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蛋白质的调节

• 批准号: 7558928
• 负责人: JAN-MICHAEL AXEL KLAPPROTH
• 金额: $ 6.98万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7558928
• 关键词: Accounting; Actins; Adherence; Adherens Junction; Affect; Apical; Bacteria; Bacterial Translocation; Cell Adhesion Molecules; Cell Culture Techniques; Chlamydia; Citrobacter rodentium; Colon; Complex; Cytoskeleton; Cytotoxin; Data; Disease; Dissociation; Distant; Enteral; Enterobacteriaceae; Enterocytes; Epithelial; Epithelial Cells; Equilibrium; Escherichia coli EHEC; Family; Foundations; Funding; Genes; Genitourinary system; Glucosyltransferase; Glucosyltransferases; Goals; Gram-Negative Bacteria; Human; Immune response; Immunocompetent; In Vitro; Infection; Intestinal Mucosa; Intestines; Investigation; Lung; Lymphocyte; Maintenance; Mediating; Mesentery; Mitochondria; Modeling; Morbidity - disease rate; Mucous Membrane; Mus; Mutation; Organ; Pathogenesis; Pathogenicity Island; Pathway interactions; Peptide Hydrolases; Proteins; Regulation; Research Project Grants; Resistance; Signal Transduction; Spleen; Submucosa; Tight Junctions; Toxin; Transaminases; Yersinia; cytokine; enteropathogenic Escherichia coli; equilibration disorder; factor A; glycosyltransferase; in vivo; insertion/deletion mutation; lymph nodes; member; microbial; mortality; pathogenic Escherichia coli; prevent; protein complex; receptor; response; rho; rho GTP-Binding Proteins

DESCRIPTION (provided by applicant): The goal of this proposal is to characterize the effect of a large toxin from Gram negative bacteria and its effect on 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, Enteropathogenic E. coli, and Citrobacter rodentium. lifA encodes for two critical enzymatic activities that have been implicated in microbial pathogenesis: a glycosyltransferase (1.6kb) and protease motif (4.5 - 4.8kb). We have generated two stable, specific in- frame insertion-deletion mutations inactivating both motifs. 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 from tight junctions by preventing activation of Cdc42. In addition, we provide evidence that the protease activity leads to dissociation of ¿-catenin from adherens junctions by activating RhoA. Hypothesis 1: LS is critical for the disassembly of proteins forming the apical junctional complex (AJC), resulting in decreased transepithelial resistance (TER), promoting bacterial translocation and systemic dissemination. Aim 1: To study the effect of C. rodentium LS glycosyltransferase and protease activity on proteins constituting the AJC in differentiated epithelial cell cultures in vitro and in mice in vivo. Further, the effect of LS on members of the AJC appears to be mediated by causing an imbalance in Rho GTPase activation. We show that the protease motif regulates activation of Rho and suppresses Cdc42, whereas the glycosyltransferase is only involved in inhibition of Cdc42. Hypothesis 2: LS glycosyltransferase and protease activity are both critical for the disassembly of AJC components by regulating Rho GTPases. Aim 2: To investigate the activation cascade of Rho GTPases signaling in response to C. rodentium WT lifA, and inactivated protease- and glycosyltransferase motif in vitro and in vivo. The proposed research project will contribute to our understanding of how bacteria regulate epithelial barrier function during enteric infection and gain access to mucosa and submucosa with eventually systemic consequences. Lymphostatin is a large toxin from Gram negative bacteria, including pathogenic E. coli and Chlamydia spp. that account for significant morbidity and mortality by infecting gut, lungs, and the genitourinary tract. Our experimental results suggest that lymphostatin inhibits the immune response and regulates intestinal barrier function. The current proposal investigates the specific host proteins affected by lymphostatin and deregulated pathways.

描述（由申请人提供）： 该提案的目的是表征革兰氏阴性菌的一种大毒素的作用及其对体外和体内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)（一种存在的大基因）编码。在革兰氏阴性细菌（包括肠出血性大肠杆菌和啮齿类柠檬酸杆菌）中，编码两种与微生物有关的关键酶活性。发病机制：糖基转移酶 (1.6kb) 和蛋白酶基序 (4.5 - 4.8kb) 我们已经生成了两个稳定的、特定的框内插入-缺失突变，使这两个基序失活。看来，糖基转移酶活性通过阻止 ZO-1 的激活而导致 ZO-1 从紧密连接上解离。此外，我们提供了蛋白酶活性导致 ¿ 解离的证据。假设 1：LS 对于形成顶端连接复合物 (AJC) 的蛋白质分解至关重要，从而导致跨上皮阻力 (TER) 降低，促进细菌易位和全身传播。啮齿类动物LS糖基转移酶和蛋白酶活性对分化上皮细胞AJC组成蛋白的影响此外，LS 对 AJC 成员的影响似乎是通过引起 Rho GTPase 激活失衡来介导的，我们表明蛋白酶基序调节 Rho 的激活并抑制 Cdc42，而糖基转移酶。仅参与 Cdc42 的抑制。 假设 2：LS 糖基转移酶和蛋白酶活性对于 AJC 成分的分解都至关重要。目标 2：研究 Rho GTPases 信号传导在体外和体内对 C. rodentium WT lifA 和失活蛋白酶和糖基转移酶基序的激活级联反应。淋巴抑素在肠道感染过程中调节上皮屏障功能，并进入粘膜和粘膜下层，最终产生全身性后果。细菌，包括致病性大肠杆菌和衣原体，它们通过感染肠道、肺部和泌尿生殖道而导致显着的发病率和死亡率。我们的实验结果表明淋巴抑素抑制免疫反应并调节肠道屏障功能。受淋巴细胞抑制素和失调途径影响的特定宿主蛋白。