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

基于 RNAi 的血吸虫 CD59 样补体调节剂功能分析

• 批准号: 8384985
• 负责人: Greice Krautz-Peterson
• 金额: $ 8.25万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8384985
• 关键词: Adult; Agriculture; Antibodies; Blood; Blood Circulation; Cell membrane; Cells; Cessation of life; Chinese Hamster Ovary Cell; Chronic; Complement; Complement Activation; Complement Inactivators; Complement Membrane Attack Complex; Complement component C8; Cytolysis; Defense Mechanisms; Development; Disease; Environment; Focus Groups; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genome; Homologous Gene; Human; Immunologic Surveillance; In Vitro; Incidence; Knowledge; Leukocytes; Life; Mammalian Cell; Mediating; Membrane; Methods; Morbidity - disease rate; Parasites; Platyhelminths; Predisposition; Proteins; Proteomics; RNA; RNA Interference; Resistance; Role; Schistosoma; Schistosomatidae; Schistosomiasis; Sequence Homology; Staging; Surface; Testing; Vaccines; Variant; World Health; base; cellular engineering; complement system; genetic regulatory protein; human tissue; inhibitor/antagonist; killings; prevent; therapeutic target

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.

描述（由申请人提供）：本申请提议在功能上表征血吸虫中一组假定的补体调节蛋白（CRP），以通过通过RNA干扰（RNAI）抑制其基因表达来评估其作为治疗靶标的潜力。血吸虫是扁虫寄生虫，引起长期和使人衰弱的疾病，遭受了全世界超过2亿人的折磨，每年造成25万多人死亡。这些蠕虫非常适应其脊椎动物宿主的脉管系统数十年，显然已经制定了逃避宿主免疫监视的策略，包括有效的补体系统。我们假设血吸虫在其tegument中表达CRP（一种覆盖整个蠕虫表面的合成膜，直接与宿主接触）是一种补体逃避的机制。使用基因组和转录组分析，确定了七个与人CD59具有显着序列同源性同源性的假定的实质组CRP。这些同源物在这里被指定为SMCD59-1、2、2、3、4、5、6、7。CD59在人体组织中具有广泛的分布。它是孔形成的有效抑制剂 通过与最终补体组件C8和C9相互作用，膜攻击复合物（MAC或C5B-9）。这里要检验的假设是，这些SMCD59基因中的一个或多个保护血吸虫免受补充杀戮，就像CD59保护宿主的细胞一样。首先，将进行研究，以确定哪种SMCD59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，并测试它们防止对转染细胞的补体损害的能力。然后，RNAi将在血吸虫中抑制选定的SMCD59基因库的表达，以评估这些寄生虫是否更容易受到体外补体杀伤的影响。在初步结果中，检测到血吸虫和成人蠕虫中所有SMCD59同源物的基因表达（两个适合于血液中生存的生命阶段）和每个SMCD59靶标的基因表达，或在长达4个基因靶标中有效地抑制了schistosomula。如果RNAi研究成功，这将是有力的证据表明，在Tegument中的SMCD59表达是反对宿主补体​​的主要寄生虫防御机制。然后可以轻松地将该项目扩展到一个更大的项目中，以探索SMCD59补体抑制机制，并采用在开发新疗法或疫苗的开发中利用这些知识的策略，旨在减少血吸虫病的发病率和/或发病率。 公共卫生相关性：血块是血液居住的蠕虫，造成世界卫生和农业的主要问题，尤其是在发展中国家。该项目建议采用现代的RNA抑制方法来评估补体抑制蛋白是否保护血吸虫免受宿主补体系统的杀戮。这些蛋白质作为针对血吸虫病的新疗法和/或疫苗的靶标具有极好的希望。