Regulation of Proteins in the Apical Junctional Complex by Citrobacter rodentium

描述(由申请人提供)： 本研究的目的是研究革兰氏阴性杆菌产生的一种大毒素在体外和体内对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是革兰氏阴性菌中存在的一个大基因，包括肠出血性、肠源性大肠杆菌和轮状柠檬酸杆菌。LifA编码两种与微生物致病有关的关键酶活性：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、特异的框内插入-删除突变，使两个基序都失活。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通过阻止CDC42的激活而导致ZO-1从紧密连接中解离。此外，我们提供的证据表明，蛋白酶活性通过激活RhoA而导致β-连环蛋白从粘着连接上解离。假设1：LS对形成顶端连接复合体(AJC)的蛋白质的分解至关重要，导致跨上皮阻力(TER)降低，促进细菌易位和全身传播。目的：在体外和体内研究轮状乳杆菌LS糖基转移酶和蛋白水解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导致Rho GTPase激活的失衡来调节的。我们发现，蛋白酶基序调节Rho的激活并抑制CDc42，而糖基转移酶只参与抑制CDc42。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：在体内外研究Rho GTP酶信号转导通路在轮齿冠状芽孢杆菌WT Lifa和失活的蛋白水解酶和糖基转移酶模体中的激活情况。拟议的研究项目将有助于我们理解细菌如何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，最终导致全身后果。淋巴抑制素是革兰氏阴性菌产生的一种大毒素，包括致病性大肠杆菌和衣原体。这是通过感染肠道、肺部和泌尿生殖道而导致严重发病率和死亡率的原因。我们的实验结果表明，淋巴抑制素抑制免疫反应，调节肠道屏障功能。目前的方案研究了受淋巴抑制素和非调控途径影响的特定宿主蛋白。

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.