Enteropathogenic E. coli effector proteins in intestinal tight junction recovery

肠病性大肠杆菌效应蛋白在肠道紧密连接恢复中的作用

• 批准号: 8435483
• 负责人: Lila Gollogly Glotfelty
• 金额: $ 3.39万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-18 至 2017-02-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8435483
• 关键词: Address; Adult; Affect; Apical; Back; Bacterial Proteins; Biological Assay; Biotinylation; Brefeldin A; Calcium; Cell Polarity; Cell membrane; Cell physiology; Cells; Cellular Structures; Cytoplasm; Cytoskeleton; Cytosol; Developing Countries; Development; Diarrhea; Diffuse; Epithelial; Epithelial Cells; Epithelium; Escherichia coli Infections; Golgi Apparatus; Health; Homeostasis; In Vitro; Infantile Diarrhea; Infection; Integral Membrane Protein; Intestines; Ion Transport; Ions; Link; Maintenance; Malnutrition; Measures; Membrane; Membrane Proteins; Microtubules; Minor; Morbidity - disease rate; Movement; Mus; Nocodazole; Pathogenesis; Play; Prevention; Process; Proteins; Recovery; Recurrence; Recycling; Role; Structure; System; Tight Junctions; Toxin; Transgenic Mice; Transgenic Organisms; Type III Secretion System Pathway; Water; anterograde transport; apical membrane; basolateral membrane; chelation; early childhood; enteropathogenic Escherichia coli; in vivo; infancy; mortality; mutant; novel therapeutics; occludin; pathogen; prevent; protein transport; reconstruction; repaired; trafficking

DESCRIPTION (provided by applicant): Enteropathogenic E. coli (EPEC) infection is a leading cause of morbidity and mortality from diarrhea in developing countries. The mechanisms underlying its pathogenesis are not completely understood. Epithelial tight junctions (TJs) restrict the passage of water and ions across the epithelium. TJs are not static; proteins constantly recycle between the plasma membrane and the cytoplasm with contributions of newly synthesized TJ proteins. EPEC induce TJ disruption in a type III secretion system dependent manner. The hypothesis of this proposal is that specific EPEC effector molecules block homeostatic movement of TJ proteins that maintain and restore TJ structure and barrier function. EPEC effectors EspG1/2 destroy microtubule (MT) networks. We hypothesize that these networks are essential for recycling TJ proteins back to the membrane. NleA interferes with anterograde transport through the ER and Golgi, potentially preventing newly synthesized TJ proteins from reaching the TJ. Together, these effectors likely contribute to disruption of TJ structure and function by impeding repair mechanisms. The following specific aims will address this hypothesis. 1: To determine if EPEC infection delays recovery of TJ structure and barrier function following disruption. The impact of EPEC effectors on TJ recovery after calcium chelation will be determined. The duration of effector expression in infected intestinal epithelial cells (IEC) will be defined using myc-tagged effectors. It is anticipated that effector proteins will persist during TJ recovery and that recovery will be significantly delayed. 2: To investigate the effect of EPEC- induced microtubule disruption on the homeostatic maintenance and recovery of TJs. Biotinylation assays will be used to determine the rate of TJ protein recycling after infection with EPEC WT or DespG1/2. IEC will be transfected to express EspG1, or treated with nocodazole to disrupt MTs. Effects on TJ recovery will be determined. In vivo studies will be performed using a transgenic EGFP-occludin mouse. The rates of intestinal epithelial TJ recovery following infection with wild-type (WT) EPEC versus DespG1/2 will be compared. It is anticipated that more TJ proteins will recycle and TJ recovery will be more rapid in the absence of EspG1/2. 3: To investigate the contribution of EPEC-induced interference of ER-Golgi trafficking of newly synthesized proteins on TJ maintenance and reconstruction and to determine the combined effect of EPEC effectors on TJ recovery. IEC will be transfected to express NleA, or treated with brefeldin A, and the effects on TJ recovery following calcium chelation will be measured. In vivo studies using EGFP-occludin transgenic mice will be performed to compare the rate of TJ recovery after infection with WT EPEC and DnleA. A triple DespG1/2/nleA mutant will be created and its effects on recovery of TJ structure and function will be determined. We hypothesize that NleA will significantly impact TJ recovery structurally and functionally, but that the triple mutant will recover the most rapidly. This proposal is the first to investigate TJ recovery as a pathogenic target. It directly addresses a need for greater understanding of how EPEC affects the intestinal epithelial barrier.

描述（由申请人提供）：肠病大肠杆菌（EPEC）感染是发展中国家腹泻发病和死亡率的主要原因。其发病机理的基础机制尚未完全理解。上皮紧密连接（TJS）限制了水和离子在上皮细胞中的通过。 TJ不是静态的；蛋白质不断在质膜和细胞质之间回收，并具有新合成的TJ蛋白的贡献。 EPEC以III型分泌系统依赖性方式诱导TJ破坏。该提议的假设是特定的EPEC效应分子阻止了维持和恢复TJ结构和屏障功能的TJ蛋白的稳态运动。 EPEC效应器ESPG1/2破坏微管（MT）网络。我们假设这些网络对于将TJ蛋白回收回膜至关重要。 NLEA干扰了通过ER和高尔基体的顺行转运，有可能阻止新合成的TJ蛋白到达TJ。这些效应子在一起可能会通过阻碍修复机制来破坏TJ结构和功能。以下具体目标将解决这一假设。 1：确定EPEC感染是否会延迟破坏后TJ结构和屏障功能的恢复。将确定EPEC效应子对钙螯合后TJ恢复的影响。感染肠上皮细胞（IEC）中效应子表达的持续时间将使用MYC标记的效应子定义。预计效应蛋白将在TJ恢复期间持续存在，并且该恢复将大大延迟。 2：研究诱导的微管破坏对TJ的稳态维持和恢复的影响。生物素化测定将用于确定用EPEC WT或DESPG1/2感染TJ蛋白回收的速率。 IEC将被转染以表达ESPG1，或用Nocodazole处理以破坏MT。将确定对TJ恢复的影响。体内研究将使用转基因EGFP-Occludin小鼠进行。将比较野生型（WT）EPEC与DESPG1/2感染后肠上皮TJ恢复的速率。可以预计，在没有ESPG1/2的情况下，更多的TJ蛋白会回收，而TJ恢复会更快。 3：研究EPEC诱导的新合成蛋白对TJ维持和重建的ER-Golgi运输的干扰的贡献，并确定EPEC效应子对TJ恢复的综合作用。 IEC将被转染以表达NLEA或用Brefeldin A处理，并测量钙螯合后对TJ恢复的影响。使用EGFP-蛋白酶转基因小鼠进行体内研究，将进行与WT EPEC和DNLEA感染后TJ恢复速率的研究。将创建一个三重DESPG1/2/NLEA突变体，并确定其对TJ结构和功能恢复的影响。我们假设NLEA将在结构和功能上显着影响TJ恢复，但是三重突变体的恢复最快。该提案是第一个研究TJ恢复作为致病靶标的提案。它直接解决了对EPEC如何影响肠上皮屏障的需求。