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: 2887038
• 负责人: Alison Davis O'Brien
• 金额: $ 21.6万
• 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 2002-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887038
• 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(摘自申请者摘要)：大肠杆菌是 年尿路感染(UTIs)的最常见原因 其他健康的个体。泌尿系致病性大肠埃希菌 导致这些感染的特征是 血清分型并表达P菌毛、α-溶血素和需氧蛋白。 来自欧洲的最新发现表明，细胞毒性坏死性疾病 1型因子(CNF1)，一种在美国很少研究的毒素，也是 通常由致泌尿系统的大肠杆菌产生。CNF1和 免疫相关的CNF2是110-115 kDa的多肽 在真核细胞中诱导多核和肌动蛋白聚合 一些动物的坏死和死亡。我们最近克隆并测序了 CNF2，发现CNF2的N-末端半部分与 多杀性巴氏杆菌的皮肤坏死毒素，是一种 介导猪进行性鼻炎的病理过程。我们也 观察到CNF2修饰一个小的GTP结合蛋白 参与应力纤维组装的Rho。长期目标 这项提议的目的是审查CNF1在 尿路致病性大肠埃希氏菌的毒力及其精确测定 CNF1修饰Rho的机制。具体目标是 旨在实现这些目标的目的是：1)分析CNF1的作用 构建J96菌株对J96菌株致病性的研究 Cnf1-该菌株的负衍生物，并将突变体与 上升性尿路感染小鼠模型中的野生型毒力 人肾和膀胱细胞；2)研究人肾和膀胱细胞 CNF1对Rho的化学修饰并阐明这种修饰是如何 导致肌动蛋白聚合；3)解剖二者之间的关系 CNF1的结构及其通过产生一组突变体的功能 通过删除、区域定向和站点特定的CNF1 诱变及其单抗和单特异性抗体的制备 作为毒素结构完整性的探针；4)尝试识别 CNF1的功能性受体。