RNAi-based functional analysis of schistosome CD59-like as complement regulators

本申请提出功能性地表征一组推定的补体调节蛋白， 通过抑制蛋白质（CRP）的表达，评估其作为治疗靶点的潜力。 通过RNA干扰（RNAi）进行基因表达。血吸虫是扁形虫寄生虫， 慢性和使人衰弱的疾病困扰着全世界2亿多人， 每年超过25万人死亡。这些蠕虫非常适合生活在它们的脉管系统中， 几十年来，脊椎动物宿主，显然已经发展出了逃离宿主的策略。 免疫监视，包括有效的补体系统。我们假设， 在它们的被膜（直接覆盖整个蠕虫表面的合胞体膜）中表达CRP 与宿主接触）作为补体逃避的机制。 使用基因组和转录组分析，七个假定的染色体CRPs具有显著的 鉴定了与人CD 59的序列同源性。这些同系物在此被指定为 SmCD59-1、2、3、4、5、6、7。CD 59在人体组织中广泛分布。它是一种有效的抑制剂， 通过与最终补体相互作用形成孔膜攻击复合物（MAC或C5 b-9） 成分，C8和C9。这里要检验的假设是，一个或多个SmCD 59 这些基因保护补体不被补体杀死，就像CD 59保护宿主细胞一样。第一、 将进行研究，以确定哪些SmCD 59蛋白是补体抑制剂， 在哺乳动物细胞中单独表达每种同源物，并测试它们预防 对转染细胞的补体损伤。然后，选择SmCD 59基因库的表达 将通过RNAi在童虫中被抑制，以评估这些寄生虫是否变得更 在体外易受补体杀伤。在初步结果中，检测到所有基因表达， SmCD 59同源物在童虫和蠕虫中（两个生命阶段都适应在环境中生存）， 血流）和每个SmCD 59靶点的基因表达，或在多达4个基因的库中 索马里境内的目标已被有效镇压。 如果RNAi研究成功，这将有力地证明SmCD 59在细胞中的表达。 被膜是寄生虫对抗宿主补体的主要防御机制。这个项目可能是 然后很容易扩展到一个更大的项目，以探索SmCD 59补体抑制机制 并在新疗法或疫苗的开发中利用这些知识 旨在降低血吸虫病的发病率和/或发病率。

This application proposes to functionally characterize a group of putative complement regulatory proteins (CRPs) in schistosomes to assess their potential as therapeutic targets by suppressing their gene expression through RNA interference (RNAi). Schistosomes are flatworm parasites that cause a chronic and debilitating disease afflicting more than 200 million people worldwide and causing more than 250,000 deaths/per year. These worms are well adapted to live in the vasculature of their vertebrate hosts for decades, and apparently have developed strategies to escape from the host's immune surveillance, including the potent complement system. We hypothesize that schistosomes express CRPs in their tegument (a syncytial membrane that covers the entire worm surface in direct contact with the host) as a mechanism of complement evasion. Using genome and transcriptome analysis, seven putative schistosome CRPs with significant sequence homology to human CD59 were identified. These homologues are designated here as SmCD59-1, 2, 3, 4, 5, 6, 7. CD59 has broad distribution in human tissues. It is a potent inhibitor of the pore-forming membrane attack complex (MAC or C5b-9) through interaction with the final complement components, C8 and C9. The hypothesis to be tested here is that one or more of these SmCD59 genes protects schistosomes from complement killing just as CD59 protects the host's cells. First, studies will be conducted to determine which of the SmCD59 proteins are complement inhibitors by expressing each homologue individually in mammalian cells and testing for their ability to prevent complement damage to transfected cells. Then, the expression of selected pools of SmCD59 genes will be suppressed in schistosomula by RNAi to assess whether these parasites become more vulnerable to complement killing in vitro. In preliminary results, gene expression was detected for all SmCD59 homologues in schistosomula and adult worms (both life stages adapted to survive in the bloodstream) and the gene expression of each SmCD59 target individually, or in pools of up to 4 gene targets have effectively been suppressed in schistosomula. If the RNAi studies are successful, this will be strong evidence that SmCD59 expression in the tegument is a major parasite defense mechanism against the host complement. This project could be then readily expanded into a larger project to explore the SmCD59 complement inhibition mechanism and to pursue strategies that exploit this knowledge in the development of new therapies or vaccines aiming to reduce the incidence and/or morbidity of schistosomiasis.