Rho-modifying Cytotoxic Necrotizing Factor of E. coli

细胞毒性坏死因子1（CNF 1）是细菌毒素家族的成员， 使RhoA、Rac和Cdc 42中的单个谷氨酰胺残基脱酰胺，从而组成性地 激活这些小GTP酶。这些脱酰胺事件引发了对细胞的无数影响。 靶细胞，如肌动蛋白细胞骨架重排、细胞周期异常和改变 在信号通路中。CNF 1和一种膜溶解毒素溶血素（Hly），通常是共 由引起尿路感染（UTI）的大肠杆菌菌株表达，即，膀胱炎或 肾盂肾炎或急性前列腺炎。事实上，cnf 1基因座和hly操纵子是共转录的。 并受原型尿路致病性E.大肠杆菌（UPEC）菌株的体外培养。的 与此建议特别相关的是，产生CNF 1/Hly的UPEC分离株更常见于 分离自人类血液和高水平的某些促炎细胞因子， 尿液中的毒素比UPEC阴性。后一个观察结果与我们先前的发现一致 CNF 1 + UPEC菌株比同基因CNF 1-UPEC菌株引起更强烈的炎症反应， 在上行性UTI的小鼠模型和急性前列腺炎的大鼠模型中， CNF 1阳性UPEC菌株CP 9在人体中的存活率优于其cnf 1同基因突变体 和小鼠多形核白细胞（PMNs）。我们还报告说，Hly引起的损失， 表面尿路上皮细胞的培养和3-D类器官模型，我们最近发现， 24小时后， 尿道内接种CP 9。因此，我们推测CNF 1和Hly增强了 UPEC菌株的致病性通过：I.）促进泌尿上皮细胞脱落和组织 出血（Hly）; ii.）引起潜在的组织损伤性PMN（CNF 1和 Hly）同时保护细菌免于吞噬细胞介导的杀伤（CNF 1）， 和; iii.）引起粘膜下水肿（CNF 1）。旨在测试这一点的具体目标 假设是：1.）描述CNF 1和Hly单独和一起对 在第一个24小时内，小鼠的血液、中性粒细胞和尿液中的选定促炎细胞因子 用CP 9及其cnf 1和hlyA 1单突变体和双突变体尿道内感染后24小时， 为了通过微阵列更广泛地比较宿主对这些同基因菌株的反应， 感染膀胱的转录分析; 2.）监测CNF 1的表达动力学， 通过真实的时间RT-PCR在CP 9攻击的小鼠的尿和/或膀胱中检测连续hly操纵子 小鼠以询问CNF 1和连接的hly操纵子是否在体内共转录; 3.）确定 CNF 1是否真的在体内修饰小GTP酶，通过测量 CP 9及其CNF感染小鼠膀胱上皮细胞中Rho、Rac和Cdc 42的表达 突变体;以及，4.）试图减少炎症和损伤的程度引起的CP 9 通过用CNF 1/Hly类毒素混合物对小鼠进行肠胃外和/或粘膜免疫。

Cytotoxic necrotizing factor type 1 (CNF1) is a member of a family of bacterial toxins that deamidate single glutamine residues in RhoA, Rac, and Cdc42 and thereby constitutively activate these small GTPases. These deamidation events trigger a myriad of effects on the target cells such as actin cytoskeleton rearrangements, cell cycle abnormalities, and alterations in signaling pathways. CNF1 and a membrane-lytic toxin, hemolysin (Hly), are often co- expressed by Escherichia coli strains that cause urinary tract infections (UTIs), i.e., cystitis or pyelonephritis, or acute prostatitis. In fact, the cnf1 locus and a hly operon are co-transcribed and co-regulated from a prototypic uropathogenic E. coli (UPEC) strain during culture in vitro. Of particular relevance to this proposal, CNF1/Hly-producing UPEC isolates are more frequently isolated from humans with blood and high levels of certain pro-inflammatory cytokines in their urine than are toxin-negative UPEC. The latter observation is consistent with our prior findings that CNF1+ UPEC strains elicit a more intense inflammatory response than do isogenic CNF1- mutants in a mouse model of ascending UTI and in a rat model of acute prostatitis and that the CNF1-positive UPEC strain CP9 survives better than does its cnf1 isogenic mutant in human and mouse polymorphonuclear leukocytes (PMNs). We also reported that Hly provokes loss of surface uroepithelial cells in culture and a 3-D organoid model, and we recently found that Hly damages the uroepithelium and evokes hemorrhage in the bladders of mice 24 hours after intraurethral inoculation with CP9. We therefore theorize that CNF1 and Hly enhance the pathogenicity of UPEC strains by: I.) promoting uroepithelial cell shedding and tissue hemorrhage (Hly); ii.) evoking a large influx of potentially tissue-damaging PMNs (CNF1 and Hly) while simultaneously protecting the bacterium from phagocyte-mediated killing (CNF1), and; iii.) eliciting submucosal edema (CNF1). The specific aims designed to test this hypothesis are to: 1.) delineate the impact of CNF1 and Hly alone and together on the levels of blood, PMNs, and selected pro-inflammatory cytokines in the urine of mice during the first 24 hours after intraurethral infection with CP9 and its cnf1 and hlyA1 single and double mutants and to more broadly compare the host response to these isogenic strains through microarray transcriptional analyses of infected bladders; 2.) monitor expression kinetics of cnf1 and the contiguous hly operon by real time RT-PCR in the urine and/or bladders of CP9-challenged mice to ask whether cnf1 and the linked hly operon are co-transcribed in vivo; 3.) determine whether CNF1 actually modifies small GTPases in vivo by measuring the extent of activation of Rho, Rac and Cdc42 in uroepithelial cells from bladders of mice infected with CP9 or its CNF mutant; and, 4.) attempt to reduce the extent of inflammation and damage evoked by CP9 through parenteral and/or mucosal immunization of mice with a CNF1/Hly toxoid cocktail.