Type IV pili are extracellular bacterial filaments required for attachment to surfaces and for pathogenesis in many plant, animal, and human infections (including bacterial meningitis caused by Neisseria meningitidis, gonorrhea caused by N. gonorrhoeae, and lung infections of cystic fibrosis patients caused by Pseudomonas aeruginosa). Through the work of the intracellular ATPase PilT, pili are retracted into the cell from which they emerge. Retraction leads to motility on surfaces and activates signaling pathways in eukaryotic target cells. Based on structural and mutagenesis data we suggest that ATP binding to PilT induces large-scale movements of individual domains, which adjusts the interface between subunits and promotes synergistic motor movements. Because unchecked retraction leads to non-piliated cells, the force of retraction is counterbalanced by other proteins with similar N-terminal sequences to Type IV prepilins. The three specific aims of this proposal are designed to improve our understanding of the mechanism of pilus retraction and counterbalance. We will take a simultaneous bottom-up &top-down approach that capitalizes on our expertise in protein structure/function analysis. Specifically, we will (1) carry out X-ray crystallography and small angle scattering studies of P. aeruginosa and N. gonorrhoeae PilT bound to various nucleotide analogues plus structure-based mutagenesis and phenotypic analysis of the resulting strains (2) identify the minimal set of pilus biogenesis proteins by expression in a non-piliated organism and (3) carry out protein crystallography and site directed mutagenesis of the low abundance prepilin-like proteins. The overall result of these experiments will be a better understanding of the molecular mechanism of retraction of the widespread bacterial virulence factor Type IV pili. Ultimately this knowledge will be used in designing approaches to control or block this pathway, thereby preventing the spread of many microbial diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059721-09
Application #
8054733
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sakalian, Michael
Project Start
2000-06-01
Project End
2013-03-31
Budget Start
2011-04-01
Budget End
2013-03-31
Support Year
9
Fiscal Year
2011
Total Cost
$295,116
Indirect Cost
Name
University of Wisconsin Madison
Department
Microbiology/Immun/Virology
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Park, Hyunjun; Kevany, Brian M; Dyer, David H et al. (2014) A polyketide synthase acyltransferase domain structure suggests a recognition mechanism for its hydroxymalonyl-acyl carrier protein substrate. PLoS One 9:e110965
Aly, Khaled A; Beebe, Emily T; Chan, Chi H et al. (2013) Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD. Microbiologyopen 2:94-104
Auldridge, Michele E; Satyshur, Kenneth A; Anstrom, David M et al. (2012) Structure-guided engineering enhances a phytochrome-based infrared fluorescent protein. J Biol Chem 287:7000-9
Mortenson, David E; Satyshur, Kenneth A; Guzei, Ilia A et al. (2012) Quasiracemic crystallization as a tool to assess the accommodation of noncanonical residues in nativelike protein conformations. J Am Chem Soc 134:2473-6
McLaughlin, Lorraine S; Haft, Rembrandt J F; Forest, Katrina T (2012) Structural insights into the Type II secretion nanomachine. Curr Opin Struct Biol 22:208-16
Franz, Laura P; Douzi, Badreddine; Durand, Eric et al. (2011) Structure of the minor pseudopilin XcpW from the Pseudomonas aeruginosa type II secretion system. Acta Crystallogr D Biol Crystallogr 67:124-30
Cordes, Timothy J; Worzalla, Gregory A; Ginster, Aaron M et al. (2011) Crystal structure of the Pseudomonas aeruginosa virulence factor regulator. J Bacteriol 193:4069-74
Misic, Ana M; Satyshur, Kenneth A; Forest, Katrina T (2010) P. aeruginosa PilT structures with and without nucleotide reveal a dynamic type IV pilus retraction motor. J Mol Biol 400:1011-21
Hansen, Johanna K; Demick, Karen P; Mansfield, John M et al. (2007) Conserved regions from Neisseria gonorrhoeae pilin are immunosilent and not immunosuppressive. Infect Immun 75:4138-47
Satyshur, Kenneth A; Worzalla, Gregory A; Meyer, Lorraine S et al. (2007) Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility. Structure 15:363-76

Showing the most recent 10 out of 15 publications