Project Summary Gram-positive bacteria are surrounded by a thick peptidoglycan cell wall that is covalently affixed with a diverse array of macromolecules that maintain cell integrity and promote effective interactions with the environment. In clinically important bacterial pathogens, these macromolecules function as essential virulence factors. Our research will investigate how bacteria elaborate their surfaces with adhesive pili and cell wall glycopolymers that are critically important for microbial pathogenicity and growth. Both are attached to the same site on the cell surface and their relative abundance is balanced to maintain homeostasis. We will study the prototypical SpaA- pilus from Corynebacterium diphtheriae, the causative agent of the severe respiratory disease, diphtheria. The SpaA-pilus and related pili in >1,800 bacterial species are assembled by polymerases that covalently link pilus protein subunits (pilins) via lysine-isopeptide bonds that confer enormous tensile strength. By synergistically employing structural, biochemical, cellular, computational, and proteomic methods, we aim to learn how these polymerases select and crosslink their pilin substrates to build pili. We will also determine the first-ever atomic level structure of a crosslinked pilus and uncover the full array of pilus structures that inhabit the surface of C. diphtheriae. In complementary studies, we seek to identify small molecules that selectively disrupt the synthesis of glycopolymers that coexist with pili on the cell surface. Using a newly developed cell-based assay we will screen for compounds that inhibit the synthesis of an important cell wall glycopolymer in methicillin-resistant Staphylococcus aureus (MRSA). These inhibitors have the potential to be developed into novel therapeutics as they would impair MRSA's ability to cause infections and re-sensitize it to existing β-lactam antibiotics. Combined, our comprehensive studies of pilus and cell wall glycopolymer biogenesis will provide fundamental insight into the chemistry underlying polymer assembly on the surface of gram-positive bacteria and could lead to new antibiotics to treat infections caused by MRSA and other drug-resistant bacteria.