E. COLI HEMOLYSIN KILLS HUMAN RENAL EPITHELIAL CELLS

大肠杆菌溶血素杀死人肾上皮细胞

• 批准号: 3802428
• 负责人: JOHN W WARREN
• 金额: --
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3802428
• 关键词: Escherichia coli; aging; bacterial cytopathogenic effect; cell adhesion; cytotoxicity; disease /disorder model; fluorescent dye /probe; hemolysin; human old age (65+); human tissue; human very old age (85+); immunoelectron microscopy; laboratory mouse; membrane channels; membrane permeability; microspectrophotometry; mutant; nosocomial infections; pilus; pyelonephritis; renal tubule; site directed mutagenesis; tissue /cell culture; urinary bladder epithelium; urinary catheterization; virulence; young adult human (21-34)

Among the aged, E.coli is the most common organism causing acute pyelonephritis (pyelo) and bacteremia in the community, hospital, and nursing home. Fifty percent of pyelo E. coli are hemolytic versus 5-10% of fecal E. coli. The pathogenesis of hemolytic E. coli infection is obscure. This project's overall hypothesis is that E. coli fimbriae bind to the target cell cytoplasmic membrane, thus allowing effective delivery of secreted hemolysin to the membrane with subsequent pore formation, ion and molecular flux, and cell death. Resources used to test this hypothesis will include: 1) a prototypic hemolytic E. coli, strain CFT073, virulent in humans and mice; and 2) a putative target cell for pyelo, the human renal proximal tubular epithelial cell (HRPTEC). CFT073 binds in large numbers to HRPTEC by fimbriae and is cytotoxic. By transposon mutagenesis, the cytotoxicity was demonstrated to be mediated by hemolysin. This Project will test the following hypotheses: 1) That the HRPTEC-binding fimbriae enhance the cytotoxicity of hemolysin. Deletion for transposon mutagenesis of CFT073 to the four permutations of +/- hemolysin and +/- HRPTEC-binding fimbriae will be performed. The ability of CFT073 and these three mutants to cause cytotoxicity to HRPTEC and pyelo in the mouse model will be compared. 2) That the cytotoxicity of hemolytic e. coli varies among human proximal tubular, collecting duct, and pelvic epithelial cells from the same human donors. A target cell for pyelo E. coli must be an epithelial cell behind which is the renal interstitium. These three are such cells and will be collected from each of 10 donors and exposed to CFT073, its hemolysin- negative mutant, and a fecal strain; adherence and cytotoxicity will be compared. 3) That the cytotoxicity of HRPTEC by hemolytic E. coli is greater in cells from aged than from younger humans. Membrane alterations are among the changes of aging. Preliminary findings suggest an increased susceptibility of cells from older than younger donors. This project will examine CFT073 adherence and cytotoxicity to HRPTEC of more than 100 donors spanning nine decades of life. Detection of change as little as one percentage point per decade and of the degree of contribution of adherence to cytotoxicity by age will be possible. 4) That hemolysin kills HRPTEC by formation of transmembrane pores with subsequent fluxes of ions and small molecules. Ion fluxes in HRPTEC as a result of hemolysin-induced pore formation will be measured radiometrically and spectrophotometrically with fluorescent probes sensitive to nanomolar concentrations of ions to determine if fluxes occur and precede cell death.

在老年人中，大肠杆菌是最常见的生物，引起急性 社区，医院和 疗养院。 pyelo大肠杆菌的百分之五十是溶血性，而5-10％ 粪便大肠杆菌。 溶血大肠杆菌感染的发病机理晦涩。 该项目的总体假设是大肠杆菌纤维化与 靶细胞细胞质膜，从而有效递送 分泌的血素到膜，随后形成孔，离子和 分子通量和细胞死亡。 用于检验该假设的资源 将包括：1）原型溶血大肠杆菌，CFT073菌株 在人类和老鼠中； 2）Pyelo，人类的推定目标细胞 肾近端管状上皮细胞（HRPTEC）。 CFT073大约结合 通过Fimbriae到HRPTEC的数字，具有细胞毒性。 通过转座子诱变， 细胞毒性已证明是由血素蛋白介导的。 该项目将检验以下假设： 1）HRPTEC结合纤维膜增强了血素蛋白的细胞毒性。 CFT073转座子诱变的删除至四个排列 将进行+/- hemolysin和+/- hrptec结合纤维化。 这 CFT073和这三个突变体对HRPTEC引起细胞毒性的能力 将比较小鼠模型中的pyelo。 2）溶血e的细胞毒性。大肠杆菌在人类近端有所不同 来自同一人的管状，收集管和骨盆上皮细胞 捐助者。 大肠杆菌的目标细胞必须是后面的上皮细胞 这是肾脏间质。 这三个是这样的细胞，将是 从10个捐助者中的每个人中收集并暴露于CFT073，其helsolysin- 负突变体和粪便菌株；依从性和细胞毒性将是 比较的。 3）溶血大肠杆菌对HRPTEC的细胞毒性在细胞中更大 来自年龄比年轻人的年龄。 膜改变是 衰老的变化。 初步发现表明易感性提高 来自年轻捐助者的细胞。 该项目将检查CFT073 遵守和细胞毒性对100多个捐助者的HRPTEC 数十年的生活。 检测更改的时间仅为一个百分点 十年和对细胞毒性依从性的贡献程度 年龄将有可能。 4）血解素通过与跨膜孔形成的杀死HRPTEC 离子和小分子的随后通量。 Hrptec中的离子通量为 溶血素诱导的孔形成的结果将通过放射线测量 分光光度法，荧光探针对纳摩尔敏感 离子的浓度确定通量是否发生并在细胞死亡之前发生。