Rho-modifying Cytotoxic Necrotizing Factor of E. coli

细胞毒性坏死性因子1(CNF1)是细菌毒素家族中的一员， 去酰胺化RhoA、RAc和CdC42中的单一谷氨酰胺残基，从而结构性地 激活这些小的GTP酶。这些去酰胺化事件会对 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。CNF1和一种膜溶解毒素溶血素(HLY)通常是共同作用的。 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，cnf1基因和一个hly操纵子是共转录的。 并在体外培养过程中由一株典型的尿路致病性大肠杆菌(UPEC)共同调节。的 与这一建议特别相关的是，产生CNF1/HLY的UPEC分离株更频繁 从血液中分离出来，并在其体内有高水平的某些促炎细胞因子 尿液都是毒素阴性的UPEC。后一种观察结果与我们先前的发现是一致的 CNF1+UPEC株比同基因CNF1-株诱导更强烈的炎症反应 UTI上升的小鼠模型和急性前列腺炎大鼠模型中的突变 CNF1阳性的UPEC株CP9在人体内的存活率高于其cnf1等基因突变株 和小鼠多形核白细胞(PMN)。我们还报道了HLY引起的损失 在培养的表面肾上皮细胞和三维类器官模型中，我们最近发现HLY 24小时后损伤小鼠膀胱上皮细胞并引起出血 经尿路接种CP9。因此，我们推测CNF1和HLY增强了 UPEC菌株的致病性由：I.)促进尿路上皮细胞脱落和组织 出血(HLY)；引起大量潜在的组织破坏性PMN的流入(CNF1和 Hly)，同时保护细菌免受吞噬细胞介导的杀伤(CNF1)， 以及；iii.)引起粘膜下水肿(CNF1)。为测试这一点而设计的具体目标 假设为：1)描述CNF1和HLY单独和共同对以下水平的影响 前24天小鼠的血液、中性粒细胞和尿液中选定的促炎细胞因子 CP9及其cnf1和hlyA1单、双突变株尿路感染后数小时 通过微阵列更广泛地比较宿主对这些同基因菌株的反应 受感染膀胱的转录分析；2)监测cnf1和cnf1的表达动力学 CP9激发后尿液和/或膀胱中连续HLY操纵子的实时RT-PCR检测 小鼠询问cnf1和连接的HLY操纵子是否在体内共转录；确定 CNF1在体内是否真的通过测量小GTP酶的激活程度来修饰小的GTP酶 CP9及其CNF感染小鼠膀胱上皮细胞中Rho、Rac和CDc42的表达 突变；以及，4.)尝试减少CP9引起的炎症和损害的程度 通过CNF1/HLY类毒素鸡尾酒对小鼠的非肠道和/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.