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Interactions of Enteropathogenic E Coli with Intestinal Epithelial Cells

致病性大肠杆菌与肠上皮细胞的相互作用

基本信息

批准号：
8597372
负责人：
Gail A Hecht
金额：
--
依托单位：
JESSE BROWN VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-10-01 至 2014-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8597372
关键词：
Address Amino Acids Animals Anti-Inflammatory Agents Anti-inflammatory Area Attenuated Bacteria Bacterial Proteins Cell physiology Cells Cessation of life Child Chromosomes Colitis Country Crohn&apos s disease Cytoplasm DNA Data Detection Developing Countries Development Diarrhea Disease Dose Elements Enterocytes Epithelial Cells Escherichia coli Infections Flagellin Funding Gastroenteritis Gastrointestinal tract structure Genes Goals Healthcare Homologous Gene Human Immune response In Vitro Infection Infectious Agent Inflammation Inflammatory Inflammatory Bowel Diseases Inflammatory Response Inflammatory disease of the intestine Intestines Investigation Life MAPK3 gene MAPK8 gene Military Personnel Mission Mitogen-Activated Protein Kinases Modeling Morbidity - disease rate Mus NF-kappa B Needles Odds Ratio Pathogenesis Pathogenicity Island Pathway interactions Phenotype Prevention strategy Production Productivity Proteins Recruitment Activity Research Resources Risk Role Signal Pathway Signal Transduction Sterility Stimulus Structure Syringes System Testing Therapeutic Agents Time Toll-like receptors Transfection Virulence Factors Water Work cytokine enteric pathogen enteritis enteropathogenic Escherichia coli experience extracellular foodborne foodborne pathogen in vitro Model in vivo mitogen-activated protein kinase p38 mutant novel therapeutics pathogen prevent public health relevance response therapeutic development transcription factor waterborne

项目摘要

DESCRIPTION (provided by applicant): The host response to infection by enteric pathogens is intestinal inflammation however many bacterial pathogens possess strategies to suppress this inflammatory response. Some enteric pathogens accomplish this by injecting bacterial effector molecules into the host cell cytoplasm via a syringe-like type III secretory system (T3SS). Enteropathogenic Escherichia coli (EPEC) is a foodborne non-invasive pathogen that causes diarrhea. EPEC virulence factors include a T3SS and secreted effector molecules. The hypothesis of this proposal is that the pro-inflammatory response of host cells to EPEC is driven by extracellular bacterial factors and that suppression of this host response depends on delivery of anti-inflammatory bacterial molecules into host cells by the T3SS. Compelling preliminary data suggest that the non-LEE encoded EPEC effectors, NleH1 and NleH2 (NleH1/2), possess anti-inflammatory activity and inhibit two pro-inflammatory signaling pathways, NF-:B and MAP kinase (MAPK). Additional preliminary data suggest that suppression of inflammation promotes colonization of the pathogen The overall goal of this proposal is to define the role of NleH1 and NleH2 in EPEC-induced anti-inflammatory activity, elucidate the underlying mechanism(s), and explore the role of these proteins in EPEC pathogenesis. This work is relevant to the VA healthcare mission as our troops, especially those stationed in remote areas overseas, are exposed to multiple enteric pathogens that cause significant morbidity thus reducing productivity. In addition, military recruits who have suffered infectious enteritis are at increased risk for the development of inflammatory bowel disease, especially Crohn's disease. Furthermore, defining the strategies by which bacterial pathogens modulate the host inflammatory response provides opportunities for the development of novel therapeutic agents for other intestinal inflammatory diseases. The following Specific Aims will address this hypothesis: Specific Aim 1. To define the roles of NleH1 and NleH2 in EPEC-induced anti-inflammatory activity. Specific Aim 2. To define the mechanism(s) by which NleH1/2 inhibit host inflammatory responses. Specific Aim 3. To determine the role of NleH1/2 in EPEC pathogenesis in a murine model of infection. Two independent in vitro models, infection and transfection, and an in vivo murine model of EPEC infection will be used to address these aims. Human and mouse intestinal epithelial cells will be infected with wildtype EPEC or deletion mutants nleH1, nleH2, or nleH1/2 and the effects on NF-:B and MAPK activation and on the expression of inflammatory cytokines will be tested. Cells will also be transfected to express bacterial proteins, NleH1, NleH2, or both, and then challenged with host cytokines, bacterial pro-inflammatory molecules, or live pathogens and the impact on activation of host signaling and production of inflammatory proteins will be analyzed. The in vitro findings will be correlated in an in vivo murine model of EPEC infection. Animals will be infected with wildtype EPEC or deletion mutant strains nleH1, nleH2, or nleH1/2, and the effects on intestinal inflammation and signaling and bacterial colonization will be determined. Additional mechanistic studies will be performed to identify specific amino acid residues or motifs in NleH1 and NleH2 that are responsible for the anti-inflammatory activity. The long-term goal of this proposal is to identify specific anti-inflammatory bacterial effectors and their mechanism(s) of action that would guide the development of therapeutic EPEC strains that attach to host intestinal epithelial cells and inject anti-inflammatory proteins but not interfere with other epithelial cell functions. PUBLIC HEALTH RELEVANCE: Infection with food- and water-borne pathogens increased 93% between 1979 and 2004 and deaths from GI infections tripled recently)(1). Infectious diarrhea diminishes significantly productivity especially of military personnel serving in resource-deficient countries. In addition, active duty military personnel who experienced infectious gastroenteritis had an increased risk (odds ratio, 1.4) for the development of inflammatory bowel disease (2). The mechanisms of pathogenesis of many of these infectious organisms are not understood. Studies focused on delineating how these bacteria cause disease will result in the development of new strategies for prevention, treatment, and avoidance of the sequelae that result from these infections. This research will focus on enteropathogenic E. coli (EPEC) and how these bacteria control the inflammatory response of the host by injecting proteins into intestinal cells through a needle-like structure, which block inflammation allowing the bacteria to persist for a longer time in the intestine.

描述（由申请人提供）：宿主对肠道病原体感染的反应是肠道炎症，但许多细菌病原体具有抑制这种炎症反应的策略。一些肠道病原体通过注射器样 III 型分泌系统 (T3SS) 将细菌效应分子注射到宿主细胞的细胞质中来实现这一点。肠致病性大肠杆菌 (EPEC) 是一种引起腹泻的食源性非侵入性病原体。 EPEC毒力因子包括T3SS和分泌的效应分子。该提议的假设是，宿主细胞对 EPEC 的促炎反应是由细胞外细菌因子驱动的，而对这种宿主反应的抑制取决于 T3SS 将抗炎细菌分子递送到宿主细胞中。令人信服的初步数据表明，非 LEE 编码的 EPEC 效应子 NleH1 和 NleH2 (NleH1/2) 具有抗炎活性，并抑制两条促炎信号通路 NF-:B 和 MAP 激酶 (MAPK)。其他初步数据表明，抑制炎症会促进病原体定植。该提案的总体目标是确定 NleH1 和 NleH2 在 EPEC 诱导的抗炎活性中的作用，阐明潜在机制，并探索这些蛋白质在 EPEC 发病机制中的作用。这项工作与退伍军人管理局的医疗保健任务相关，因为我们的部队，特别是驻扎在海外偏远地区的部队，暴露于多种肠道病原体，这些病原体会导致严重的发病率，从而降低生产力。此外，患有传染性肠炎的新兵患炎症性肠病，特别是克罗恩病的风险增加。此外，确定细菌病原体调节宿主炎症反应的策略为开发其他肠道炎症疾病的新型治疗剂提供了机会。以下具体目标将解决这一假设：具体目标 1. 明确 NleH1 和 NleH2 在 EPEC 诱导的抗炎活性中的作用。具体目标 2. 明确 NleH1/2 抑制宿主炎症反应的机制。具体目标 3. 确定 NleH1/2 在小鼠感染模型中 EPEC 发病机制中的作用。两个独立的体外模型（感染和转染）以及 EPEC 感染的体内小鼠模型将用于实现这些目标。人类和小鼠肠上皮细胞将被野生型 EPEC 或缺失突变体 nleH1、nleH2 或 nleH1/2 感染，并测试对 NF-:B 和 MAPK 激活以及炎症细胞因子表达的影响。细胞还将被转染以表达细菌蛋白、NleH1、NleH2 或两者，然后用宿主细胞因子、细菌促炎分子或活病原体进行攻击，并分析对宿主信号传导激活和炎症蛋白产生的影响。体外研究结果将与 EPEC 感染的体内小鼠模型相关。动物将被野生型 EPEC 或缺失突变菌株 nleH1、nleH2 或 nleH1/2 感染，并确定对肠道炎症和信号传导以及细菌定植的影响。将进行额外的机制研究，以确定 NleH1 和 NleH2 中负责抗炎活性的特定氨基酸残基或基序。该提案的长期目标是确定特定的抗炎细菌效应物及其作用机制，以指导开发治疗性 EPEC 菌株，这些菌株附着于宿主肠上皮细胞并注入抗炎蛋白，但不干扰其他上皮细胞功能。公共卫生相关性： 1979 年至 2004 年间，食源性和水源性病原体感染增加了 93%，胃肠道感染导致的死亡人数最近增加了两倍）(1)。感染性腹泻会显着降低生产力，尤其是在资源匮乏国家服役的军事人员的生产力。此外，患有传染性胃肠炎的现役军人患炎症性肠病的风险增加（优势比为 1.4）(2)。许多这些传染性生物体的发病机制尚不清楚。研究重点是描述这些细菌如何引起疾病，这将有助于制定预防、治疗和避免这些感染引起的后遗症的新策略。这项研究将重点关注致病性大肠杆菌（EPEC），以及这些细菌如何通过针状结构将蛋白质注入肠道细胞来控制宿主的炎症反应，从而阻止炎症，从而使细菌在肠道中存活更长时间。