worcester, Western Cape, South Africa, July 2014 Identification of Binding Partners for a Putative P. falciparum Inhibitor of Apoptosis (IAP) Protein Dale Liebenberg and Thérèsa L. Coetzer Poster presentation at: • The Molecular Biosciences Research Thrust (MBRT) Research Day, University of the Witwatersrand, Johannesburg, Gauteng, South Africa, December 2013 v Research Outputs Expression of a Putative P. falciparum Inhibitor of Apoptosis (IAP) Protein Dale Liebenberg and Thérèsa L. Coetzer Oral presentation at: • The Faculty of Health Sciences Research Day and Postgrad Expo, University of the Witwatersrand, Johannesburg, Gauteng, South Africa, September 2012 Poster presentation at: • The Molecular Biosciences Research Thrust (MBRT) Research Day, University of the Witwatersrand, Johannesburg, Gauteng, South Africa, December 2012 Study of a Putative P. falciparum Inhibitor of Apoptosis (IAP) Protein Dale Liebenberg, Pierre M. Durand and Thérèsa L. Coetzer Poster presentation at: • The Molecular Biosciences Research Thrust (MBRT) Research Day, University of the Witwatersrand, Johannesburg, Gauteng, South Africa, December 2011 vi Research Outputs ABSTRACT The majority of the worldwide malaria deaths are caused by the Plasmodium falciparum parasite and unfortunately parasite strains are emerging that are resistant to not only artemisinin, but also the partner drugs used in current antimalarial combination therapy. The intraerythrocytic P. falciparum life stage is characterised by exponential, asexual proliferation that could cause the premature death of the human host before the sexual gametocytes have had enough time to develop and be taken up by the mosquito vector to continue its lifecycle. It is hypothesised that P. falciparum maintains its population at a level low enough to allow for the transmission of these gametocytes by using a form of regulated cell death (RCD). The molecular members of this cell death pathway are currently unclear, but a putative P. falciparum inhibitor of apoptosis protein (PfIAP; PF3D7_0519600) has previously been identified. Metazoan IAP proteins play anti-apoptotic roles in cells by interacting and inhibiting pro-apoptotic caspases, but also perform other functions. Analysis of the PfIAP protein using bioinformatic tools revealed that it contains one conserved baculoviral IAP repeat (BIR) domain and that this P. falciparum BIR domain is structurally similar to the BIR domains of various human IAP proteins. mRNA extracted from asexual P. falciparum parasites was used to construct a biotin-tagged phage display library, which was used in biopanning experiments with two regions of the PfIAP protein, expressed as recombinant GST-tagged proteins. Four binding partners were identified for the N-terminal BIR domain of the protein, while two proteins were identified as interacting partners for the C-terminal region of PfIAP. Of these, a double C2-like domain-containing protein (PfDOC2) and the high molecular weight rhoptry protein 3 (PfRhopH3) were expressed as recombinant His-tagged proteins and verified as PfIAP binding partners by in vitro binding assays. Transgenic P. falciparum parasites were generated expressing a GFP- tagged PfIAP BIR domain, which localised to the cytoplasm under both normal and high temperature conditions which mimic febrile malaria, a physiological trigger of RCD. Knockout experiments of the pfiap gene using the CRISPR-Cas9 genome editing tool suggested that this gene could be essential for the survival of asexual P. falciparum parasites. This study offers the first details of a putative P. falciparum inhibitor of apoptosis protein and suggests that it could have non-apoptotic roles in the parasite, given the diverse functions of the binding partners that comprise the PfIAP protein-protein network. vii Abstract ACKNOWLEDGEMENTS This thesis would not have been possible without the help of an innumerable number of people. Trying to name everyone involved would be an impossible task and I would invariably do someone a disservice by leaving them off this list. The following people though, deserve a very special thank you: Prof. Thérèsa L. Coetzer for her guidance and enthusiasm throughout this project. Dr. Sonja Lauterbach and Dr. Kuben Naidoo for their invaluable assistance and for sharing their formidable knowledge with me. Dr. Alisje Churchyard, Dr. Warren Vieira, Melanie Wepener, Serena Shunmugam, Dr. Belinda Bezuidenhout, Dr. Dewaldt Engelbrecht and the other members of the Plasmodium Molecular Research Unit (PMRU) for their camaraderie and constant encouragement throughout this adventure. Dr. Tristan Scott for helping me with the CRISPR-Cas9 vectors, which were kindly provided by Dr. Mehdi Ghorbal and Dr. Jose-Juan Lopez-Rubio (Institut Pasteur, France). Most importantly though, I have to thank my parents who have supported me unconditionally over these many years. Funding None of this work would have been possible without contributions from the following funding bodies: National Health Laboratory Service (NHLS) National Research Foundation (NRF) University of the Witwatersrand viii Acknowledgements “The time will come when diligent research over long periods will bring to light things which now lie hidden. A single lifetime, even though entirely devoted to the sky, would not be enough for the investigation of so vast a subject... And so this knowledge will be unfolded only through long successive ages. There will come a time when our descendants will be amazed that we did not know things that are so plain to them... Many discoveries are reserved for ages still to come, when memory of us will have been effaced. Our universe is a sorry little affair unless it has in it something for every age to investigate... Nature does not reveal her mysteries once and for all.” ― Seneca, Natural Questions Book 7, c. first century ETHICS CLEARANCE Ethics clearance was granted by the University of the Witwatersrand Human Research Ethics Committee (Medical). Clearance certificate number: M1703102. ix Ethics Clearance TABLE OF
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