Impact of the Heat Shock Protein 100 on the Intracellular Survival of Leishmania Amastigote Stages

进入哺乳动物宿主后，利什曼原虫被巨噬细胞吞噬。在吞噬体内部，细长的、有鞭毛的promastigoate阶段转化为圆形的、有鞭毛的无尾无纺体阶段。作为无尾线虫，利什曼原虫持续存在并增殖，从而破坏其宿主细胞。利什曼原虫在吞噬溶酶体内生存和逃避破坏的能力是其致病性的关键因素。利什曼原虫的100 kda热休克蛋白Hsp100在这一过程中起着选择性的关键作用。缺乏Hsp100的δ clpB基因替代突变体在无菌培养条件下完全存活。相比之下，在小鼠巨噬细胞内或小鼠实验感染期间，这些基因替代菌株表现出明显的毒力下降和细胞内存活受损。在Hsp100突变体中，一些无尾线虫特异性蛋白的表达受到影响。我们计划鉴定受影响的蛋白质和/或其相应的基因，并确定它们对细胞内存活和毒力的重要性。为此，我们提出了一种双管齐下的方法：i)野生型和三角洲型clpB突变体在无尾轴分化期间和之后的蛋白质组比较分析，ii)功能遗传互补。本研究的目的是深入了解利什曼原虫在巨噬细胞内逃脱破坏并在恶劣的细胞内环境中增殖的过程。

Following entry into a mammalian host organism, Leishmania spp. are phagocytised by macrophages. Inside the phagosome, the elongated, flagellated promastigote stage converts into the round, aflagellated amastigote stage. As amastigotes, Leishmania parasites persist and proliferate, thereby destroying their host cells. The ability of Leishmania amastigotes to survive within the phagolysosome and to escape destruction is a key factor to their pathogenicity. The 100-kDa heat shock protein, Hsp100, of Leishmania plays a selective and crucial role in this process. delta clpB gene replacement mutants that lack Hsp100 are perfectly viable under axenic culture conditions. By contrast, inside mouse macrophages or during experimental infection of mice, these gene replacement strains show a marked decrease of virulence and an impaired intracellular survival. The expression of some amastigote-specific proteins is affected in the Hsp100-- mutants. We plan to identify the affected proteins and/or their corresponding genes and establish their importance for intracellular survival and virulence. To this end we propose a two-pronged approach: i) comparative proteome analysis of wild type and delta clpB mutants during and after amastigote differentiation, and ii) functional genetic complementation. The goal of this study is to gain insight into the processes that allow Leishmania species to escape destruction inside the macrophages and to proliferate within a hostile intracellular environment.