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Investigation Into the Major Surface Proteases of African Trypanosomes

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Investigation Into the Major Surface Proteases of African Trypanosomes Investigation into the Major Surface Proteases of African Trypanosomes by Alexandré Marie Chaplin Delport BSc. (Hons) Biochemistry (cum laude) Submitted in fulfilment of the academic requirements for the degree of Master of Science in Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg. 2016 i Preface The experimental work described in this dissertation was carried out in the School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg, from January 2014 to October 2016, under the supervision of Professor THT Coetzer. The studies represent original work by the author and have not otherwise been submitted in any other form to another University. Where use has been made of the work of others, it has been duly acknowledged in the text. / /2017 Miss AMC Delport As the candidate’s supervisor I agree to the submission of this dissertation. / /2017 Prof. THT Coetzer ii Declaration - Plagiarism I, Alexandré Marie Chaplin Delport, declare that: 1. The research reported in this dissertation, except where otherwise indicated, is my original research. 2. This dissertation has not been submitted for any degree or examination at any other university. 3. This dissertation does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This dissertation does not contain other persons' writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written but the general information attributed to them has been referenced b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This dissertation does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the dissertation and in the references sections. / /2017 Miss AMC Delport iii Abstract The unicellular parasite of the genus Trypanosoma infects a number of mammalian species including livestock and humans. In sub-Saharan Africa three main parasitic species cause disease: Trypanosoma brucei, T. congolense and T. vivax. The lack of sensitive diagnosis and increased drug resistance leaves an avenue in trypanosome research for exploring novel virulence factors as diagnostic and chemotherapeutic agents. The work reported in this dissertation involved investigation of the identified virulence factor, the Major Surface Protease (MSP), of African trypanosomes. The MSPs comprise a group of metalloproteases which have been found in over 13 Leishmanian species, T. cruzi, T. brucei and T. congolense as well as other Kinetoplastids. In this study, putative M8 metalloprotease sequences were also identified in the T. vivax genome. These putative sequences were grouped into four classes of protein, TvMSP-A, -C, -D and -E by phylogenetic comparison with other MSPs. Three-dimensional modelling showed high structural identity with leishmanolysin from Leishmania major. The T. vivax MSP sequences were used, in conjunction with T. brucei and T. congolense sequences, to select immunogenic peptide regions to produce anti-peptide antibodies. Three peptides were selected with the intention to 1) detect both TbMSP-B and TcoMSP-B (peptide Tb/TcoMSP:303-314, cross-species), 2) detect only TbMSP-C (peptide TbMSP:400-412) and 3) detect only TvMSP-C (peptide TvMSP:686-697). They were used to generate two types of detection molecules: complete IgY anti-peptide antibodies and single chained fragments (scFvs), with the capability to detect the peptide in an ELISA format. Single scFv expressing E. coli colonies were successfully selected and shown to detect two (Tb/TcoMSP:303-314 and TbMSP:400-412) of the three peptides. The anti-peptide antibodies, produced in chickens, were used to successfully detect native MSP within T. brucei and T. congolense parasite lysates; however, cross-reactivity between species was seen. The T. brucei MSP-C class of protease was successfully cloned, expressed and purified, although, numerous truncated proteins and gene mutations occurred [truncated (t)TbMSP-C]. The expression constructs rTbMSP-C and rTcoMSP-C were hence synthesised. These two enzymes were successfully expressed and purified and were shown to form high molecular weight multimers. Furthermore, the enzymes were able to cleave the peptide substrate H-Suc-Leu-Tyr-AMC with acidic pH optima and activity was inhibited with metalloprotease inhibitors, EDTA and 1,10 phenanthroline. iv The successful detection of rTcoMSP-C by T. congolense infected cattle sera was also observed. tTbMSP-C, rTbMSP-C and rTcoMSP-C were detected with the chicken anti- peptide antibodies and it was again found that these antibodies cross-reacted with different species MSPs. The high identity shared between MSPs from all Trypanosoma species made selecting species-specific antibodies difficult. Further work to detect native MSP-C protease within infected sera or blood would give a definitive conclusion of its use in diagnostics. Moreover, antibodies that detect just T. brucei and T. congolense still need to be produced. Preliminary activity assays were performed on rTbMSP-C and rTcoMSP-C but, additional research on the kinetics of these proteases is still needed. In summary, it was shown that MSPs have the potential to be novel diagnostic markers especially for cross-trypanosomal species detection. Furthermore, activity of rTbMSP-C and rTcoMSP-C has substrate cleavage specificity and pH optima that are comparable with leishmanolysin. v Acknowledgements I would like to extend my gratitude and thanks to the following people and institutes: Firstly, a very special thank you to my supervisor, Prof. Coetzer for all her help, assistance, support and guidance in completing my MSc (especially reading and correcting this dissertation). We finally got there!! I extend this thank you to the other academics within our department, especially Prof Goldring, Prof. Niesler and Dr Hewer. To my colleagues in Lab 44 for all the support and laughs over the three years. A special thanks to Lauren Eyssen whom without, the laboratory would not run as it does. To the other laboratories within out department of biochemistry, thank you for your constant friendliness, help and advice. I would also like to thank everyone who forms the driving force of our department and university: the technicians, financial division and cleaning staff. All of you make it pleasant and easy to work at UKZN. I would like to thank the National Research Foundation (NRF) for funding my three years of study, without which this degree would have not been possible. I would like to thank the University of KwaZulu-Natal for their financial support in my first year of masters. I dedicate this dissertation to my dad who taught me to always be curious and that, without mistakes you can never learn. To my mom who taught me to never give up and always believe in myself. Above all I dedicate this to my beautiful sister Jodi, who reminds me every day that things are never as bad as they seem, to always follow my heart and in her strength she gives me strength. You are forever my inspiration. vi Contents Contents Preface .................................................................................................................................. ii Declaration - Plagiarism ..................................................................................................... iii Abstract ............................................................................................................................... iv Acknowledgements ............................................................................................................ vi Contents ............................................................................................................................. vii List of Figures ..................................................................................................................... xi List of Tables ..................................................................................................................... xiv Abbreviations ..................................................................................................................... xv Chapter 1 Literature Review: The Trypanosome Parasites and their Major Surface Proteases .............................................................................................................................. 1 1.1 History of Trypanosomiasis ......................................................................................... 1 1.2 Classification of Trypanosomes ................................................................................... 2 1.3 Biology of the Trypanosome ........................................................................................ 5 1.3.1 The Genome of African Trypanosomes ................................................................ 5 1.3.2 Morphology of Trypanosomes .............................................................................. 5 1.3.3 Antigenic Variation ............................................................................................... 7 1.4 Life Cycle of the African Trypanosome ........................................................................ 7 1.5 African Trypanosomiasis ............................................................................................. 9 1.5.1
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