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

基本信息

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
项目类别：
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

项目摘要

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）：本申请提出对血吸虫中一组假定的补体调节蛋白（CRP）进行功能表征，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，会引起一种慢性衰弱性疾病，影响全世界 2 亿多人，每年造成超过 25 万人死亡。这些蠕虫非常适合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。我们假设血吸虫在其体皮（覆盖与宿主直接接触的整个蠕虫表面的合胞膜）中表达 CRP，作为补体逃避的机制。通过基因组和转录组分析，鉴定出七种与人类 CD59 具有显着序列同源性的假定血吸虫 CRP。这些同源物在此被指定为 SmCD59-1、2、3、4、5、6、7。CD59 在人体组织中广泛分布。它是一种有效的成孔抑制剂通过与最终补体成分 C8 和 C9 相互作用，膜攻击复合物（MAC 或 C5b-9）。这里要测试的假设是这些 SmCD59 基因中的一个或多个可以保护血吸虫免受补体杀伤，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转染细胞的补体损伤的能力来确定哪些SmCD59蛋白是补体抑制剂。然后，通过 RNAi 抑制血吸虫中选定的 SmCD59 基因库的表达，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，检测到血吸虫和成虫（这两个生命阶段都适应在血液中生存）中所有 SmCD59 同源物的基因表达，并且每个 SmCD59 靶标单独或最多 4 个基因靶标池中的基因表达已被有效地检测到。血吸虫中受到抑制。如果 RNAi 研究成功，这将有力地证明体被中的 SmCD59 表达是寄生虫针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到更大的项目，以探索 SmCD59 补体抑制机制，并寻求利用这些知识开发新疗法或疫苗的策略，旨在降低血吸虫病的发病率和/或发病率。公共卫生相关性：血吸虫是一种血液蠕虫，给世界健康和农业造成重大问题，特别是在发展中国家。该项目建议采用现代 RNA 抑制方法来评估补体抑制蛋白是否可以保护血吸虫免受宿主补体系统的杀伤。这些蛋白质作为血吸虫病新疗法和/或疫苗的靶标具有良好的前景。