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: 6733544
• 负责人: Alison Davis O'Brien
• 金额: $ 29.64万
• 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/6733544
• 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 型 (CNF1) 是细菌毒素家族的一员，针对哺乳动物细胞中的小 GTP 结合蛋白 Rho 家族。 CNF1 使 RhoA、Cdc42 和 Rac1 中的单个谷氨酰胺残基脱酰胺，但不使 Ras 中的单个谷氨酰胺残基脱酰胺。这种脱酰胺作用会导致这些 GTP 酶的组成型激活，从而触发肌动蛋白应力纤维形成、多核或细胞死亡，具体取决于靶细胞和毒素剂量。 CNF1 经常由引起尿路感染 (UTI) 的大肠杆菌菌株产生，例如膀胱炎、前列腺炎和肾盂肾炎。 为了支持这种流行病学联系，我们最近表明，与 CNF1 阴性同基因突变体相比，CNF1 不仅能诱导 5637 个人类尿路上皮细胞凋亡，而且还能为上行性尿路感染小鼠模型中的尿路致病性大肠杆菌 (UPEC) 提供生长优势。 。 此外，我们发现，CNF1 会增强受感染小鼠膀胱的炎症程度以及由此产生的组织损伤，以及在尿道内接受产生 CNF1 的 UPEC 攻击的大鼠前列腺中的炎症程度。 最后，我们发现在人类多形核白细胞 (PMN) 存在的情况下，产生 CNF1 的 UPEC 比 CNF1 阴性等基因突变体存活得更好。综上所述，这些发现使我们提出以下假设。 CNF1 通过以下方式增强 UPEC 的致病性： i.) 促进尿上皮细胞脱落； ii.) 引起大量中性粒细胞涌入，同时提供产毒大肠杆菌保护，防止中性粒细胞介导的杀伤作用，以及； iii.) 促进感染菌株更深入地侵入膀胱或前列腺。 测试这一理论的具体目的是：1.) 通过分析 CNF1 或表达 CNF1 的 UPEC 与 PMN 的相互作用，进一步明确 CNF1 在 UPEC 介导的小鼠膀胱炎和大鼠前列腺炎发病机制中的作用。来自这些动物并通过定义引起 PMN 流入的 CNF1 介导的细胞因子反应； 2) 研究人类膀胱类器官对 CNF1 或产生 CNF1 的 UPEC 菌株的细胞和细胞因子反应； 3.) 通过连续的生化和分子方法鉴定 CNF1 的功能受体； 4.) 通过表征中和单克隆抗体识别的 CNF1 表位并分析由 CNF1 部分和相关毒素 CNF2、多杀性巴斯德氏菌毒素和皮肤坏死性博德特氏菌毒素组成的嵌合分子，继续评估 CNF1 结构和功能。