Pili or fimbriae provide adhesive functions for the pathogenesis of bacterial infections and play important roles during the establishment of respiratory, urinary, and intestinal infectious diseases. The mechanism of pilus assembly in gram-negative bacteria has been well characterized; however, an assembly pathway for pili of gram-positive pathogens has not yet been revealed. The long-term goal of this proposal is to elucidate the mechanisms of pillus assembly in gram-positive microbes. Our specific hypothesis is that gram-positive bacterial pili are covalently linked by sortase to the bacterial cell wall. This hypothesis is based on the following observations: i) Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, a mechanism that requires sorting signals of surface protein precursors and sortase, which cleaves sorting signals at the LPXTG motif and links the C-terminus of surface proteins via an amide bond to the peptidoglycan cross-bridge; ii) Pilus assembly in Corynebacterium diphtheriae occurs by a mechanism of ordered cross-linking, whereby pilin-specific sortases cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits; iii) Muramidase treatment releases pili into the medium; iv) Sortase and pilin genes with sorting signal and pilin motif are found in many gram-positive pathogens. Based on these observations, we propose the following specific aims: 1. Determination of the molecular architecture of corynebacterial pili. The proposed experiments will characterize the biochemical linkages between subunits of corynebacterial pili and the requirements of sortases and pilin motif sequences for assembly. 2. Biochemical analysis of pilus assembly in corynebacteria. 3. Determination of the universality of pilus architecture and assembly mechanisms in gram-positive bacteria. We will determine, by biochemical and microscopic methods, whether Streptococcus agalactiae employes the same mechanism for pilus assembly. We will also evaluate the role of pilus-specific genes in S. agalactiae pathogenesis using rodent models of infection based on the observation that corynebacterial pili plays an important role in bacterial adherence. Together these studies will reveal the assembly mechanism of pili in gram-positive bacteria and the contribution of these organelles during the pathogenesis of infection. [unreadable] [unreadable]