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: 8142639
• 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/8142639
• 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; 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)： 这是我第三次申请荣誉奖，目的是表征来自革兰氏阴性细菌的一种大型毒素的影响及其在体外和体内对肠上皮屏障功能的影响。淋巴抑制素(LS)由淋巴细胞抑制因子A(LifA)编码，LifA是革兰氏阴性菌中的一个大基因，存在于肠出血性(EHEC)、肠源性大肠杆菌和轮状柠檬酸杆菌等细菌中。LifA基因已被确定为与EPEC引起的腹泻和EHEC引起的溶血性尿毒症综合征具有最强统计学相关性的单基因。LifA编码一个与微生物致病有关的关键酶活性：1.6kb的葡萄糖基转移酶基序。我们已经产生了新的稳定的、特异的框内插入-删除突变，使这个基序失活。我们的初步数据表明，LS参与了上皮屏障功能的解体。糖基转移酶的活性似乎是导致ZO-1和occludin从紧密连接(TJ)中解离的原因，它通过阻止CDC42的激活来实现。假设1：LS对形成TJS的蛋白质的分解至关重要，导致细胞旁通透性增加，促进细菌易位和全身传播。目的1：探讨淋巴组织抑制素葡萄糖基转移酶基序在体外损伤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激活的失衡来介导的。我们发现葡萄糖转移酶参与了对CDC42途径的抑制。假设2：LS中存在的葡萄糖转移酶活性对于通过调节Rho GTPase CDC42途径来分解TJS组分是至关重要的。目的：研究轮齿乳杆菌Lifa糖基转移酶基序在体外对小分子GTP酶CDC42信号通路的影响。最后，我们计划在动物模型中测试淋巴抑素葡萄糖转移酶活性与TJS完整性的相关性。假设3：LS在体内诱导TJ的分解，导致粘膜、粘膜下层和肠外器官的侵袭和定植。目的：探讨葡萄糖转移酶模体在体内对肠上皮屏障功能的影响机制。拟议的研究项目将有助于我们了解早期的病理生理事件，阐明细菌在肠道感染期间如何调节肠道上皮屏障功能，并进入粘膜和粘膜下层，最终造成毁灭性的全身后果。 公共卫生相关性： 感染革兰氏阴性细菌，如大肠杆菌，是一种世界性的健康威胁，导致腹泻，在儿童、老年人和部署的部队中具有显著的发病率和死亡率。淋巴抑制素是一种存在于革兰氏阴性菌中的新型大毒素，包括致病性大肠埃希菌和轮状柠檬酸杆菌，可感染人和小鼠的肠道和其他器官。我们的实验结果表明，淋巴抑制素调节肠上皮屏障功能，破坏其完整性。目前的建议是研究淋巴抑制素对构成肠上皮屏障的特定蛋白及其调节途径的影响。