Williams, Bryony Alice Peris; (2003) Molecular evolution and the fate of the mitochondrion in microsporidia parasites. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Microsporidia are important obligate intracellular parasites of a variety of eukaryotes and increasingly important pathogens of human AIDS patients. They have a highly complex and unique infection apparatus but otherwise appear structurally simple. In particular, typical eukaryotic organelles such as mitochondria and peroxisomes have never been identified. This absence was widely interpreted as evidence that these peculiar eukaryotes diverged prior to the mitochondrial endosymbiosis, making microsporidia potentially one of the earliest offshoots in eukaryotic evolution. However, microsporidial nuclear genes encoding homologues of mitochondrial heat shock proteins (Hsp) were subsequently detected and interpreted as evidence of secondary loss of organelle or endosymbiont. In this project the mitochondrial Hsp70 protein from the microsporidian Trachipleistophora hominis was characterised. The full-length gene was amplified, cloned and sequenced. A detailed phylogenetic analysis of this protein to ascertain its relationship to other eukaryotic Hsp70 proteins was carried out, which demonstrated a strong phylogenetic relationship to other mitochondrial isoforms and placed microsporidia as sister group to the Fungi. However, typical mitochondrial targeting signals within this protein were not found using either in silico or in cellulo detection methods. A highly specific polyclonal antibody to a fusion protein of this gene was prepared. Western blotting of T. hominis proteins with this antibody showed the protein to be expressed in both spore and intracellular stages of T. hominis and that it was not upregulated in response to heat shock. The antibody was used in immunocytochemistry experiments to detect the presence of numerous discrete compartments in proliferative forms (meronts) of this parasite. The ultrastructure of these compartments was investigated further using immuno-electron microscopy. This revealed that they are small double membrane bound organelles of probable mitochondrial origin. These data provide the first direct link between a protein of demonstrable mitochondrial ancestry and otherwise strongly conserved mitochondrial function, to a putative microsporidian organelle.

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