Regulation of proteins in the apical junctional complex by Citrobacter rodentium

啮齿类柠檬酸杆菌对顶端连接复合体中蛋白质的调节

• 批准号: 8244939
• 负责人: JAN-MICHAEL AXEL KLAPPROTH
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8244939
• 关键词: Adherens Junction; Animal Model; Apical; Award; Bacteria; Bacterial Translocation; Cessation of life; Child; Citrobacter rodentium; Clinical; Complex; Data; Diarrhea; Dissociation; Elderly; Enteral; Enterobacteriaceae; Epithelial; Epithelium; Escherichia coli; Escherichia coli EHEC; Event; Exhibits; Genes; Glucosyltransferase; Goals; Gram-Negative Bacteria; Guanosine Triphosphate Phosphohydrolases; Health; Hemolytic-Uremic Syndrome; Human; Immunosuppressive Agents; In Vitro; Individual; Infection; Intestines; Investigation; Iraq; Laboratories; Lead; Lymphocyte; Mediating; Modeling; Monomeric GTP-Binding Proteins; Morbidity - disease rate; Mucous Membrane; Mus; Organ; Pathogenesis; Pathway interactions; Permeability; Play; Proteins; Regulation; Research Project Grants; Role; Signal Pathway; Signal Transduction; Soldier; Structure; Submucosa; Testing; Tight Junctions; Toxin; design; enteropathogenic Escherichia coli; factor A; glycosyltransferase; in vivo; insertion/deletion mutation; member; microbial; mortality; mutant; novel; occludin; pathogenic Escherichia coli; prevent; public health relevance; rho GTP-Binding Proteins

DESCRIPTION (provided by applicant): This is the third submission for my first Merit Award application with the goal to characterize the effect of a large toxin from Gram negative bacteria and its effect on intestinal epithelial barrier function in vitro and in vivo. Lymphostatin (LS) is encoded by lymphocyte inhibitory factor A (lifA), a large gene present in Gram negative bacteria including Enterohemorrhagic (EHEC), Enteropathogenic E. coli, and Citrobacter rodentium. lifA has been identified as the single gene with the strongest statistical association with EPEC induced diarrhea and hemolytic-uremic syndrome caused by EHEC. lifA encodes for a critical enzymatic activity that have been implicated in microbial pathogenesis: a glucosyltransferase motif at 1.6 kb. We have generated new stable, specific in-frame insertion-deletion mutations inactivating this motif. Our preliminary data suggest that LS is involved in disassembly of epithelial barrier function. It appears that the glycosyltransferase activity is responsible for the dissociation of ZO-1 and occludin from tight junctions (TJs) by preventing activation of Cdc42. Hypothesis 1: LS is critical for the disassembly of proteins forming the TJs, resulting in increased paracellular permeability, promoting bacterial translocation and systemic dissemination. Aim 1: To define the mechanisms by which the lymphostatin glucosyltransferase motif impairs intestinal epithelial barrier function in vitro. Further, the effect of LS on TJs protein members appears to be mediated by causing an imbalance in Rho GTPase activation. We show that the glucosyltransferase is involved in inhibition of the Cdc42 pathway. Hypothesis 2: The glucosyltransferase activity present in LS is critical for the disassembly of TJs components by regulating Rho GTPase Cdc42 pathway. Aim 2: To investigate the effect of C. rodentium lifA glucosyltransferase motif on the small GTPase Cdc42 signaling cascade in vitro. Finally, we plan to test the relevance of lymphostatin glucosyltransferase activity for TJs integrity in an animal model. Hypothesis 3: LS induces disassembly of TJs in vivo, leading to invasion and colonization of mucosa, submucosa and extra-intestinal organs. Aim 3: To investigate the mechanism by which glucosyltransferase motif compromises intestinal epithelial barrier function in vivo. The proposed research project will contribute to our understanding of early pathophysiological events that clarify in how bacteria regulate intestinal epithelial barrier function during enteric infection and gain access to mucosa and submucosa with eventually devastating systemic consequences. PUBLIC HEALTH RELEVANCE: Infection with Gram negative bacteria, like Escherichia coli, is a worldwide health threat resulting in diarrhea with significant morbidity and mortality among children, elderly, and deployed troops. Lymphostatin is a large, novel toxin present in Gram negative bacteria, including pathogenic Escherichia coli strains and Citrobacter rodentium infect the intestine and other organs in humans and mice. Our experimental results suggest that lymphostatin regulates intestinal epithelial barrier function, disrupting its integrity. The current proposal investigates the effect of lymphostatin on specific proteins constituting the intestinal epithelial barrier and their regulating pathways.

描述（由申请人提供）： 这是我的第一个优异奖申请的第三次提交，目的是描述革兰氏阴性菌的大毒素的作用及其对体外和体内肠上皮屏障功能的影响。淋巴抑素 (LS) 由淋巴细胞抑制因子 A (lifA) 编码，这是革兰氏阴性菌中存在的一个大基因，包括肠出血性 (EHEC)、肠病性大肠杆菌和啮齿类柠檬酸杆菌。 lifA 已被确定为与 EPEC 诱发的腹泻和 EHEC 引起的溶血尿毒症综合征具有最强统计相关性的单基因。 lifA 编码与微生物发病机制有关的关键酶活性：1.6 kb 的葡萄糖基转移酶基序。我们已经生成了新的稳定的、特定的框内插入删除突变，使该基序失活。我们的初步数据表明 LS 参与上皮屏障功能的分解。糖基转移酶活性似乎通过阻止 Cdc42 的激活来导致 ZO-1 和 occludin 从紧密连接 (TJ) 上解离。假设 1：LS 对于形成 TJ 的蛋白质分解至关重要，导致细胞旁通透性增加，促进细菌易位和全身传播。目标 1：明确淋巴抑制素葡萄糖基转移酶基序在体外损害肠上皮屏障功能的机制。此外，LS 对 TJs 蛋白成员的影响似乎是通过引起 Rho GTPase 激活失衡来介导的。我们表明葡萄糖基转移酶参与 Cdc42 途径的抑制。假设2：LS中存在的葡萄糖基转移酶活性通过调节Rho GTPase Cdc42途径对于TJs组分的分解至关重要。目标 2：体外研究 C. rodentium lifA 葡萄糖基转移酶基序对小 GTPase Cdc42 信号级联的影响。最后，我们计划在动物模型中测试淋巴抑素葡萄糖基转移酶活性与 TJ 完整性的相关性。假设3：LS在体内诱导TJ解体，导致粘膜、粘膜下层和肠外器官的侵袭和定植。目标 3：研究葡萄糖基转移酶基序损害体内肠上皮屏障功能的机制。拟议的研究项目将有助于我们了解早期病理生理学事件，阐明细菌如何在肠道感染期间调节肠上皮屏障功能，并进入粘膜和粘膜下层，最终造成毁灭性的系统后果。 公共卫生相关性： 大肠杆菌等革兰氏阴性菌感染是一种全球性的健康威胁，会导致腹泻，在儿童、老年人和部署的部队中具有显着的发病率和死亡率。淋巴抑素是革兰氏阴性细菌中存在的一种大型新型毒素，包括致病性大肠杆菌菌株和啮齿类柠檬酸杆菌，可感染人类和小鼠的肠道和其他器官。我们的实验结果表明淋巴抑素调节肠上皮屏障功能，破坏其完整性。目前的提案研究淋巴抑素对构成肠上皮屏障的特定蛋白质的影响及其调节途径。