: Pathogenic bacteria must assemble and secrete virulence factors in order to interact with their hosts and cause disease. Gram-negative bacteria have an outer membrane in addition to a cytoplasmic membrane and must secrete virulence factors across both these barriers. The mechanisms by which this occurs can be quite complex and are not well understood. The chaperone/usher pathway is a virulence protein secretion pathway that requires two components for secretion across the outer membrane: a periplasmic chaperone and an outer membrane protein termed an usher. The chaperone directs proper folding of the secreted proteins and prevents their engagement in non-productive interactions. The usher serves as an assembly platform at the outer membrane and provides a secretion channel to the cell surface. The chaperone/usher pathway is required for assembly and secretion of a superfamily of adhesive structures in a broad range of Gram-negative pathogens. The prototypical organelles assembled by this pathway are the P and type 1 pili expressed by uropathogenic Escherichia coli, the primary causative agent of urinary tract infections. P and type 1 pili are critical virulence factors, allowing binding and colonization of the kidney and bladder, respectively. The long-term goal of this proposal is to use pilus biogenesis by uropathogenic E. coli as a model system with which to understand virulence factor secretion in Gram-negative bacteria. More specifically, the structure and function of the usher will be investigated to elucidate the molecular mechanisms governing secretion across the outer membrane. The first specific aim is to create a detailed model of the structural arrangement of the usher in the outer membrane using computer analysis and epitope mapping techniques. The second specific aim is to probe function of the usher through generation and analysis of mutants. The third specific aim is to establish a cell-free system for pilus biogenesis based on reconstitution of the usher into liposomes. Such a system will provide an invaluable tool for studying the chaperone/usher pathway and analyzing mutants. The work described in this proposal will elucidate mechanisms of virulence factor secretion and create opportunities for the development of novel antimicrobial agents to treat not only urinary tract infections, but also a broad range of infectious diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Chin, Jean
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
State University New York Stony Brook
Schools of Medicine
Stony Brook
United States
Zip Code
Du, Minge; Yuan, Zuanning; Yu, Hongjun et al. (2018) Handover mechanism of the growing pilus by the bacterial outer-membrane usher FimD. Nature 562:444-447
Werneburg, Glenn T; Thanassi, David G (2018) Pili Assembled by the Chaperone/Usher Pathway in Escherichia coli and Salmonella. EcoSal Plus 8:
Sarowar, Samema; Hu, Olivia J; Werneburg, Glenn T et al. (2016) The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution. J Bacteriol 198:2360-9
Pham, Thieng; Werneburg, Glenn T; Henderson, Nadine S et al. (2016) Effect of chaperone-adhesin complex on plug release by the PapC usher. FEBS Lett 590:2172-9
Chahales, Peter; Hoffman, Paul S; Thanassi, David G (2016) Nitazoxanide Inhibits Pilus Biogenesis by Interfering with Folding of the Usher Protein in the Outer Membrane. Antimicrob Agents Chemother 60:2028-38
Chahales, Peter; Thanassi, David G (2015) Structure, Function, and Assembly of Adhesive Organelles by Uropathogenic Bacteria. Microbiol Spectr 3:
Werneburg, Glenn T; Henderson, Nadine S; Portnoy, Erica B et al. (2015) The pilus usher controls protein interactions via domain masking and is functional as an oligomer. Nat Struct Mol Biol 22:540-6
Chahales, Peter; Thanassi, David G (2015) A more flexible lipoprotein sorting pathway. J Bacteriol 197:1702-4
Pham, Thieng; Henderson, Nadine S; Werneburg, Glenn T et al. (2015) Electrostatic networks control plug stabilization in the PapC usher. Mol Membr Biol 32:198-207
Farabella, Irene; Pham, Thieng; Henderson, Nadine S et al. (2014) Allosteric signalling in the outer membrane translocation domain of PapC usher. Elife 3:

Showing the most recent 10 out of 34 publications