Molecular Mechanisms to Attenuate Leishmania Parasite

利什曼原虫引起人类疾病，其临床症状从自愈的皮肤病变到致命的内脏感染。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，潜伏感染可在感染艾滋病毒后重新激活。由于缺乏对这种寄生虫的细胞生物学和致病机制的了解，控制这种严重的全球健康风险的任务变得困难。在美国本土，美国军事人员、他们的家人以及其他前往或居住在疫区的旅行者尤其担心。为了寻找新的方法来控制这种病原体，我们已经开始研究寄生虫从无毒（promastigote）到有毒（amastigote）的分化机制。利什曼原虫的膜或分泌蛋白与其防御宿主杀死的机制有关。人们对利什曼原虫等锥虫寄生虫的分泌途径知之甚少，特别是对糖基化/折叠和膜和分泌蛋白的细胞内运输至关重要。已知内质网常驻伴侣蛋白在这些过程中起重要作用。我们认为，通过调节伴侣蛋白的表达改变分泌过程可能导致利什曼原虫毒力的衰减。我们从多诺瓦利什曼原虫中克隆了钙网蛋白和蛋白二硫异构酶（PDI）等伴侣蛋白的同源物。利什曼原虫钙调蛋白p结构域的过度表达导致寄生虫分泌酸性磷酸酶的分泌显著减少，这是一种假定的毒力因子。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降低了存活率。另一种重要的内质网伴侣蛋白PDI的活性位点突变和表达也影响了s-酸性磷酸酶（sAcP）的分泌。除了伴侣蛋白活性外，PDI对维持内质网内还原环境和形成二硫键很重要。这些研究表明，伴侣蛋白表达的改变可以调节利什曼推定毒力因子的分泌，从而导致其传染性的减弱。此外，利什曼原虫PDI被证明比典型的哺乳动物PDI（如人类宿主）的尺寸要小得多，因此，我们已经开始研究阐明寄生虫酶的3D结构。为此，在天然条件下制备了毫克量的表达利什曼原虫PDI的大肠杆菌，目前正在进行纯化以达到均质性。这样的制备将受到蛋白质结晶和x射线衍射分析，以确定利什曼原虫PDI的结构。将寄生虫的PDI与人类的PDI进行比较，可能会发现显著的差异，这可以用于开发新的抗利什曼原虫药物。

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.