Complement mediated lysis resistance genes of Leishmania

利什曼原虫通过白蛉传播给人类和其他脊椎动物宿主。这些寄生虫对人类和动物健康有巨大影响，特别是在发展中国家。寄生虫对补体介导的裂解（CML）的抗性对于脊椎动物的感染是重要的。CML是一种微生物诱导的宿主防御机制，涉及血清的无细胞部分。在白蛉中，利什曼原虫从对脊椎动物没有感染性的前环前鞭毛体发展成高度感染性的后环前鞭毛体。对CML的抗性在前环前鞭毛体中较低，但在大多数利什曼原虫种的后环前鞭毛体中较高。本研究的长期目标是了解 利什曼原虫会传染。该提案的总体目标是识别和表征在对CML的抗性中起作用的基因。查加斯利什曼原虫的低代次环前鞭毛体对CML具有抗性，并具有高水平的表面蛋白GP 46，而高代次前鞭毛体和低代次前鞭毛体对CML敏感，并下调GP 46的表达。用编码GP 46的质粒转染的高传代细胞是CML抗性的，表明高传代细胞可用于筛选赋予CML抗性的特异性利什曼原虫基因/编码序列。用高传代细胞筛选利什曼原虫基因组DNA片段（粘粒中32-35 kb片段 载体）以获得CML抗性的能力。五个基因组等同物的筛选导致选择十二个互补序列。当重新转染到CML敏感的前鞭毛体中时，所选择的cosmetic赋予CML抗性。该项目的具体目标是确定和表征赋予CML抗性的粘粒插入物内的基因，并表征该粘粒赋予CML抗性的机制。将通过序列分析和筛选亚克隆插入片段来鉴定赋予CML抗性的基因 并且通过使用北方和蛋白质印迹分析表征其基因表达谱来支持/测试其生物学相关性。表征的机制，其中的cosestrin（或基因）的行动将确定点的CML-级联是由cosestrin扰动，并将研究补体蛋白和利什曼原虫蛋白之间可能的相互作用，赋予CML-抗性。

Leishmania are transmitted to human and other vertebrate hosts by sand flies. These parasites have a tremendous impact on human and animal health, particularly in developing countries. Resistance of the parasite to complement-mediated lysis (CML) is important for infection of vertebrates. CML is a microbe-induced host defense mechanism involving the cell free fraction of serum. In the sand fly, Leishmania develop from procyclic promastigotes that are not infectious to vertebrates into highly infectious metacyclic promastigotes. Resistance to CML is low in procyclic promastigotes, but is high in metacyclic promastigotes for most Leishmania species. The long term objective of this research is to understand the process by which Leishmania become infectious. The overall aim of this proposal is to identify and characterize genes that function in resistance to CML. Low passage metacyclic promastigotes of Leishmania chagasi are CML-resistant and have high levels of surface protein GP46, while high passage promastigotes, and low passage procyclic promastigotes are sensitive to CML and downregulate GP46 expression. High passage cells transfected with plasmids encoding GP46 were CML-resistant, indicating that high passage cells can be used to screen for specific Leishmania genes/coding sequences that confer resistance to CML. The high passage cells were used to screen Leishmania genomic DNA segments (32-35 kb fragments in cosmid vectors) for the ability to confer CML-resistance. A screen of five genomic equivalents resulted in selection of twelve cosmids. When re-transfected into CML-sensitive promastigotes, the selected cosmids conferred CML-resistance. The specific aims of this project are to determine and characterize the genes within the cosmid inserts that confer CML-resistance, and to characterize the mechanism by which the cosmids confer resistance to CML. Genes conferring CML resistance will be identified by sequence analysis and by screening subcloned inserts for functionality, and their biological relevance will be supported/tested by characterizing their gene expression profiles by using northern and western blot analyses. Characterization of the mechanism by which the cosmids (or genes) act will identify the point in the CML-cascade that is perturbed by the cosmids, and will study possible interactions between complement proteins and Leishmania proteins that confer CML-resistance.