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.
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