MAPK-mediated defense pathways against pore-forming toxins

描述(申请人提供)：本申请的广泛目标是了解宿主细胞如何防御由病原菌产生的形成毛孔的毒素。长期目标是利用这些信息开发新的治疗方法，通过增强细胞对毛孔形成毒素的防御来保护人类免受细菌感染。形成孔洞的毒素是细菌毒力因子中最大的一组，占细菌产生的所有蛋白质毒素的25%。它们对包括金黄色葡萄球菌和链球菌在内的许多人类主要细菌病原体的体内毒力都很重要。秀丽隐杆线虫已经成为一种重要的遗传易控的活体系统，用于研究动物细胞如何以与哺乳动物相关的方式防御形成毛孔的毒素。C-Jun氨基末端酶(JNK)丝裂原活化蛋白激酶(MAPK)是线虫细胞对致孔毒素诱导防御的中心调节因子，调节着PFT诱导的半数防御。具体目的I将利用正向和反向遗传学、遗传和生化测试、定量中毒分析和表达分析来尽可能完整地组装线虫JNK PFT诱导的防御途径，使其上游的JNK形成初始孔，下游的JNK进入受调控的防御亚途径。特殊目的II涉及使用经医学上相关的成孔毒素处理的哺乳动物细胞来确定线虫的防御在哺乳动物细胞中保存得如何。特殊目的III使用在线虫中开发的一套独特和强大的分析方法来研究JNK PFT诱导的防御途径保护细胞免受PFT攻击的实际机制。第三个特定目标的另一个目标是发现PFT防御的新细胞机制。 与公共卫生相关：在感染期间，许多致病细菌会分泌形成毛孔的毒素，这些毒素会在细胞膜上穿孔，以此作为破坏和使我们的细胞瘫痪的手段。在这项应用中，我们将研究我们的细胞如何反击并保护自己免受这些毒素的伤害。我们的目标是利用这些信息设计新的药物，增强我们对细菌毒素和疾病的防御能力。

DESCRIPTION (provided by applicant): The broad objective of this application is to understand how host cells defend themselves against pore-forming toxins made by pathogenic bacteria. The long term objective is to use this information for development of novel therapeutics that protects humans against bacterial infection by boosting cellular defenses against pore-forming toxins. Pore-forming toxins are the largest single group of bacterial virulence factors, comprising >25% of all protein toxins made by bacteria. They are important for the in vivo virulence of many major bacterial pathogens of humans, including Staphylococcus aureus and Streptococci. Caenorhabditis elegans has emerged as an important genetically-tractable in vivo system for studying how animal cells defend against pore-forming toxins in a manner relevant to mammals. c-JUN N-terminal Kinase (JNK) mitogen-activated protein kinase (MAPK) is a central regulator of induced cellular defenses to pore-forming toxins in C. elegans, regulating half of all PFT-induced defenses. Specific Aim I will use forward and reverse genetics, genetic and biochemical tests, quantitative intoxication assays, and expression analyses to assemble as completely as possible the C. elegans JNK PFT-induced defense pathway, heading both upstream of JNK towards initial pore-formation and downstream of JNK into regulated defense subpathways. Specific Aim II involves the use of mammalian cells treated with a medically relevant pore-forming toxin to ascertain how well C. elegans defenses are conserved in mammalian cells. Specific Aim III uses a unique and powerful set of assays developed in C. elegans to study the actual mechanisms by which the JNK PFT-induced defense pathway protects cells against PFT attack. An additional goal of Specific Aim III is to uncover new cellular mechanisms for PFT defenses. PUBLIC HEALTH RELEVANCE: During infection, many disease-causing bacteria secrete pore-forming toxins that punch holes in cell membranes as a means towards damaging and disabling our cells. In this application, we will study how our cells fight back and protect themselves against these toxins. Our goal is to use this information to design new drugs that boost our own defenses against bacterial toxins and disease.