Mechanism of the Usher in Assembly and Secretion of Pili

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PROJECT SUMMARY Pathogenic bacteria must assemble and secrete virulence factors to interact with host tissues and cause disease. Gram-negative bacteria have an outer membrane (OM) in addition to a cytoplasmic membrane and must secrete virulence factors across both these barriers. The mechanisms by which this occurs are not well understood. This project uses the chaperone-usher (CU) pathway as a model system with which to probe mechanisms of protein secretion and virulence factor biogenesis in Gram-negative bacteria. The CU pathway is a conserved secretion system dedicated to the assembly of virulence-associated surface structures termed pili or fimbriae. Pili are hair-like polymers that typically function as adhesive organelles and have roles in colonization of surfaces, biofilm formation, interactions with host cells, and pathogenesis. The prototype structures assembled by the CU pathway are the type 1 and P pili expressed by uropathogenic Escherichia coli. E. coli is the primary causative agent of urinary tract infections, a major healthcare burden and source of antibiotic resistance. Type 1 and P pili are critical virulence factors that mediate colonization of the bladder and kidneys, respectively. The CU pathway requires two components for secretion across the OM: a periplasmic chaperone and an integral OM protein termed the usher. The chaperone directs proper folding of pilus subunit proteins and maintains the proteins in an assembly-competent state. The usher is a dynamic molecular machine that catalyzes the formation of subunit-subunit interactions, promotes ordered polymerization of the pilus fiber, and provides the channel for secretion of the pilus to the cell surface. Pili are critical for initiating and sustaining infection, and the CU pathway represents an attractive target for the development of anti-virulence therapeutics, particularly for infection of the urinary tract. The goals of this proposal are to probe the structure and function of the usher to gain a complete understanding of the molecular mechanisms governing pilus biogenesis at the bacterial OM, and to exploit the knowledge and expertise gained over the course of this project to develop small molecule inhibitors of the CU pathway. This proposal will test the hypothesis that the usher distinguishes among and organizes pilus subunits to guarantee the assembly of adhesive organelles, and that the usher and usher-chaperone-subunit interfaces are targets for small molecule inhibition. The Specific Aims of this proposal are to: 1) Determine how the usher is activated and initiates pilus biogenesis; 2) Define molecular details of the subunit incorporation and pilus extension cycle; and 3) Identify small molecule inhibitors of usher function in pilus biogenesis. The proposed studies will answer fundamental questions of protein secretion and the regulated assembly of complex organelles at the bacterial OM, and will provide a foundation for the development of novel therapeutic agents that selectively disrupt virulence factor biogenesis.