Molecular Mechanisms to Attenuate Leishmania Parasite

利什曼原虫引起人类疾病，临床症状从自愈性皮肤损伤到致命的内脏感染。此外，在流行地区，感染艾滋病毒的人特别容易感染利什曼原虫，潜伏感染可在获得艾滋病毒后重新激活。对这种寄生虫的细胞生物学和致病机制缺乏了解，使得控制这种严重的全球健康风险的任务变得困难。在离家更近的地方，美国军事人员、他们的家人和其他访问或居住在流行地区的旅行者尤其关注这一问题。为了找到控制这种病原体的新方法，我们已经开始研究寄生虫从无毒力（前鞭毛体）到有毒力（无鞭毛体）形式的分化机制。利什曼原虫的膜或分泌蛋白与其抵抗宿主杀伤的防御机制有关。锥虫寄生虫如利什曼原虫的分泌途径知之甚少，特别是糖基化/折叠和细胞内转运的膜和分泌蛋白。ER驻留伴侣蛋白在这些过程中发挥重要作用。我们认为，通过调节伴侣蛋白表达的分泌过程的改变可能会导致利什曼原虫毒力的减弱。我们已经从杜氏利什曼原虫中克隆了几个这样的伴侣蛋白的同源物，如钙网蛋白和蛋白质二硫键异构酶（PDI）。利什曼原虫钙网蛋白P结构域的过表达导致寄生虫分泌性酸性磷酸酶（一种推定的毒力因子）的分泌显着减少。这种效应与寄生虫体内活性酶的细胞内积累有关。这些寄生虫在人类巨噬细胞内的存活率降低。另一种必需的ER分子伴侣PDI的活性位点和表达的突变也影响了s-酸性磷酸酶（sAcP）的分泌，PDI对于维持ER内的还原环境和除了分子伴侣活性之外的二硫键的形成非常重要。这些研究表明，伴侣蛋白表达的改变可以调节利什曼原虫推定毒力因子的分泌，这可能导致其感染性的减弱。此外，利什曼原虫PDI被证明在尺寸上显著小于典型的哺乳动物PDI（例如人类宿主），因此，我们已经开始研究以阐明寄生虫酶的3D结构。为此，已经在天然条件下制备了毫克量的大肠杆菌表达的利什曼原虫PDI，并且目前正在纯化至均一。将对此类制剂进行蛋白质结晶和X射线衍射分析，以确定利什曼原虫PDI的结构。寄生虫PDI与人PDI的比较可能导致显著差异，其可用于开发新型抗利什曼原虫药物。

Leishmania parasite causes human disease with clinical symptoms ranging from-self healing cutaneous lesions to a fatal visceral infection. Additionally, in endemic areas, people infected with HIV are especially prone to Leishmania infection and latent infections can reactivate upon acquisition of HIV. The lack of understanding of cell biology and pathogenic mechanisms of this parasite makes the task of controlling this grave, worldwide health risk difficult. Closer to home, it is particularly of concern to U.S. military personnel, their families and other travelers visiting or living in the endemic areas. To find novel methods for control of this pathogen, we have initiated study to understand the mechanism of parasite differentiation from the avirulent (promastigote) to virulent (amastigote) form. Membrane or secretory proteins of Leishmania have been implicated for its defense mechanism against killing by the host. Very little is known about the secretory pathway of trypanosomatids parasites such as Leishmania in general with particular importance to glycosyation/folding and intracellular transport of membrane and secretory proteins. The ER resident chaperone proteins are known to play an essential role in these processes. We argued that alteration of the secretion process via modulating the expression of chaperone proteins might result in attenuation of virulence in Leishmania. We have cloned several homologues of such chaperone proteins such as calreticulin and protein disulfide isomerase (PDI) from Leishmania donovani. Overexpression of the P-domain of Leishmania calreticulin resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases, a putative virulent factor. This effect is associated with an intracellular accumulation of active enzyme inside the parasite. Such parasites where shown to have decreased survival inside human macrophages. Mutations in the active site and expression of another essential ER chaperone PDI, which is important for maintaining reduced environment inside the ER and for the formation of disulphide bonds in addition to chaperone activity also affected the secretion of s-acid phosphatase (sAcP). These studies suggest that alteration in the chaperone protein expression can modulate the secretion of Leishmanial putative virulent factors, which can result in the attenuation of its infectivity. Further, the Leishmania PDI proved to be significantly smaller in size than a typical mammalian PDI (e.g. human host), therefore, we have initiated studies to elucidate the 3D structure of the parasite enzyme. To that end, milligram quantities of E.coli expressed Leishmania PDI have been prepared under native conditions and are currently being purified to homogeneity. Such preparation will be subjected to protein crystallization and X-ray diffraction analyses in order to determine structure of the Leishmania PDI. Comparison of the parasite PDI with the human PDI may results in significant differences that could be exploited for the development of novel anti-leishmanial drugs.