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

描述(申请人提供)：本申请建议对血吸虫中一组假定的补体调节蛋白(CRP)进行功能鉴定，以通过RNA干扰(RNAi)抑制其基因表达，以评估它们作为治疗靶点的潜力。血吸虫是一种扁虫寄生虫，会引起一种慢性的、使人衰弱的疾病，困扰着全球2亿多人，每年造成超过25万人死亡。这些蠕虫很好地适应了在脊椎动物宿主的血管系统中生活了几十年，显然已经制定了逃避宿主免疫监视的策略，包括强大的补体系统。我们假设，血吸虫在其被膜(覆盖与宿主直接接触的整个蠕虫表面的合胞膜)中表达CRP，作为一种补体逃避机制。通过基因组和转录组分析，鉴定出7个与人CD59有显著序列同源性的血吸虫CRP。这些同源物在这里被命名为SmCD59-1，2，3，4，5，6，7。CD59在人体组织中分布广泛。它是一种有效的孔道形成抑制剂。 膜攻击复合体(MAC或C5b-9)通过与最终补体成分C8和C9相互作用而产生。这里要检验的假设是，这些SmCD59中的一个或多个基因保护血吸虫免受补体杀死，就像CD59保护宿主细胞一样。首先，将通过在哺乳动物细胞中单独表达每个同源物并测试它们防止补体对转基因细胞的损害的能力，来确定哪些SmCD59蛋白是补体抑制剂。然后，通过RNAi在血吸虫幼虫中抑制选定的SmCD59基因库的表达，以评估这些寄生虫在体外是否更容易受到补体的杀伤。在初步结果中，在血吸虫和成虫(两个适应于在血液中生存的生命阶段)中检测到所有SmCD59同源物的基因表达，并且每个SmCD59靶点的基因表达单独或在多达4个基因靶点的池中有效地在血吸虫中被抑制。如果RNAi研究成功，这将是一个强有力的证据，证明SmCD59在被膜中的表达是寄生虫对宿主补体的主要防御机制。然后，该项目可以很容易地扩展为一个更大的项目，以探索SmCD59补体抑制机制，并寻求在开发旨在减少血吸虫病发病率和/或发病率的新疗法或疫苗方面利用这一知识的策略。 公共卫生相关性：血吸虫是血吸虫，对世界卫生和农业造成重大问题，特别是在发展中国家。该项目建议使用现代RNA抑制方法来评估补体抑制蛋白是否能保护血吸虫免受宿主补体系统的杀伤。这些蛋白质很有希望成为血吸虫病新疗法和/或疫苗的靶标。

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.