Complement mediated lysis resistance genes of Leishmania

利什曼原虫通过沙蝇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)的抵抗力是脊椎动物感染的重要因素。慢性粒细胞白血病是一种微生物诱导的宿主防御机制，涉及血清中的游离部分。在沙蝇中，利什曼原虫从对脊椎动物不具传染性的原环前鞭毛体发展成高度具有传染性的后环前鞭毛体。对于大多数利什曼原虫来说，对CML的抵抗力在原循环前鞭毛体中较低，但在后循环前鞭毛体中却很高。这项研究的长期目标是了解 利什曼病会传染。这项建议的总体目标是识别和表征与慢性粒细胞白血病耐药相关的基因。查加西利什曼原虫的低传代后循环前鞭毛体对慢性粒细胞白血病具有抗性，并且具有高水平的表面蛋白GP46，而高传代前鞭毛体和低传代前周期前鞭毛体对CML敏感，并下调GP46的表达。高传代细胞中携带GP46基因的高传代细胞对慢性粒细胞白血病耐药，表明高传代细胞可用于筛选对慢性粒细胞白血病耐药的利什曼原虫基因/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32-35kb片段 载体)用于赋予慢性粒细胞白血病抗性的能力。五个基因组等价物的筛选结果是选择了12个宇宙体。当再次将其导入CML敏感的前鞭毛体时，所选的粘粒产生了CML抗性。该项目的具体目标是确定和鉴定粘粒插入片段中赋予CML抗性的基因，并表征粘粒赋予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与慢性粒细胞白血病耐药相关的基因 对于功能，将通过使用Northern和Western印迹分析表征它们的基因表达谱来支持/测试它们的生物学相关性。对粘粒(或基因)作用机制的表征将确定CML级联中被粘粒干扰的点，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，从而赋予CML耐药性。

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.