Rho-modifying Cytotoxic Necrotizing Factor of E. coli

大肠杆菌的 Rho 修饰细胞毒性坏死因子

基本信息

批准号：
8060497
负责人：
Alison Davis O'Brien
金额：
$ 37.49万
依托单位：
HENRY M. JACKSON FDN FOR THE ADV MIL/MED
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-09-30 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8060497
关键词：
3-Dimensional Actins Acute Acute Prostatitis Adult Affect Animals Bacteria Bacterial Infections Bacterial Toxins Bladder Blood CCL2 gene Cell Cycle Cells Cystitis Cytoplasmic Protein Cytoskeleton DNA Sequence Rearrangement Disease Edema Escherichia coli Event Family GTP Binding GTP-Binding Proteins Genes Glutamine Goals Guanosine Triphosphate Phosphohydrolases Health Hemolysin Hemorrhage Hour Human IL8 gene Image Immune response Immunization In Vitro Infection Inflammation Inflammatory Inflammatory Response Kinetics Lead Link Lytic Mammalian Cell Manuscripts Measures Mediating Membrane Modeling Molecular Monitor Monomeric GTP-Binding Proteins Mus Nuclear Operon Organism Organoids Pathogenicity Pathogenicity Island Phagocytes Plasmids Positioning Attribute Production Pyelonephritis Rattus Reaction Relative (related person)Reporting Research Personnel Reverse Transcriptase Polymerase Chain Reaction Signal Pathway Signal Transduction Signaling Pathway Gene Surface System Testing Therapeutic Time Tissues Toxin Toxoids Urinary tract infection Urine Uropathogenic E. coli Vaccinated Vesicle Woman ascending urinary tract infection cell injury cytokine cytotoxic cytotoxic necrotizing factor type 1 deamidation design fimbria in vivo killings member men mouse model mutant neutrophil novel prevent prostatitis public health relevance rho stress-activated protein kinase 1 tissue culture transcription factor urinary

项目摘要

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.

描述（由申请人提供）：细胞毒性坏死因子 1 型 (CNF1) 是细菌毒素家族的成员，其使 RhoA、Rac 和 Cdc42 中的单个谷氨酰胺残基脱酰胺，从而组成型激活这些小 GTP 酶。这些脱酰胺事件会引发对靶细胞的多种影响，例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。 CNF1 和溶膜毒素溶血素 (Hly) 通常由大肠杆菌菌株共表达，导致尿路感染 (UTI)，即膀胱炎或肾盂肾炎或急性前列腺炎。事实上，cnf1 基因座和 hly 操纵子是在体外培养过程中由原型尿路致病性大肠杆菌 (UPEC) 菌株共同转录和共同调节的。与该提议特别相关的是，与毒素阴性 UPEC 相比，产生 CNF1/Hly 的 UPEC 分离株更频繁地从血液和尿液中某些促炎细胞因子水平较高的人类中分离出来。后一个观察结果与我们之前的发现一致，即在上行性尿路感染小鼠模型和急性前列腺炎大鼠模型中，CNF1+ UPEC 菌株比同基因 CNF1- 突变体引起更强烈的炎症反应，并且 CNF1 阳性 UPEC 菌株 CP9 存活下来比其 cnf1 同基因突变体在人和小鼠多形核白细胞 (PMN) 中的效果更好。我们还报道了 Hly 会引起培养物和 3D 类器官模型中表面尿上皮细胞的损失，并且我们最近发现 Hly 会损伤尿道上皮并在尿道内接种 CP9 24 小时后引起小鼠膀胱出血。因此，我们推测 CNF1 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.) 引起大量潜在的组织损伤性中性粒细胞（CNF1 和 Hly）涌入，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)。测试这一假设的具体目的是：1.) 描述尿道内注射后 24 小时内，CNF1 和 Hly 单独和共同对小鼠尿液中的血液、PMN 和选定的促炎细胞因子水平的影响。 CP9及其cnf1和hlyA1单突变体和双突变体的感染，并通过受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.)通过实时RT-PCR监测CP9攻击小鼠的尿液和/或膀胱中cnf1和连续hly操纵子的表达动力学，以询问cnf1和连接的hly操纵子是否在体内共转录； 3.) 通过测量感染 CP9 或其 CNF 突变体的小鼠膀胱的尿上皮细胞中 Rho、Rac 和 Cdc42 的激活程度，确定 CNF1 是否实际上在体内修饰小 GTPase； 4.)尝试通过用CNF1/Hly类毒素混合物对小鼠进行胃肠外和/或粘膜免疫来减少CP9引起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，女性受感染的比例高于男性。该项目将有助于更好地了解 CNF1 和 Hly 在 UPEC 介导的疾病中的相对重要性，并可能为针对 UTI 的新型预防和治疗策略的设计铺平道路。