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: 6874967
• 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/6874967
• 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.

细胞毒性坏死型因子1(CNF1)是针对哺乳动物细胞中的Rho小GTP结合蛋白家族的细菌毒素家族的成员。CNF1可使RhoA、Cdc42和rac1中的单一谷氨酰胺残基去胺化，但不能使RAS中的谷氨酰胺残基失效。这种脱酰胺作用会导致这些GTP酶的结构性激活，从而触发肌动蛋白应激纤维的形成、多核或细胞死亡，这取决于目标细胞和毒素的剂量。CNF1通常是由引起尿路感染(UTI)的大肠杆菌菌株产生的，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，与CNF1阴性的等基因突变体相比，CNF1不仅可以诱导5637人尿路上皮细胞的凋亡，而且在UTI上升的小鼠模型中，CNF1不仅能诱导5637人尿路上皮细胞的凋亡，而且还能为UPEC提供生长优势。此外，我们发现CNF1增强了感染小鼠膀胱的炎症程度和由此导致的组织损伤，以及经尿路注射产生CNF1的UPEC挑战的大鼠的前列腺。最后，我们发现，在人多形核白细胞(PMN)存在下，产生CNF1的UPEC比CNF1阴性的同基因突变株存活得更好。综上所述，这些发现让我们提出了以下假设。CNF1通过以下途径增强UPEC的致病性：i.)促进尿路上皮细胞脱落；ii.)引起大量PMN的涌入，同时提供产生毒素的大肠杆菌对PMN介导的杀戮的保护，以及；促进感染菌株对膀胱或前列腺的更深层次的侵袭。检验这一理论的具体目的是：1.)通过分析表达CNF1或表达CNF1的UPEC与这些动物的中性粒细胞的相互作用，以及通过定义CNF1介导的细胞因子反应，进一步确定CNF1在UPEC介导的小鼠膀胱炎和大鼠前列腺炎的发病机制中所起的作用；2)研究人膀胱有机物对CNF1或产生CNF1的UPEC菌株的细胞和细胞因子反应；通过连续的生化和分子方法鉴定CNF1的功能受体，以及。通过鉴定中和单抗识别的CNF1表位，以及分析CNF1部分与相关毒素CNF2、多杀性巴氏杆菌毒素和皮肤波氏杆菌毒素的嵌合分子，继续评估CNF1的结构和功能。