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

近年来越来越清楚的是，除了宿主的遗传易感性外，寄生虫种群内的克隆表达特征对利什曼原虫感染的结果有重大影响。利什曼原虫主要毒力标志物P46的表达增加，增强了巨噬细胞的感染性，并将宿主谱扩展到正常耐药的实验宿主。在感染过程中，蛋白质被输出到巨噬细胞细胞质中，在那里它被怀疑干扰巨噬细胞的防御机制。我们建议使用蛋白质组学和转录组学分析巨噬细胞感染P46过表达寄生虫或其他暴露于P46后基因表达的变化。暴露于P46下的巨噬细胞将用其他细胞内病原体进行挑战，以确定P46效应的特异性。P46的寡聚体结构将通过各种生物化学方法进行探索，包括天然蛋白质和重组表达的P46。出口途径将使用亚细胞分级分离方案，诱变和荧光显微镜技术，以测试另一个毒力标记，HSP 100的可能作用解开。各种缺失突变体的表达将有助于鉴定负责增加感染性的结构域，而P46无效突变体的产生将揭示P46对寄生虫生存力和毒力的总体影响。

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.