RHO MODIFYING CYTOTOXIC NECROTIZING FACTOR OF E COLI

RHO修饰大肠杆菌细胞毒性坏死因子

• 批准号: 2457831
• 负责人: Alison Davis O'Brien
• 金额: $ 19.97万
• 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 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2457831
• 关键词: Escherichia coli; Escherichia coli infections; actins; bacterial toxins; cytotoxicity; disease /disorder model; gene deletion mutation; kidney cell; laboratory mouse; monoclonal antibody; pathologic process; polymerization; site directed mutagenesis; urinary bladder; urinary tract infection; virulence

DESCRIPTION (Adapted from applicant's abstract): Escherichia coli is the most common cause of urinary tract infections (UTIs) in otherwise healthy individuals. The uropathogenic E. coli isolates that cause these infections are characteristically of certain serotypes and express P fimbriae, alpha- hemolysin, and aerobactin. Recent findings from Europe indicate that the cytotoxic necrotizing factor type 1 (CNF1), a toxin little studied in the U.S., is also frequently produced by uropathogenic E. coli. CNF1 and the immunologically related CNF2 are 110-115 kDa polypeptides that induce multinucleation and actin polymerization in eukaryotic cells and necrosis and death of some animals. We recently cloned and sequenced cnf2 and found that the N-terminal half of CNF2 is homologous to the dermonecrotic toxin of Pasteurella multocida, a toxin that mediates the pathology of progressive rhinitis in pigs. We also observed that CNF2 modifies a small GTP-binding protein designated Rho that is involved in stress fiber assembly. The long term goals of this proposal are to examine the role that CNF1 plays in the virulence of uropathogenic E. coli and to determine the precise mechanism by which CNF1 modifies Rho. The specific aims are designed to achieve these goals are to: 1) analyze the role of CNF1 in the pathogenicity of E. coli strain with J96 by constructing a cnf1-negative derivative of that strain and comparing the mutant with the wild-type for virulence in a mouse model of ascending UTI and in human kidney and bladder cells; 2) investigate the nature of the chemical modification of Rho by CNF1 and clarify how that modification leads to actin polymerization; 3) dissect the relationship between the structure of CNF1 and its function by generating a set of mutant CNF1s through deletion, regionally-directed, and site-specific mutagenesis and preparing monoclonal and monospecific antibodies as probes for toxin structural integrity; 4) attempt to identify the functional receptor for CNF1.

描述（改编自申请人的摘要）：大肠杆菌是 尿路感染 (UTI) 的最常见原因 其他方面健康的人。尿路致病性大肠杆菌分离株 导致这些感染的原因有一定的特征 血清型并表达 P 菌毛、α-溶血素和需氧菌素。 欧洲最近的研究结果表明，细胞毒性坏死 1 型因子 (CNF1) 是一种在美国很少被研究的毒素，也是 经常由尿路致病性大肠杆菌产生。 CNF1 和 免疫学相关的 CNF2 是 110-115 kDa 的多肽， 诱导真核细胞中的多核和肌动蛋白聚合， 一些动物坏死和死亡。我们最近克隆并测序了 cnf2 并发现 CNF2 的 N 端一半与 多杀性巴氏杆菌的皮肤坏死毒素，一种毒素 介导猪进行性鼻炎的病理学。 我们也 观察到 CNF2 修饰了一个小的 GTP 结合蛋白，称为 Rho 参与应力纤维组装。 长期目标 该提案的目的是检查 CNF1 在 尿路致病性大肠杆菌的毒力并确定精确的 CNF1 修饰 Rho 的机制。具体目标是 旨在实现这些目标的是：1）分析CNF1的作用 通过构建一个 J96 大肠杆菌菌株的致病性 该菌株的 cnf1 阴性衍生物并将突变体与 野生型在上行性尿路感染小鼠模型中的毒力 人类肾脏和膀胱细胞； 2）调查事物的性质 CNF1 对 Rho 进行化学修饰并阐明该修饰是如何进行的 导致肌动蛋白聚合； 3）剖析之间的关系 通过生成一组突变体来了解CNF1的结构及其功能 通过删除、区域定向和位点特异性的 CNF1 诱变和制备单克隆和单特异性抗体 作为毒素结构完整性的探针； 4）尝试识别 CNF1 的功能受体。