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: 8445152
• 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/8445152
• 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.

描述（由申请人提供）： 这是我第一个值得奖励应用程序的第三次提交，目的是表征革兰氏阴性细菌的大毒素的影响及其对体外和体内肠上皮屏障功能的影响。淋巴结蛋白（LS）由淋巴细胞抑制因子A（LIFA）编码，这是革兰氏阴性细菌中存在的大基因，包括肠hap虫（EHEC）（EHEC），肠病毒大肠杆菌和cit虫杆菌。 LIFA已被确定为与EPEC诱导的腹泻和溶血性尿毒症综合征最强统计相关的单基因。 LIFA编码与微生物发病机理有关的关键酶活性：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框架内插入缺失突变，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拆卸。看来，糖基转移酶活性通过防止CDC42的激活来使ZO-1和occludin从紧密连接（TJ）中解离。假设1：LS对于形成TJ的蛋白质的拆卸至关重要，从而导致细胞细胞通透性增加，从而促进细菌易位和全身传播。目标1：定义淋巴结蛋白葡萄糖基转移酶基序会损害体外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导致Rho GTPase激活的失衡而介导的。我们表明，葡萄糖基转移酶参与CDC42途径的抑制。假设2：通过调节Rho GTPase CDC42途径，LS中存在的葡萄糖基转移酶活性对于TJS成分的拆卸至关重要。目的2：研究啮齿动物C. rudentium lifa葡萄糖基转移酶基序对体外GTPase CDC42信号级联的影响。最后，我们计划测试淋巴结蛋白葡萄糖基转移酶活性与动物模型中TJS完整性的相关性。假设3：LS诱导体内TJ的拆卸，从而导致粘膜，粘膜下和肠外器官的浸润和定殖。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对早期病理生理事件的理解，这些事件阐明了细菌在肠道感染期间如何调节肠上皮屏障功能，并获得粘膜和粘膜粘膜，并最终具有毁灭性的系统性后果。