E. COLI HEMOLYSIN KILLS HUMAN RENAL EPITHELIAL CELLS

大肠杆菌溶血素杀死人肾上皮细胞

基本信息

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
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

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引起急性急性呼吸道疾病的微生物。社区、医院和医院的肾盂肾炎（pyelo）和菌血症疗养院。 50% 的幽门大肠杆菌具有溶血性，而 5-10% 的粪便大肠杆菌。溶血性大肠杆菌感染的发病机制尚不清楚。该项目的总体假设是大肠杆菌菌毛与靶细胞的细胞质膜，从而可以有效地递送将溶血素分泌到膜上，随后形成孔，离子和分子流动和细胞死亡。用于检验这一假设的资源将包括：1) 原型溶血大肠杆菌，菌株 CFT073，剧毒在人类和小鼠中； 2) 人类肾盂肾盂的假定靶细胞肾近端肾小管上皮细胞（HRPTEC）。 CFT073 结合大通过菌毛对 HRPTEC 进行计数，并且具有细胞毒性。通过转座子诱变，细胞毒性被证明是由溶血素介导的。该项目将测试以下假设： 1) HRPTEC 结合菌毛增强溶血素的细胞毒性。 CFT073转座子诱变至四种排列的缺失将进行 +/- 溶血素和 +/- HRPTEC 结合菌毛。这 CFT073 和这三个突变体对 HRPTEC 造成细胞毒性的能力与小鼠模型中的 pyelo 进行比较。 2) 溶血e的细胞毒性。大肠杆菌在人类之间存在差异来自同一个人的肾小管、集合管和盆腔上皮细胞捐助者。幽门大肠杆菌的靶细胞必须是背后的上皮细胞这是肾间质。这三个细胞就是这样的细胞，并且将是从 10 名捐赠者中收集并暴露于 CFT073（其溶血素）阴性突变体和粪便菌株；粘附性和细胞毒性将比较的。 3) 溶血性大肠杆菌对HRPTEC的细胞毒性更大来自老年人的比来自年轻人的。膜改变是其中之一衰老的变化。初步研究结果表明易感性增加来自年长捐赠者的细胞比来自年轻捐赠者的细胞。该项目将检查 CFT073 九个地区 100 多名捐赠者对 HRPTEC 的依从性和细胞毒性几十年的人生。检测到的变化只有一个百分点十年以及坚持细胞毒性的贡献程度年龄将成为可能。 4) 溶血素通过形成跨膜孔杀死 HRPTEC 随后的离子和小分子通量。 HRPTEC 中的离子通量作为溶血素诱导的孔隙形成的结果将通过辐射测量来测量并使用对纳摩尔敏感的荧光探针进行分光光度分析离子浓度以确定通量是否发生以及细胞死亡之前是否发生。