MOLECULAR MECHANISM OF LEISHMANIASIS

利什曼原虫引起人类疾病（利什曼病），临床症状从自愈性皮肤损伤到致命的内脏感染。由于对利什曼原虫致病机制缺乏了解，在全球范围内，特别是对美国，构成了严重的公共卫生风险。S.在疫区居住或旅行的军人、家属和游客。 作为了解利什曼原虫发病机制的分子机制的第一步，我们已经开始分析参与寄生虫生命周期从无毒到有毒形式的转化过程。我们已经确定了几个差异调节基因的利什曼原虫，内脏寄生虫，使用各种分子生物学技术，如差异杂交和任意引物聚合酶链反应（AP-PCR）。 我们的特点是这样一个基因，编码的同源物的哺乳动物伴侣蛋白calretciculin（CR），这是差异调节。此外，CR沿着其他分子伴侣已被证明是重要的糖基化和细胞内转运的膜和分泌蛋白在高等真核生物。 关于寄生虫的这一过程，特别是利什曼原虫，我们知之甚少。此外，利什曼原虫的膜蛋白或分泌蛋白被认为是其抵抗宿主杀伤的防御机制，我们试图改变利什曼原虫CR基因的表达。donovani通过基因敲除，以改变这些蛋白质的生物学，创造attenauted寄生虫。CR基因的缺失对该虫是致命的。因此，我们试图通过过表达CR的完整编码区或部分编码区来改变内源性CR的表达。我们获得了过度表达CR不同区域的寄生虫。 目前，我们正在分析CR突变体寄生虫改变利什曼原虫膜和分泌蛋白表达的能力。这种分析将使我们能够测试CR突变体作为利什曼原虫疫苗减毒寄生虫的潜力。

Leishmania parasite causes human disease (Leishmaniasis) with clinical symptoms ranging from self healing cutaneous lesions to fatal visceral infection. The lack of understanding of the mechanism by which Leishmania parasite causes disease poses a serious public health risk worldwide and in particular for U. S. military personnel their families and tourists either living or travelling in endemic areas. As a first step towards understanding the molecular mechanism of Leishmania pathogenesis, we have began to analyze the processes that are involved in parasite life cycle in transformation from an avirulent to virulent form. We have identified several differentially regulated genes of Leishmania donovani, a viscerotropic parasite, using various molecular biology techniques such as differential hybridization and arbitarily primed polymerase chain reaction (AP-PCR). We have characterized one such gene that codes for a homologue of a mammalian chaperone protein calretciculin (CR) which is differentially regulated. In addition,CR along with other chaperones has been shown to be important for glycosylation and intracellular transport of membrane and secretory proteins in the higher eukaroyotes. Very little is known about this process in parasites in general and Leishmania in particular. Further, membrane proteins or secretory proteins of Leishmania have been implied for its defense mechanism against killing by the host, we attempted to alter the expression of CR gene in L. donovani by gene-knock out so as to alter the biology of these proteins to create attenauted parasites. Deletion of CR gene was lethal for the parasite. Therefore, we attempted to alter the expression of endogenous CR by overexpressing complete coding region or portions of the coding region of CR. We obtained parasites which are overexpressing vaious regions of CR. Currently, we are analyzing the CR mutant parasites for their ability to alter the expression of Leishmanial membrane and secretory proteins. Such an analysis will allow us to test the potential of CR mutants as attenuated parasites for Leishmania vaccine.