Rho-modifying Cytotoxic Necrotizing Factor of E. coli

描述（由申请人提供）：1型细胞毒性坏死因子（CNF 1）是细菌毒素家族的一员，可使RhoA、Rac和Cdc 42中的单个谷氨酰胺残基脱酰胺，从而组成性激活这些小GTP酶。这些脱酰胺事件触发了对靶细胞的无数影响，如肌动蛋白细胞骨架重排、细胞周期异常和信号通路的改变。CNF 1和膜溶解毒素溶血素（Hly）通常由引起尿路感染（UTI）的大肠杆菌菌株共表达，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操纵子是从原型尿路致病性大肠杆菌共转录和共调节的。大肠杆菌（UPEC）菌株的体外培养。与该提议特别相关的是，产生CNF 1/Hly的UPEC分离物比毒素阴性的UPEC更频繁地从血液和尿液中高水平的某些促炎细胞因子的人中分离。后一个观察结果与我们先前的发现一致，即在上行性UTI小鼠模型和急性前列腺炎大鼠模型中，CNF 1 + UPEC菌株比同基因CNF 1突变体引起更强烈的炎症反应，并且CNF 1阳性UPEC菌株CP 9在人类和小鼠多形核白细胞（PMN）中的存活率优于其cnf 1同基因突变体。我们还报道了Hly在培养和3-D类器官模型中引起表面尿路上皮细胞的损失，我们最近发现Hly在尿道内接种CP 9后24小时损害了尿路上皮并引起小鼠膀胱出血。因此，我们推测CNF 1和Hly通过以下方式增强UPEC菌株的致病性：I.）促进尿路上皮细胞脱落和组织出血（Hly）; ii.）引起潜在的组织损伤性PMN（CNF 1和Hly）的大量流入，同时保护细菌免受吞噬细胞介导的杀伤（CNF 1），和; iii.）引起粘膜下水肿（CNF 1）。为检验这一假设而设计的具体目标是：1）。描述在用CP 9及其cnf 1和hlyA 1单突变体和双突变体尿道内感染后的前24小时期间，CNF 1和Hly单独和一起对血液、PMN和小鼠尿液中选定的促炎细胞因子水平的影响，并通过感染膀胱的微阵列转录分析更广泛地比较宿主对这些同基因菌株的反应; 2.）通过真实的时间RT-PCR监测CP 9攻击小鼠的尿和/或膀胱中cnf 1和邻接的hly操纵子的表达动力学，以询问cnf 1和连接的hly操纵子是否在体内共转录; 3.）通过测量来自感染CP 9或其CNF突变体的小鼠膀胱的尿路上皮细胞中Rho、Rac和Cdc 42的活化程度来确定CNF 1是否实际上在体内修饰小GTP酶;和4.）尝试通过用CNF 1/Hly类毒素混合物对小鼠进行肠胃外和/或粘膜免疫来降低由CP 9诱发的炎症和损伤的程度。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，女性受影响的比例高于男性。该项目将导致更好地了解CNF 1和Hly在UPEC介导的疾病中的相对重要性，并可能为设计针对UTI的新型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.