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。我们最近还克隆了一个蛋白质二硫异构酶（PDI）基因，它参与内质网中蛋白质二硫键的形成。在高等真核生物中，CR和其他伴侣蛋白（如PDI）对糖基化/折叠以及膜和分泌蛋白的细胞内转运具有重要作用。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，利什曼原虫的膜蛋白或分泌蛋白与其防御宿主杀伤的机制有关。我们试图改变多诺瓦氏L. CR基因的表达，以操纵该蛋白的生物学特性，并创建一个减毒寄生虫。CR基因的缺失对寄生虫是致命的。接下来，我们过表达完整的编码区或假设的CR结构域，如N端(N)，脯氨酸丰富(P)和C端(C)区域。表达N-， P-和c -结构域的突变体寄生虫的一种必需分泌蛋白，s-酸性磷酸酶（sAcP）显著降低。sAcP分泌减少是由于内质网中蛋白质的积累。进一步的分析表明，这些突变体对巨噬细胞的杀伤抵抗力较弱。因此，这些研究表明，改变分泌的利什曼假定的毒力因子可以导致其传染性的衰减。

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. We have also recently cloned a gene for protein disulfide isomerase (PDI) that is involved in formation of disulfide bond of proteins in the ER. CR along with other chaperones such as PDI have been shown to be important for glycosylation/folding and intracellular transport of membrane and secretory proteins in 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 implicated for its defense mechanism against killing by the host. We attempted to alter the expression of CR gene in L. donovani so as to manipulate the biology of this protein and to create an attenauted parasite. Deletion of CR gene was lethal for the parasite. Next, we overexpressed the complete coding region or the putative domains of CR such as N-terminal(N), Proline rich (P), and C-terminal (C) regions. Mutant parasites expressing N-, P- and C-domains had a siginificnt decrease in one of the essential secretory proteins, s-acid phosphatase (sAcP). Reduction in the sAcP secretion was due to the accumulation of the protein in the ER. Further analysis showed that these mutants were less resitant to killing by the macrophages. Thus, these studies suggest that alteration in the secretion of leishmanial putative virulent factors can result in the attenuation of its infectivity.