Variant surface glycoprotein diversification in Trypanosoma brucei

项目摘要 布氏锥虫是人类和动物非洲锥虫病的病原体， 影响撒哈拉以南非洲的疾病。一种致密的变异表面糖蛋白（VSG）外壳覆盖着它。 细胞外寄生物，并有效地掩盖细胞表面上的所有其他抗原。T.布鲁塞逃脱淘汰 哺乳动物宿主的免疫系统通过其VSG外壳的抗原变异：它“切换” 表达VSG，利用~2000个VSG编码基因的基因组库。然而，寄生虫 群体在体内表达一组不同的VSG编码基因。表达的VSG数量超过30 感染的天数可以接近寄生虫基因组内功能性VSG库的三分之一， 突出了一个有趣的问题：如果现有的基因组库可以在几个月内用完， 在野外感染能持续数年吗此应用程序的目标是调查 T.布鲁氏菌使其基因组VSG库多样化以维持感染。初步 数据表明，血管外空间可能为T. brucei使其VSG档案多样化 在没有免疫压力的情况下。在这项建议的第一个目的，这个血管外生态位在VSG中的作用， 将研究多样化。第二个目标将利用CRISPR/Cas9技术结合 用一种新的高通量测序方法来研究片段基因转换事件， 从而产生新颖的“马赛克”VSG。在第三个目标中，将研究单个VSG的突变，以便 了解控制寄生虫基因组内VSG档案演变的机制， 感染的过程。这些研究将开始阐明T.布鲁塞长期以来， 长期感染在其宿主，这将是重要的发展新的战略，用于治疗这一点 被忽视的热带病为实现这些目标，提出了三个目标：1）阐明 VSG多样化的血管外空间; 2）确定节段性基因的机制 产生镶嵌VSG的转化事件; 3）量化VSG的超突变率。

PROJECT SUMMARY Trypanosoma brucei is a causative agent of both human and animal African trypanosomiasis, devastating diseases affecting sub-Saharan Africa. A dense variant surface glycoprotein (VSG) coat covers this extracellular parasite and effectively masks all other antigens on the cell surface. T. brucei evades elimination by the mammalian host's immune system through antigenic variation of its VSG coat: it “switches” the expressed VSG, taking advantage of a genomic repertoire of ~2000 VSG-encoding genes. However, parasite populations in vivo express a diverse set of VSG-encoding genes. The number of expressed VSGs over 30 days of infection can approach one-third of the functional VSG repertoire within the parasite genome, which highlights an interesting question: if the existing genomic repertoire can be used up in a matter of months, how can an infection be sustained for years in the field? The goal of this application is to investigate the mechanisms by which T. brucei diversifies its genomic VSG repertoire in order to sustain infection. Preliminary data suggest that extravascular spaces may provide an important area for T. brucei to diversify its VSG archive in the absence of immune pressure. In the first aim of this proposal, the role of this extravascular niche in VSG diversification will be investigated. The second aim will take advantage of CRISPR/Cas9 technology combined with a novel high-throughput sequencing method to investigate the segmental gene conversion events resulting in novel “mosaic” VSGs. In the third aim, the mutation of individual VSGs will be studied, in order to understand the mechanisms that govern the evolution of the VSG archive within the parasite genome over the course of infection. These studies will begin to elucidate the mechanisms by which T. brucei sustains a long- term infection in its host, which will be important for the development of new strategies for treatment of this neglected tropical disease. Three aims are proposed to accomplish these goals: 1) To elucidate the role of extravascular spaces in VSG diversification; 2) To identify the mechanisms responsible for the segmental gene conversion events that generate mosaic VSGs; 3) To quantify the rate of hypermutation of VSGs.