Rho-Modifying Cytotoxic Necrotizing Factor of E. coli

大肠杆菌 Rho 修饰细胞毒性坏死因子

• 批准号: 6542190
• 负责人: Alison Davis O'Brien
• 金额: $ 29.16万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6542190
• 关键词: Escherichia coli; Escherichia coli infections; actins; amidation /deamidation; apoptosis; bacterial toxins; cell migration; chimeric proteins; cytokine; cytotoxicity; glutamine; immunoprecipitation; interstitial cystitis; laboratory mouse; laboratory rat; microorganism immunology; neutralizing antibody; neutrophil; prostatitis; protein structure function; receptor; tissue /cell culture; urinary bladder; urinary tract infection; western blottings

Cytotoxic necrotizing factor type 1 (CNF1) is a member of a family of bacterial toxins that target the Rho family of small GTP-binding proteins in mammalian cells. CNF1 deamidates a single glutamine residue in RhoA, Cdc42, and Rac1 but not in Ras. This deamidation results in the constitutive activation of these GTPases which can trigger actin stress fiber formation, multinucleation, or cell death, depending on the target cell and dose of toxin. CNF1 is frequently produced by Escherichia coli strains that cause urinary tract infections (UTIs), such as cystitis, prostatitis, and pyelonephritis. In support of this epidemiological connection, we recently showed that CNF1 not only induces apoptosis in 5637 human uroepithelial cells but also provides a growth advantage to uropathogenic E. coli (UPEC) in a mouse model of ascending UTI when compared to CNF1-negative isogenic mutants. Additionally, we found that CNF1 enhances the degree of inflammation and resulting tissue damage in bladders of infected mice and in prostates of rats challenged intraurethrally with CNF1-producing UPEC. Finally, we discovered that CNF1- producing UPEC survive better than CNF1-negative isogenic mutants in the presence of human polymorphonuclear leukocytes (PMNs). Taken together, these findings have led us to propose the following hypothesis. CNF1 enhances the pathogenicity of UPEC by: i.) promoting uroepithelial cell shedding; ii.) evoking a large influx of PMNs while providing toxin-producing E. coli protection against PMN-mediated killing, and; iii.) facilitating deeper invasion of the bladder or prostate by the infecting strain. The specific aims designed to test this theory are to: 1.) further define the role that CNF1 plays in the pathogenesis of UPEC-mediated cystitis in the mouse and prostatitis in the rat by analyzing the interaction of CNF1 or CNF1-expressing UPEC with PMNs from these animals and by defining the CNF1-mediated cytokine response that evokes PMN influx; 2) investigate the cellular and cytokine responses of a human bladder organoid to CNF1 or a CNF1-producing UPEC strain; 3.) identify the functional receptor for CNF1 by sequential biochemical and molecular approaches, and; 4.) continue to evaluate CNF1 structure and function by characterizing the CNF1 epitopes recognized by neutralizing monoclonal antibodies and by analyzing chimeric molecules comprised of portions of CNF1 and the related toxins CNF2, Pasteurella multocida toxin, and the Bordetella dermonecrotic toxin.

细胞毒性坏死因子1（CNF 1）是一个细菌毒素家族的成员，靶向哺乳动物细胞中的Rho家族的小GTP结合蛋白。 CNF 1在RhoA、Cdc 42和Rac 1中脱酰胺化单个谷氨酰胺残基，但在Ras中不脱酰胺化。这种脱酰胺作用导致这些GTP酶的组成性激活，其可触发肌动蛋白应力纤维形成、多核化或细胞死亡，这取决于靶细胞和毒素剂量。 CNF 1通常由引起尿路感染（UTI）的大肠杆菌菌株产生，如膀胱炎、前列腺炎和肾盂肾炎。 为了支持这种流行病学联系，我们最近发现CNF 1不仅诱导5637人尿路上皮细胞凋亡，而且还为尿路致病性大肠杆菌提供了生长优势。大肠杆菌（UPEC）在上升型UTI小鼠模型中的CNF 1阴性同基因突变体相比。 此外，我们发现CNF 1增强了感染小鼠膀胱和用产生CNF 1的UPEC经尿道内攻击的大鼠前列腺的炎症程度和导致的组织损伤。 最后，我们发现，CNF 1生产的UPEC生存更好的CNF 1阴性的同基因突变体在人类多形核白细胞（PMN）的存在下。综上所述，这些发现使我们提出了以下假设。 CNF 1通过以下方式增强UPEC的致病性：i.）促进尿路上皮细胞脱落; ii.）引起大量PMN流入，同时提供产毒素E.大肠杆菌针对PMN介导的杀伤的保护，和; iii.）促进感染菌株对膀胱或前列腺的更深侵入。 测试这一理论的具体目标是：1）。通过分析CNF 1或表达CNF 1的UPEC与来自这些动物的PMN的相互作用并通过定义引起PMN流入的CNF 1介导的细胞因子应答，进一步定义CNF 1在小鼠中UPEC介导的膀胱炎和大鼠中前列腺炎的发病机制中发挥的作用; 2）研究人膀胱类器官对CNF 1或产生CNF 1的UPEC菌株的细胞和细胞因子应答; 3.）通过连续的生物化学和分子方法鉴定CNF 1的功能性受体，和; 4.）通过鉴定由中和单克隆抗体识别的CNF 1表位，并通过分析由CNF 1部分和相关毒素CNF 2、多杀性巴氏杆菌毒素和博德特氏杆菌皮肤坏死毒素组成的嵌合分子，继续评价CNF 1的结构和功能。