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

描述（由申请人提供）：本申请提出在功能上表征一组假定的补体调节蛋白（CRP），以通过RNA干扰（RNAi）抑制其基因表达来评估其作为治疗靶点的潜力。血吸虫是一种扁形虫寄生虫，可引起慢性和使人衰弱的疾病，困扰着全世界2亿多人，每年造成25万多人死亡。这些蠕虫很好地适应了在脊椎动物宿主的脉管系统中生活数十年，并且显然已经发展出逃避宿主免疫监视的策略，包括有效的补体系统。我们假设，作为补体逃避的一种机制，补体小体在其被膜（覆盖整个蠕虫表面与宿主直接接触的合胞体膜）中表达CRP。使用基因组和转录组分析，鉴定了7种与人CD 59具有显著序列同源性的推定染色体CRPs。这些同源物在此被指定为SmCD 59 -1、2、3、4、5、6、7。CD 59在人体组织中广泛分布。它是一种有效的成孔抑制剂， 膜攻击复合物（MAC或C5 b-9）通过与最终补体成分C8和C9相互作用。这里要检验的假设是，这些SmCD 59基因中的一个或多个保护线粒体免受补体杀伤，就像CD 59保护宿主细胞一样。首先，将进行研究，以确定哪些SmCD 59蛋白是补体抑制剂，通过在哺乳动物细胞中单独表达每个同源物，并测试其防止补体损伤转染细胞的能力。然后，通过RNAi抑制选定的SmCD 59基因库在童虫中的表达，以评估这些寄生虫是否在体外更容易受到补体杀伤。在初步结果中，检测到所有SmCD 59同源物的基因表达在童虫和蠕虫（这两个生命阶段都适应于在血流中生存）和每个SmCD 59目标的基因表达单独，或在多达4个基因目标池已有效地抑制在童虫。如果RNAi研究成功，这将是有力的证据表明，在皮层SmCD 59的表达是一个主要的寄生虫防御机制，对宿主补体。然后，该项目可以很容易地扩展为一个更大的项目，以探索SmCD 59补体抑制机制，并在开发旨在降低血吸虫病发病率和/或发病率的新疗法或疫苗时利用这一知识寻求策略。 公共卫生相关性：血吸虫是血液寄生蠕虫，对世界卫生和农业造成重大问题，特别是在发展中国家。该项目提出采用现代RNA抑制方法来评估补体抑制蛋白是否保护溶酶体免受宿主补体系统的杀伤。这些蛋白质作为抗血吸虫病的新疗法和/或疫苗的靶点具有很好的前景。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Schistosomes are blood dwelling worms causing major problems for world health and agriculture, particularly in the developing world. This project proposes to employ modern RNA suppression methods to assess whether complement inhibitory proteins protect schistosomes from killing by the host complement system. These proteins have excellent promise as targets for new therapies and/or vaccines against schistosomiasis.