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

• 批准号: 8391593
• 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
• 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/8391593
• 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' 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

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.

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，然而许多细菌病原体都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的III型分泌系统(T3SS)将细菌效应分子注入宿主细胞细胞质中，从而实现这一点。肠致病性大肠埃希菌(EPEC)是一种食源性非侵袭性致病菌，可引起腹泻。EPEC毒力因子包括T3SS和分泌的效应分子。该方案的假设是，宿主细胞对EPEC的促炎反应是由细胞外细菌因子驱动的，这种宿主反应的抑制依赖于T3SS向宿主细胞输送抗炎细菌分子。令人信服的初步数据表明，非Lee编码的EPEC效应物NleH1和NleH2(NleH1/2)具有抗炎活性，并抑制两个促炎信号通路：核因子-B和MAPK。更多的初步数据表明，抑制炎症促进了病原体的定植。本研究的总体目标是确定NleH1和NleH2在EPEC诱导的抗炎活性中的作用，阐明其潜在机制(S)，并探讨这些蛋白在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有关，因为我们的部队，特别是驻扎在海外偏远地区的部队，暴露在多种肠道病原体中，导致严重发病率，从而降低生产力。此外，患有感染性肠炎的新兵患炎症性肠病的风险增加，尤其是克罗恩病。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性疾病的新型治疗药物提供了机会。以下特定目的将解决这一假说：特定目的1.确定NleH1和NleH2在EPEC诱导的抗炎活性中的作用。明确NleH1/2抑制宿主炎症反应的机制(S)。具体目的3.在小鼠感染模型中确定NleH1/2在EPEC发病机制中的作用。两个独立的体外模型，感染和转基因，以及EPEC感染的体内小鼠模型将用于解决这些目标。用野生型EPEC或缺失突变体nleH1、nleH2或nleH1/2感染人和小鼠的肠上皮细胞，检测其对NF-：B和MAPK活化及炎性细胞因子表达的影响。细胞也将被导入表达细菌蛋白、NleH1和/或NleH2，然后用宿主细胞因子、细菌致炎分子或活的病原体攻击，并分析对宿主信号激活和炎症蛋白产生的影响。体外研究结果将与EPEC感染的小鼠体内模型相关联。动物将感染野生型EPEC或缺失突变株nleH1、nleH2或nleH1/2，并将确定其对肠道炎症、信号转导和细菌定植的影响。还将进行其他机制研究，以确定NleH1和NleH2中负责抗炎活性的特定氨基酸残基或基序。这项建议的长期目标是确定特定的抗炎细菌效应物及其作用机制(S)，以指导治疗性EPEC菌株的开发，这些菌株附着宿主肠道上皮细胞，注入抗炎蛋白，但不干扰其他上皮细胞功能。