MOLECULAR MECHANISM OF LEISHMANIASIS

利什曼原虫引起人类疾病(利什曼病)，临床症状从自我愈合的皮肤损害到致命的内脏感染。由于缺乏对利什曼原虫致病机制的了解，在世界范围内，尤其是对居住或旅行在流行地区的美国军人、他们的家人和游客，构成了严重的公共卫生风险。作为了解利什曼原虫致病分子机制的第一步，我们已经开始分析寄生虫从无毒形态向强毒形态转化的生命周期中所涉及的过程。我们利用差异杂交和随机引物聚合酶链式反应(AP-PCR)等多种分子生物学技术，鉴定了嗜内脏寄生虫杜氏利什曼原虫的几个差异调控基因。我们已经鉴定了一个这样的基因，它编码哺乳动物伴侣蛋白Calretcrulin(CR)的同源物，这种蛋白是差异调节的。此外，在高等真核生物中，CR与其他伴侣蛋白一起被证明对细胞膜和分泌蛋白的糖基化和细胞内运输具有重要作用。人们对寄生虫，特别是利什曼原虫的这一过程知之甚少。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，我们试图通过基因敲除来改变杜氏利什曼原虫CR基因的表达，从而改变这些蛋白的生物学特性，从而创造出弱化的寄生虫。CR基因缺失对寄生虫具有致死性。因此，我们试图通过过度表达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多种区的寄生虫。目前，我们正在分析CR突变寄生虫改变利什曼原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.