ANALYSIS OF ESCHERICHIA COLI VIRULENCE DETERMINANTS

大肠杆菌毒力决定因素分析

• 批准号: 3070924
• 负责人: David Alan Low
• 金额: $ 7.18万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 1993-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3070924
• 关键词: Escherichia coli; agglutination reaction; antibacterial antibody; antibody specificity; antiserum; autoradiography; bacterial antigens; bacterial genetics; bacterial proteins; binding proteins; cell adhesion; enzyme linked immunosorbent assay; gel electrophoresis; gene expression; gene mutation; genetic manipulation; genetic strain; hybridomas; immunoelectron microscopy; immunological substance; laboratory mouse; laboratory rabbit; membrane activity; membrane proteins; monoclonal antibody; pilus; plasmids; polymerization; pyelonephritis; transposon /insertion element; urinary bladder disorder; urinary tract infection; virulence

Urinary tract infections (UTI) caused by Escherichia coli are among the most common bacterial diseases, yet little is known about the pathogenic mechanisms involved. Previous work has shown that adhesion of these bacteria to host epithelium via the P blood group antigen receptor is an important virulence determinant. The pyelonephritis-associated pili (pap) gene cluster has been shown to encode P-adhesion activity as well as pilin monomers that polymerize at the bacterial cell surface to form pili fibers. Genetic evidence indicates that pilin and adhesin protein(s) may interact to form an "adhesin structure". However, the role(s) of each pap-encoded protein in forming this postulated structure and in adherence are not known. The studies proposed here are designed to provide information regarding the molecular mechanism(s) by which these bacteria that cause UTI adhere to host cells. My approach will be to first generate antisera to pap- encoded gene products that appear to play roles in adhesion. Antisera will be generated to partially-purified protein isolated from E. coli strains over-producing these proteins. These antisera and antisera I have already generated to pap-encoded pili will be used to determine the cellular location(s) of each pap-encoded protein. An important part of these studies will be the analysis of strains containing non-polar insertions within each of the pap genes. By analyzing the effect(s) of these mutations on the interactions between pap-encoded pilin and adhesin-associated proteins, the pathway for assembly of these gene products into an adhesin structure will be determined. The interactions between these pap proteins to form an adhesin structure will be determined by using colloidal gold-tagged anti-pap protein antisera and electron microscopy.

由大肠杆菌引起的尿路感染 (UTI) 是 最常见的细菌性疾病之一，但人们知之甚少 关于所涉及的致病机制。 之前的作品有 表明这些细菌通过 P 粘附到宿主上皮上 血型抗原受体是重要的毒力 决定因素。 肾盂肾炎相关菌毛 (pap) 基因簇 已被证明编码 P-粘附活性以及菌毛蛋白 在细菌细胞表面聚合形成的单体 菌毛纤维。 遗传证据表明菌毛蛋白和粘附素 蛋白质可以相互作用形成“粘附素结构”。 然而， 每个 pap 编码的蛋白质在形成该假定的过程中的作用 结构和粘附性尚不清楚。 提出的研究 这里旨在提供有关分子的信息 这些引起尿路感染的细菌粘附的机制 宿主细胞。 我的方法是首先产生抗血清 编码的基因产物似乎在粘附中发挥作用。 部分纯化的分离蛋白将产生抗血清 来自过量生产这些蛋白质的大肠杆菌菌株。 这些抗血清 我已经生成了巴氏编码菌毛的抗血清 用于确定每个 pap 编码的细胞位置 蛋白质。 这些研究的一个重要部分将是分析 每个pap内含有非极性插入的菌株 基因。 通过分析这些突变对 pap编码的菌毛蛋白和粘附素相关蛋白之间的相互作用 蛋白质，将这些基因产物组装成蛋白质的途径 将测定粘附素结构。 之间的相互作用 这些 pap 蛋白形成粘附素结构 使用胶体金标记的抗 PAP 蛋白测定 抗血清和电子显微镜。