Lynch, Amber Carol (2016) Novel mitochondrial metabolism in excavate protists. PhD thesis, UNSPECIFIED.
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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.