W Robert McMaster

Protozoan parasites of the genus Leishmania are responsible for the disease leishmaniasis, one of the major parasitic diseases in humans. The Leishmania life cycle alternates between promastigotes, present in the digestive tract of the sand fly, and amastigotes that reside in the phagolysosome of macrophages after transmission of the parasite to a mammalian host. Because the amastigote is the form under which the parasite can survive and multiply in the human host while evading the immune system and leading to the disease, Leishmania genes that are preferentially expressed in amastigotes have been investigated. Previous studies in our laboratory identified a family of four tandemly linked genes in L. mexicana, the A600 locus, which members show upregulation in amastigotes. Targeted gene deletion studies showed that these genes are essential for the successful replication of amastigotes. The aim of the present work was to elucidate the role of the A600 proteins, A600.1 and A600.4 in amastigotes development. Western blot analysis of L. mexicana promastigotes and amastigotes found that the A600.1 protein was constitutively expressed throughout the parasite life cycle, and localized to the mitochondria of amastigotes. A600-/- amastigotes exhibited impaired mitochondrial functions, characterized by low cellular ATP levels and mitochondrial membrane potential in comparison to the wild type amastigotes. This correlated with a decrease in the activity of Complexes III and IV of the oxidative phosphorylation (OXPHOS) pathway, while Complexes II and V activities were not affected by the absence of the A600 proteins. This data suggest an involvement of the A600 proteins in the optimisation of the OXPHOS pathway function in amastigotes, either by mediating interactions between Complexes III and IV or by contributing to the mitochondrial inner membrane architecture.A GST protein-protein affinity chromatography technology and a co-immunoprecipitation approach were applied to isolate and identify potential A600.1 interacting proteins. The co-immunoprecipitation approach used was successful in immunoprecipitating the A600.1 protein and identified a number of potential interacting proteins. Due to variability between replicates, the data obtained was not conclusive as to whether A600.1 was part of a protein complex and what mitochondrial process it may be associated to.

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