Biochemical and cell biological analysis of a virulence enhancing protein from Leishmania major; its impact on macrophage activity and gene expression

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It has become increasingly clear in recent years that apart from the genetic predisposition of the host, clonally expressed traits within parasite populations have a major impact on the outcome of Leishmania infections. Increased expression of a Leishmania major virulence marker, P46, boosts infectiousness in macrophages and extends the host spectrum to normally resistant experimental hosts. During infection the protein is exported into the macrophage cytoplasm, where it is suspected of interfering with the macrophage defence mechanisms. We propose to analyse macrophages for changes of gene expression after infection with P46 overexpressing parasites or other exposure to P46, using proteomics and transcriptomics. Macrophages under P46 exposure will be challenged with other intracellular pathogens to determine the specificity of the P46 effect. The oligomeric structure of P46 will be explored by various biochemical methods, both for natural protein and for recombinantly expressed P46. The export pathway will be unravelled using subcellular fractionation schemes, mutagenesis, and fluorescent microscopy techniques, to test for a possible role of another virulence marker, HSP100. Expression of various deletion mutants will help to identify the domains responsible for increased infectiousness while the generation of P46 null-mutants will reveal the overall impact of P46 on parasite viability and virulence.