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。我们最近还克隆了一个蛋白质二硫键异构酶（PDI）的基因，它参与了ER中蛋白质二硫键的形成。CR沿着其他分子伴侣如PDI已被证明对于高等真核生物中膜和分泌蛋白的糖基化/折叠和细胞内转运是重要的。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与其抵抗宿主杀伤的防御机制有关。我们试图改变L. donovani，以便操纵这种蛋白质的生物学，并创造一个attenauted寄生虫。CR基因的缺失对该虫是致命的。接下来，我们过表达CR的完整编码区或推定结构域，例如N-末端（N）、富含脯氨酸（P）和C-末端（C）区。表达N-、P-和C-结构域的突变体寄生虫的基本分泌蛋白之一S-酸性磷酸酶（sAcP）显著降低。sAcP分泌的减少是由于蛋白质在ER中的积累。进一步的分析表明，这些突变体对巨噬细胞的杀伤抗性较低。因此，这些研究表明，利什曼原虫假定毒力因子分泌的改变可导致其感染性的减弱。

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.