Molecular Basis of the Biogenesis and Function of the \u3ci\u3eEnterococcus faecalis\u3c/i\u3e Endocarditis- and Biofilm-Assiciated Pilus

In recent decades, the bacteria Enterococcus faecalis and Enterococcus faecium have emerged as leading causes of bloodstream infections and urinary tract infections (UTIs) in the health-care environment. Among the few enterococcal virulence factors identified using animal models of these diseases are surface proteins anchored to the cell wall by the house-keeping sortase SrtA and the endocarditis- and biofilm-associated (Ebp) pilus. The Ebp sortase-assembled pilus comprises three structural subunits, the minor pilins EbpA and EbpB and the major polymerizing pilin EbpC. They are assembled by the SrtC sortase enzyme. To investigate Ebp pilus biogenesis, I created a panel of E. faecalis mutants. Among these were single and combinatorial chromosomal deletion strains lacking the sortases and/or Ebp pilins and strains with point mutants i) in conserved structural subunit motifs, ii) in predicted sortase catalytic residues, and iii) in a putative EbpA functional domain. I assessed Ebp pilus fiber polymerization and morphology, minor pilin incorporation into fibers, and pilus subcellular compartmentalization using bacterial cell fractionation, immunoblotting, and immunogold electron microscopy. The role of Ebp pili in catheter-associated UTI (CAUTI), the most common hospital-acquired infection, and the molecular basis of Ebp pilus function in vivo were evaluated in a mouse model of CAUTI using a subset of these mutants. In this dissertation, I described the roles of all sortases, Ebp pilins, and their conserved sequence motifs in pilus biogenesis. My results support a model where pilus assembly is initiated by SrtC processing of EbpA, the tip pilin. Repeating subunits of EbpC are then polymerized by successive rounds of SrtC-pilus intermediate resolution by a conserved Lys in EbpC. EbpC polymerization is terminated when a conserved Lys from EbpB is incorporated at the pilus base. SrtA processing of EbpB leads to the anchoring of pili to the peptidoglycan cell wall. Animal studies revealed that EbpA\u27s metal ion-dependent adhesion site (MIDAS) motif encoded within its predicted von Willebrand factor A (VWA) domain, a common domain among sortase-assembled tip pilins, governed pilus function in vivo. Thus, these results describe molecular interactions involved in both Ebp pilus biogenesis and function in vivo, potential targets for interference with enterococcal virulence