Rho-modifying Cytotoxic Necrotizing Factor of E. coli

描述(由申请人提供)：细胞毒性坏死性因子1(CNF1)是细菌毒素家族的成员，它能使RhoA、RAC和CDC42中的单个谷氨酰胺残基去氨基，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产生一系列的影响，如肌动蛋白细胞骨架重排、细胞周期异常以及信号通路的改变。CNF1和一种膜溶解毒素溶血素(HLY)通常在引起尿路感染(UTI)的大肠杆菌菌株中共表达，即膀胱炎或肾盂肾炎，或急性前列腺炎。事实上，在体外培养过程中，cnf1基因和HLY操纵子是从一株尿路致病性大肠杆菌(UPEC)的原型菌株中共转录和共调控的。与这一建议特别相关的是，与毒素阴性的UPEC相比，产生CNF1/HLY的UPEC分离株更多地从有血液和尿液中高水平某些促炎细胞因子的人身上分离出来。后一个观察结果与我们之前的发现一致，即在UTI上升的小鼠模型和急性前列腺炎大鼠模型中，CNF1+UPEC株比同基因CNF1-突变株诱导更强烈的炎症反应，并且CNF1阳性UPEC株CP9在人和小鼠多形核白细胞(PMN)中比其CNF1等基因突变株存活得更好。我们还报道了HLY在培养和三维器官模型中引起表面尿路上皮细胞的丢失，最近我们发现HLY在小鼠经尿路接种CP9 24小时后损伤尿路上皮并引起膀胱出血。因此，我们推测CNF1和HLY通过以下途径增强UPEC菌株的致病性：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(CNF1和HLY)的涌入，同时保护细菌免受吞噬细胞介导的杀伤(CNF1)，以及；引起粘膜下水肿(CNF1)。检验这一假设的具体目的是：1.)通过对感染膀胱的微阵列转录分析，描述在CP9及其cnf1和hlyA1单突变体和双突变体经尿路感染后的24小时内，CNF1和HLY单独和共同对小鼠血液、PMN和尿液中选定的促炎细胞因子水平的影响，并更广泛地比较宿主对这些同基因菌株的反应。用实时定量RT-PCR技术监测CP9攻击小鼠尿液和/或膀胱中cnf1和邻近的Hly操纵子的表达动态，以了解cnf1和连接的hly操纵子是否在体内共转录；通过测量感染CP9或其CNF突变体的小鼠膀胱上皮细胞中Rho、Rac和CDc42的激活程度，确定CNF1是否确实在体内修饰了小的GTP酶；以及试图通过CNF1/HLY类毒素鸡尾酒对小鼠的非肠道和/或粘膜免疫来减少CP9引起的炎症和损害的程度。公共卫生相关性：尿路感染是成人最常见的细菌性疾病之一，女性比男性受影响的比例更大。该项目将有助于更好地理解CNF1和HLY在UPEC介导的疾病中的相对重要性，并可能为设计新的尿路感染预防和治疗策略铺平道路。

DESCRIPTION (provided by applicant): 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 coexpressed 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. PUBLIC HEALTH RELEVANCE: UTIs are among the most common bacterial diseases of adults, with women affected disproportionately to men. This project will lead to a better understanding of the relative importance of CNF1 and Hly in UPEC-mediated disease and may pave the way for the design of novel preventative and therapeutic strategies against UTIs.