MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF A SECRETED LIPASE IN LEISHMANIA

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这项拟议的研究旨在帮助理解利什曼原虫的生存、生长和发展机制。利什曼病的疾病谱从轻微的自限性皮肤溃疡到潜在的致命内脏感染，每种疾病都取决于传染性寄生虫的种类。利什曼原虫分泌的特定蛋白质使有机体能够感知、反应和改变环境。因此，这种分泌的蛋白质是开发预防或治疗这种疾病的新化合物的潜在目标。目前还没有疫苗可用于预防这些寄生虫的传播。有毒的锑化合物曾被使用过，但未获成功。脂肪酶是一种脂肪分解酶，可以水解甘油三酯的酯键。在其他系统中，脂肪酶活性参与膜结构的重组、细胞信号传递或营养物质的获取。这些观察结果表明，脂肪酶活性对利什曼原虫是必不可少的，尽管目前该酶的生物学意义尚未得到实验证明。脂肪酶对利什曼原虫至关重要的工作假说将得到检验。为了确定该酶在寄生虫生命周期中的作用，计划采用以下方法：1)鉴定和鉴定脂肪酶基因的全长拷贝；在寄生虫发育的整个生命周期中检测脂肪酶的表达，并确定动质体类原生动物中脂肪酶的系统发育保守性。2)将产生脂肪酶活性缺乏的零突变体，并对其活性进行测试。3)脂肪酶基因将以重组蛋白的形式表达，并进行底物选择性检测。此外，还将测试各种类型的脂肪酶有机磷抑制剂，以确定利什曼脂肪酶对这些化合物的敏感性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proposed research is designed to contribute to the understanding of mechanisms of Leishmania survival, growth and development. The disease spectrum of leishmaniasis ranges from mild self-limiting cutaneous ulcers to a potentially fatal visceral infection, each dependant on the species of infectious parasite. Specific proteins secreted by Leishmania enable the organisms to sense, respond to, and alter their environment. Such secreted proteins are thus potential targets for the development of new compounds to prevent or treat this diseases. Currently there is no vaccine available for the prevention of transmission of these parasites. Toxic antimony compounds have been used unsuccessfully. Lipases are lipolytic enzymes that hydrolyze the ester bond of triglycerides. In other systems, lipase activity is involved in the reorganization of membrane structure, cell signaling or nutrient acquisition. These observations suggest that lipase activity is essential for Leishmania although presently the biological significance of this enzyme has not been proven experimentally. The working hypothesis, that lipase is essential to Leishmania, will be tested. The following approaches to determine the role of this enzyme in the parasite life cycle are planned: 1) The full-length copy of the lipase gene will be identified and characterized; the expression of lipase will be examined though out the parasite developmental life cycle and the phylogenic conservation of lipase among kinetoplastid protozoa will be determined. 2) Null-mutants deficient in lipase activity will be generated and tested for viability. 3) The lipase gene will be expressed as a recombinant protein and assayed for substrate preference. In addition, various classes of organophosphate inhibitors of lipase will be tested to determine the sensitivity of the leishmanial lipase to these compounds.