MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF A SECRETED LIPASE IN LEISHMANIA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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，每种疾病都取决于感染的寄生虫种类。利什曼原虫分泌的蛋白质之所以令人感兴趣，是因为寄生虫正是通过这些分子能够感知、反应和改变它们的环境。这种分泌的蛋白质可以成为独特的合适的靶点，用于开发新的化合物来预防或治疗这种疾病。目前还没有预防这些寄生虫传播的疫苗，有毒的锑化合物经常被用来治疗这种寄生虫，但没有成功。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，尽管这种酶的生物学意义尚未得到实验证明。这项研究的工作假设是脂肪酶对利什曼病是必不可少的。将采取以下方法来确定该酶在寄生虫生命周期中的作用：1)鉴定和鉴定脂肪酶基因的全长拷贝。将在寄生虫发育的整个生命周期中检测脂肪酶的表达，并确定动质体原虫中脂肪酶的系统发育保守性；2)产生脂肪酶活性不足的零突变，并检测其活性；3)脂肪酶基因将作为重组蛋白表达，并进行底物选择性分析。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 leishmanias is ranges from mild self-limiting cutaneous ulcers to a potentially fatal visceral infection, each dependant on the species of parasite with which one is infected. Proteins secreted by Leishmania are of interest because it is through these molecules that the parasites are able to sense, respond to, and alter their environments. Such secreted proteins could make unique suitable targets for the development of new compounds to prevent or treat this disease. Currently there is no vaccine available for the prevention of transmission of these parasites, and toxic antimony compounds are often used unsuccessfully for its treatment. 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 the biological significance of this enzyme has not been proven experimentally. The working hypothesis of this study is that lipase is essential to Leishmania. The following approaches will be taken to determine the role of this enzyme in the parasite life cycle: 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.