Woods, Kerry Louise
Regulators of autophagy in Leishmania major.
PhD thesis, University of Glasgow.
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Autophagy is a conserved lysosomal degradation pathway for recycling long-lived proteins and organelles that is thought to be required for life cycle progression and virulence of Leishmania. ATG8 is a ubiquitin like protein that is required for the formation of autophagosomes, and Leishmania uniquely possesses a set of ATG8-like proteins in addition to ATG8, that are distributed in three multi-gene families called ATG8A, ATG8B and ATG8C. The localisation and expression of ATG8A, ATG8B and ATG8C were analysed using GFP fusion proteins and affinity purified antibodies. ATG8A exhibited a dramatic relocalisation to punctate structures under starvation conditions, suggestive of a specific role for ATG8A in starvation induced autophagy. Although ATG8 and ATG8A both participate in a response to starvation, they differed in their sensitivity to the PI(3) kinase inhibitor wortmannin, responded differently to the presence of energy sources, and labelled distinct subsets of vesicles. When the data generated in this thesis was considered together with recent analyses of the functions of the cysteine peptidases ATG4.1 and ATG4.2, evidence for distinct roles of ATG8 and ATG8A emerged. ATG8B and ATG8C localised to single punctate structures close to the flagellar pocket in a small proportion of promastigotes grown under nutrient rich conditions. The distribution of ATG8B and ATG8C labelled structures did not change during differentiation or starvation, suggestive of a role distinct from autophagy. ATG8B labelled structures appeared to be duplicated during cell division, and might be derived from endosomal membranes.
ATG8A, ATG8B and ATG8C expression was shown to be developmentally regulated with all expressed at high levels in stationary phase promastigotes. Conjugation of ATG8 to phosphatidylethanolamine (PE) is required for the association of ATG8 with autophagosome membranes, and while ATG8 was shown to be conjugated to a phospholipid, no evidence was obtained to suggest that ATG8 paralogues are modified by a lipid. High molecular weight proteins were detected by western blot with anti-ATG8, ATG8A, ATG8B and ATG8C antibodies, perhaps indicating associations with other proteins in complexes. Two ubiquitin fusion proteins and a putative SNARE were identified in co-immunoprecipitation experiments performed with anti-ATG8B antibodies, although further experiments are required to determine the validity of these interactions.
To analyse the role of a predicted presenilin-1 (PS1) homologue in L. major, Δps1 null mutants were generated. These mutants were not defective in their ability to differentiate into infective metacyclic promastigotes, and could establish infections in vivo and in vitro, demonstrating that PS1 is not essential and is not a good target for drug development. Large autophagosomes accumulated in Δps1 mutants suggesting that PS1 might be involved in the regulation of autophagy, although it seemed that the parasites could compensate for this, as autophagy was restored to normal levels in Δps1 mutants that had undergone differentiation into amastigotes. Antibodies were raised against a PS1 peptide that recognised L. major PS1 only when over-expressed, suggesting that endogenous PS1 is expressed at a low level. PS1-HA that was stably integrated into the genome localised to a structure close to the flagellar pocket, although a different localisation was observed when PS1-GFP was over-expressed, and investigation is required to clarify the subcellular localisation.
In summary, the regulation of autophagy in L. major has been investigated from two different angles, leading to the characterisation of a unique family of ATG8-like proteins and an aspartic peptidase, presenilin-1.
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