Molecular Analysis of the PfEMP1 Binding Activity

描述（由申请人提供）：由恶性疟原虫引起的疟疾是人类最重要的传染病之一。据估计，每年有3 - 6.6亿例临床恶性疟原虫疟疾发作，其中1- 200万人将死于感染。在恶性疟原虫中，var基因家族编码红细胞膜蛋白（PfEMPI），其作为毒力因子负责抗原变异和感染红细胞与内皮的结合。每个寄生虫基因组编码大约60个PfEMPI蛋白，这些蛋白在寄生虫品系之间有所不同，并结合不同的宿主受体。当受感染的红细胞在脑或胎盘中隔离时，就会发生严重的疟疾。提出的研究背后的具体假设是，特定的黏合剂类型可能由于其结合倾向而易患严重疟疾。本研究将确定PfEMPI蛋白的结合特性以及它们如何影响受感染红细胞对不同微血管内皮细胞的选择性。PfEMPI结合将从一个已经适应在实验室生长的寄生虫分离物中进行研究。通过有限稀释克隆或选择不同的宿主受体，可以产生表达特定PfEMPI变体的寄生虫系。实时PCR方法与var基因家族的基因特异性引物将用于确定var基因在不同品系中表达。每个PfEMPI蛋白的特异性粘附特性将通过表征感染红细胞对广泛的宿主受体和微血管内皮细胞的粘附来确定。这些研究将有助于详细描述寄生虫的细胞粘附特性，并使人们能够更好地了解疟疾发病机制的分子基础。

DESCRIPTION (provided by applicant): Malaria caused by Plasmodium falciparum is one of the most important infectious diseases of humankind. Each year there is an estimated 300-660 million episodes of clinical P. falciparum malaria and 1-2 million will die from the infection. In P. falciparum, the family of var genes encodes erythrocyte membrane proteins (PfEMPI), which act as virulence factors responsible for both antigenic variation and binding of infected erythrocytes to endothelium. Each parasite genome encodes approximately 60 PfEMPI proteins that differ between parasite strains and bind different host receptors. Severe malaria occurs when infected erythrocytes sequester in brain or placenta. The specific hypothesis behind the proposed research is that particular adhesive types may predispose to severe malaria because of their binding tropism. This study will determine the binding properties of PfEMPI proteins and how they affect infected erythrocyte selectivity for different micro-vascular endothelial cells. PfEMPI binding will be studied from a well characterized parasite isolate that has been adapted to grow in the laboratory. Parasite lines expressing specific PfEMPI variants will be generated by limited dilution cloning or by selection on different host receptors. Real-time PCR approaches with gene-specific primers to the family of var genes will be used to define the var genes expressed in the various lines. The specific adhesion properties of each PfEMPI protein will be determined by characterizing infected erythrocyte adhesion against an extensive panel of host receptors and micro-vascular endothelial cells. These studies will contribute to a detailed characterization of the cytoadhesive properties of a parasite and enable a greater understanding of the molecular basis of malaria pathogenesis.