CONTROL AND EXPRESSION OF TYPE 1 PILI IN E. COLI

大肠埃希菌和其他革兰氏阴性肠杆菌的1型菌毛 细菌是由丝状、蛋白质组成的附属物 主要是一种叫做皮林的亚基。I型菌毛介导 大肠杆菌对多种真核生物的甘露糖敏感黏附 细胞，并且是促进低层细胞定植的一个因素 尿路。该项目的长期目标是建立 基因控制的分子本质，组装和 1型菌毛的受体结合活性，并在此过程中 对控制和装配的理解 超分子结构和受体-配体相互作用 将军。此外，希望这项研究将导致更好的 对细菌黏附在发病机制中作用的认识 传染病的风险。本文提出的研究使用了多种 利用遗传和生化技术：1)辨别因素 影响Pila的表达。Pila基因编码了主要的 菌毛的亚基，并受至少两种类型的 转录控制。一种类型是由 相邻基因的产物，另一种类型是亚稳态(ON 或关闭)由Pila启动子的反转实现的控制。 提出了创建和分析影响这些的损伤的方法 两种类型的转录调控。2)辨别分子 菌毛组装所需的相互作用。提出了一些方法。 用来分析具有点突变的突变体，一个基因 编码受体所需的菌毛的次要成分 有约束力的。项目包括分离外源基因 补偿性突变，条件性突变的分析 和桩蛋白的定位。此外，还可以使用 分离和检测具有影响两个人的皮损的突变体 其他与组装相关的基因，其中一个影响菌毛长度， 都被提出了。3)识别所需的分子相互作用 受体结合。概述了分离和分析的方法 受体特异性改变的突变体。此外，还有几种方法 分离PAL基因产物并评估其作用 受体结合中的蛋白质被概述。此外，还包括 受体结合和菌毛长度对纤毛虫相互作用的影响 用巨噬细胞对大肠杆菌进行了检测。

Type 1 pili of Escherichia coli and other gram negative enteric bacteria are filamentous, proteinaceous appendages composed primarily of a subunit called pilin. Type 1 pili mediate the mannose-sensitive adhesion of E. coli to a variety of eucaryotic cells and are a factor in promoting colonization in the lower urinary tract. The long term goal of this project is to establish the molecular nature of the genetic control, assembly and receptor binding activity of type 1 pili and in doing so, contribute to the understanding of the control and assembly of supramolecular structures and receptor-ligand interactions in general. Also, it is hoped that this research will lead to a better understanding of the role of bacterial adhesion in the pathogenesis of infectious disease. The research herein proposed uses a variety of genetic and biochemical techniques to: 1) Discern the factors effecting pilA expression. The pilA gene encodes the major subunit of pili and is subject to at least two types of transcriptional control. One type is a repression effected by the product of an adjacent gene, the other type is a metastable (ON or OFF) control effected by the inversion of the pilA promotor. Ways are proposed to create and analyze lesions affecting these two types of transcriptional control. 2) Discern the molecular interactions required for pilus assembly. Methods are suggested for the analysis of mutants having point mutations in pilE, a gene encoding a minor component of pili that is required for receptor binding. Projects include the isolation of extrageneic compensatory mutations, analysis of conditional pilE mutations and localization of the pilE protein. In addition, methods for isolating and examining mutants having lesions affecting two other assembly related genes, one of them affecting pilus length, are proposed. 3) Discern the molecular interactions required for receptor binding. Methods are outlined for isolating and analyzing mutants with an altered receptor specificity. Also, ways for isolating the pilE gene product and assessing the role of the pilE protein in receptor binding are outlined. In addition, the role of receptor binding and pilus length on the interaction of piliated E. coli with macrophages is examined.