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: 2075275
• 负责人: Alison Davis O'Brien
• 金额: $ 19.67万
• 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/2075275
• 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（改编自申请人摘要）：大肠埃希菌是 泌尿道感染（UTIs）的最常见原因 健康的个体。泌尿系致病性E.杆菌分离株 导致这些感染的特征是 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， 因子1（CNF 1），一种在美国很少研究的毒素，也是 常由尿路致病性E.杆菌 CNF 1和 免疫相关CNF 2是110-115 kDa多肽， 在真核细胞中诱导多核化和肌动蛋白聚合， 一些动物的坏死和死亡。我们最近克隆并测序了 CNF 2的N-末端的一半是同源的， 多杀性巴氏杆菌的皮肤坏死毒素， 介导猪进行性鼻炎的病理学。 我们也 观察到CNF 2修饰了一种小的GTP结合蛋白， Rho参与应力纤维组装。 长期目标 这项建议的目的是研究CNF 1在 尿路致病性E.大肠杆菌，并确定精确的 CNF 1修饰Rho的机制。具体目标是 旨在实现这些目标是：1）分析CNF的作用1 E.大肠杆菌菌株与J 96的融合， 菌株CNF 1阴性衍生物，并将突变体与 在上行性UTI小鼠模型中的野生型毒力和在 人肾脏和膀胱细胞; 2）研究 通过CNF 1对Rho进行化学修饰，并阐明该修饰 导致肌动蛋白聚合; 3）剖析 CNF 1结构及其功能，通过产生一组突变体 CNF 1通过缺失、区域定向和位点特异性 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）尝试鉴定 CNF 1的功能性受体。