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

基本信息

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
项目类别：
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

项目摘要

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细菌的大毒素的影响及其对体外和体内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，包括肠内霍尼，肠病，肠病大肠杆菌和柠檬酸杆菌。 LIFA编码在微生物发病机理中实现的两种关键酶促活性：糖基转移酶（1.6KB）和蛋白酶基序（4.5-4.8KB）。我们已经产生了两个稳定的特异性插入插入缺失突变，使两个基序失活。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从紧密连接处解离。此外，我们还提供了证据表明，蛋白酶活性通过激活RhoA导致 - 帕宁蛋白与粘附连接的解离。假设1：LS对于形成顶端连接络合物（AJC）的蛋白质的拆卸至关重要，从而改善了旋转抗耐药性（TER），促进细菌易位和全身传播。目标1：研究啮齿动物C. rdentium ls糖基转移酶和蛋白酶活性对分化上皮细胞培养物在体外和小鼠体内构成AJC的蛋白质的影响。此外，LS对AJC成员的影响似乎是通过在Rho GTPase激活中导致失衡而介导的。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抑制Cdc42。假设2：通过调节Rho GTPases，LS糖基转移酶和蛋白酶活性对于AJC成分的拆卸至关重要。目的2：研究Rho GTPases信号的激活级联反应于啮齿动物C. wt LIFA，以及在体外和体内的非活性蛋白酶和糖基转移酶基序。拟议的研究项目将有助于我们对细菌在肠道感染期间如何调节上皮屏障功能的理解，并获得最终系统性后果的粘膜和粘膜。淋巴结蛋白是革兰氏阴性细菌的大毒素，包括致病性大肠杆菌和衣原体属。被感染的肠道，肺部和泌尿生殖道的显着发病率和死亡率。我们的实验结果表明，淋巴结蛋白抑制免疫响应并调节肠道屏障功能。当前的提案研究了受淋巴结蛋白和放松管制途径影响的特定宿主蛋白。