MOLECULAR MECHANISM OF LEISHMANIASIS

利什曼原虫引起人类疾病（利什曼病），其临床症状从自愈的皮肤病变到致命的内脏感染。由于缺乏对利什曼原虫致病机制的了解，在世界范围内造成了严重的公共卫生风险，特别是对在流行地区生活或旅行的美国军人及其家属和游客。作为了解利什曼原虫发病机制的第一步，我们已经开始分析寄生虫生命周期中从无毒形态到有毒形态转化的过程。我们利用不同的分子生物学技术，如差异杂交和任意引物聚合酶链反应（AP-PCR），鉴定了多诺瓦利什曼原虫（Leishmania donovani）的几个差异调控基因。我们已经描述了一个这样的基因，编码一个同源的哺乳动物伴侣蛋白钙环蛋白（CR），这是差异调节。此外，CR和其他伴侣蛋白已被证明对高等真核生物的膜和分泌蛋白的糖基化和细胞内运输很重要。人们对寄生虫的这一过程知之甚少，尤其是利什曼原虫。此外，由于利什曼原虫的膜蛋白或分泌蛋白具有防御宿主杀死利什曼原虫的机制，我们试图通过基因敲除来改变利什曼原虫中CR基因的表达，从而改变这些蛋白的生物学特性，从而产生减毒寄生虫。CR基因的缺失对寄生虫是致命的。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，我们获得了过表达CR各个区域的寄生虫，目前我们正在分析CR突变体寄生虫改变利什曼膜和分泌蛋白表达的能力。这样的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.