Novel Mitochondrial Metabolism in Excavate Protists Amber Carol Lynch This thesis is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Lancaster University January 2016 Abstract The Excavata super-group contains a massively diverse range of protists that inhabit a plethora of different trophic niches. Many excavates are free-living and little-studied, but there are also many examples of parasitism evolving multiple times within the group. Trypanosoma brucei is a euglenozoan parasite that is responsible for causing Human African trypanosomiasis (HAT) and Nagana in livestock. It has a complex heteroxenous life cycle that involves a variety of complex morphological and biochemical changes to the cell as it switches from a mammalian to an insect host. Within the mid-gut of its tsetse fly vector, T. brucei replicates as a procyclic trypomastigote; these cells are readily cultured and can be subjected to genetic manipulation in the laboratory. Despite the complete genome sequence being available, there are still many novel proteins within T. brucei that have yet to be assigned a function within a cell. T. brucei appears to have an extensive array of histidine phosphatases, many of which are uncharacterised (Brown, 2011). The histidine phosphatases are an ancient, ubiquitous enzyme superfamily that catalyse the dephosphorylation of a substrate, and function is critically dependent upon an active site histidine. Given that adaptation to parasitism is classically associated with metabolic stream-lining, it is intriguing that T. brucei has retained so many histidine phosphatases. Two of these histidine phosphatases appear to have arisen by gene duplication, although one appears to have degenerated and subsequently has lost the essential catalytically-active histidine. Through phylogenetic analyses and contemporary molecular characterisation techniques, I report the progress made on attempts to assign function to this paralogous gene pair, both of which localise to the mitochondria in procyclic T. brucei. My efforts to unsuccessfully delete genes encoding these histidine phosphatases from the genome of procyclic T. brucei suggests they are essential. Another excavate for which genome data is freely available is the free-living heterolobosean Naelgeria gruberi. The sequencing and annotation of the N. gruberi genome (Fritz-Laylin et al., 2010) revealed a surprising variety of anaerobic metabolic pathways potentially available to the cell, when encountering anoxic conditions. Using T. brucei as a host for heterologous expression, I tested the mitochondrial candidature of several of these N. gruberi novel proteins associated with anaerobic metabolism. Since the Heterolobosea are sister to the Euglenozoa, evidence for the expression and localisation of N. gruberi proteins in T. brucei is a good indication of where these proteins may localise within N. gruberi cells. These analyses showing mitochondrial localisation of enzymes associated with anaerobic metabolism point to a currently highly unusual repertoire of metabolic flexibility within the mitochondria of a eukaryotic heterotroph. i Declaration I declare that the content presented in this thesis is my own work, except where explicitly stated, and has not been submitted in substantially the same form for the award of a higher degree elsewhere. ii Acknowledgements I would like to kindly thank my supervisors, Dr Michael Ginger and Dr Paul McKean for all the help, guidance, support and opportunities they have provided throughout my time at Lancaster. It has been a pleasure to be a member of your labs, and I couldn’t have wished for better supervisors. I would like to give a special thanks to Dr Jane Andre and Dr Jon Moran for their invaluable help in the lab and their ongoing friendships. Thank you to Dr Fiona Benson, Dr Mick Urbaniak, Dr Derek Gatherer, Dr Jackie Parry, Janice Drinkall and Dr Steve Roberts for their help and advice, and their kind generosity with reagents and equipment. A huge thank you to some of the greatest lab friends anyone could ever wish for over the years! Gillian, Xin, Harriet, Kurimun, Laura, Hollian, you are all fabulous and are responsible for some of my best times at Lancaster… And a special thanks to Louise Kerry, my trypanosome friend. This project was partially funded by the Faculty of Health and Medicine, and this financial support was invaluable to me. However, the remaining funding was provided by my spectacular grandparents, Carol and Jerry Lynch. I wouldn’t be where I am today without you, you know how much you’ve both done for me. Thank you to you both, I love you so much. And finally thank you to my partner Dave Moran, who has been nothing but loving, patient, caring and supportive throughout these challenging years! iii Table of Contents 1. General Introduction 1 1.1 The diversity and origin of eukaryotic life 1 1.2 The radiation of eukaryotic lineages 9 1.3 Diversity of the Excavata 19 1.4 Mitochondrial diversity 25 1.5 Life cycle biology and metabolism of Trypanosoma brucei 34 1.6 Ecology of Naegleria 43 1.7 Overview of thesis results 46 2. Materials and Methods 47 2.1 Buffers and solutions 47 2.2 Antibiotic and drug stock solutions 50 2.3 Antibodies 51 2.4 Oligonucleotide primers 52 2.5 Polymerase chain reaction 53 2.6 Agarose gel electrophoresis 53 2.7 DNA ligation 54 2.8 Transformations of E. coli 54 2.9 Extraction and purification of plasmid DNA 55 2.10 Ethanol precipitation of plasmid DNA 55 2.11 Restriction digests of DNA 55 2.12 Gel extraction 56 2.13 Induction of recombinant protein expression 56 2.14 Determining protein solubility 57 2.15 Protein sample preparation for IMAC purification under denaturing conditions 57 2.16 IMAC protein purification under denaturing conditions 57 2.17 Protein sample preparation for benchtop purification 58 2.18 Benchtop protein purification under denaturing conditions 58 2.19 Polyclonal antibody production 58 2.20 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) 59 2.21 Silver staining 59 iv 2.22 Southern blot analyses 60 2.23 Western blot analyses 61 2.24 Naegleria gruberi (NEG-M) cell culture 62 2.25 Naegleria gruberi (NEG-M) protein samples 62 2.26 Trypanosoma brucei cell culture 62 2.27 Transfection of DNA into trypanosomes 63 2.28 Trypanosome protein samples 63 2.29 Isolation of T. brucei genomic DNA 64 2.30 Preparation of slides for fluorescence microscopy 64 2.31 MitoTracker® probing 65 2.32 Gene knockout in trypanosomes 65 2.33 Co-immunoprecipitation 67 2.34 Culturing of T. brucei in SILAC media 67 2.35 Sample preparation for mass spectrometry 2.35.1 BioID sample preparation 69 2.35.2 Co-immunoprecipitation sample preparation 71 2.36 Liquid chromatography–mass spectrometry 73 2.37 Analysis of mass spectrometry data 73 2.38 Analysis of protist FeFe-hydrogenases 73 2.39 Analysis of microarray data 74 2.40 Assembling an Access database for the analysis of TbHP40 and TbHP30 evolution 75 2.41 Protein targeting predictions 76 2.42 Homology modelling 76 3. Bioinformatics analysis of novel, paralogous histidine phosphatases 79 3.1 An overview of the histidine phosphatase superfamily 79 3.2 TbHP40 and TbHP30 are mitochondrial proteins 82 3.3 Phylogenomic distribution of TbHP40 and TbHP30 orthologues 83 3.4 Homology modelling 86 4. Evidence for the essentiality of TbHP40 and TbHP30 106 4.1 The generation of a TbHP40/TbHP30+/- heterozygous cell line 106 4.2 Generation of a TbHP40/TbHP30-/- - attempt 1 108 4.3 Generation of a TbHP40/TbHP30-/- - attempt 2 109 v 4.4 Generation of a TbHP40/TbHP30-/- - attempt 3 112 4.5 Progress with the generation of a conditional TbHP40-/- cell line 114 5. Identifying interacting partners for the novel histidine phosphatases TbHP40 and TbHP30 126 5.1 A proximity-dependent biotinylation approach to identifying candidate interacting partners for TbHP40 128 5.2 Identifying potential interacting partners of TbHP40 using a co- immunoprecipitation approach 133 5.3 Summary of attempts to identify interacting partners of TbHP40 and TbHP30 138 6. Heterologous expression in Trypanosoma brucei of candidate mitochondrial proteins from Naegleria gruberi 157 6.1 Bioinformatic analysis of a range of eukaryotic FeFe-hydrogenases 158 6.2 Antibody-based detection of N. gruberi FeFe-hydrogenase 160 6.3 Heterologous expression of N. gruberi enzymes in T. brucei 162 6.4 Summary of characterisation attempts of N. gruberi novel mitochondrial proteins 166 7. General Discussion 183 8. Appendices 191 8.1 Appendix I – abbreviations from Figure 1.12 191 8.2 Appendix II – restriction maps for plasmids used in this study 191 9. References 196 vi Abbreviations a.a. Amino acids ABC Ammonium bicarbonate AdoMetDC S-adenosylmethionine decarboxylase AMP Adenosine monophosphate ASCT Acetate:succinate CoA-transferase BioID Proximity-dependent biotinylation identification BLAST Basic Local Alignment Search Tool blast Blasticidin blastp Protein-protein basic local alignment search tool Bleo Bleomyin bp Base pairs BPP Benchtop purification protocol Co-IP Co-immunoprecipitation DAPI 4', 6‐diamidino‐2‐phenylindole DHS Deoxyhypusine synthase DMSO Dimethyl sulfoxide DTT Dithiothreitol EB Elution buffer ELISA Enzyme-linked immunosorbent assay euBACs Universal eukaryotic genes of bacterial origin gDNA Genomic deoxyribose nucleic acid GFP Green fluorescence protein HA Human influenza hemagglutinin HAT Human African trypanosomiasis HK Hexokinase Hmd Methylenetetrahydromethanopterin HRP Horseradish peroxidase hyg Hygromycin IAA Iodoacetamide IG In-gel IMAC Immobilised metal-ion affinity