Regulation of Proteins in the Apical Junctional Complex by Citrobacter rodentium

描述（由申请人提供）： 本提案的目的是表征革兰氏阴性菌的大毒素的作用及其对体外和体内上皮屏障功能的作用。淋巴细胞抑制因子A（lifA）是革兰氏阴性菌中的一个大基因，存在于肠出血性大肠杆菌、肠致病性大肠杆菌、大肠杆菌和大肠杆菌中。大肠杆菌和啮齿类柠檬酸杆菌。lifA基因编码两种重要的酶活性，即糖基转移酶（1.6kb）和蛋白酶基序（4.5 - 4.8kb）。我们已经产生了两个稳定的，特异性的框内插入-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分解。看来糖基转移酶活性通过阻止Cdc 42的活化而负责ZO-1从紧密连接的解离。此外，我们提供的证据表明，蛋白酶活性导致解离的<$-catenin从adherens连接激活RhoA。假设1：LS对于形成顶端连接复合物（AJC）的蛋白质的分解至关重要，导致跨上皮阻力（TER）降低，促进细菌移位和全身传播。目的1：研究C.啮齿动物LS糖基转移酶和蛋白酶活性对构成体外分化上皮细胞培养物和小鼠体内AJC的蛋白质的影响。此外，LS对AJC成员的作用似乎是通过引起Rho GT3活化失衡来介导的。我们发现，蛋白酶基序调节激活Rho和抑制Cdc 42，而糖基转移酶只参与抑制Cdc 42。假设2：LS糖基转移酶和蛋白酶活性都是通过调节Rho GTP酶来分解AJC组分的关键。目的2：研究C.啮齿类野生型lifA，和失活的蛋白酶和糖基转移酶基序在体外和体内。拟议的研究项目将有助于我们了解细菌如何在肠道感染过程中调节上皮屏障功能，并最终进入粘膜和粘膜下层并产生全身性后果。嗜磷素是一种来自革兰氏阴性菌的大毒素，包括致病性大肠杆菌。大肠杆菌和衣原体属（Chlamydia spp.）通过感染肠道、肺和泌尿生殖道导致显著的发病率和死亡率。我们的实验结果表明，淋巴抑制素抑制免疫反应和调节肠道屏障功能。目前的建议调查特定的宿主蛋白受淋巴抑制素和失调的途径。

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.