Pseudomonas aeruginosa is a major opportunistic pathogen, and this study will elucidate its pathogenic mechanisms. The ability of P. aeruginosa to secrete numerous toxic and degradative enzymes plays a major role in its pathogenesis. Among these exoenzymes are LasB protease (elastase) and LasA protease (staphylolytic protease) which act synergistically to degrade elastin and many other clinically relevant proteins. Both LasA and LasB proteases are initially formed as pre-proenzymes with three domains: a signal sequence, a propeptide and the mature sequence. The propeptide domain acts as an intramolecular chaperone that is required for both the enzyme's activity and its extracellular localization. The objective of this proposal is to understand LasA and LasB molecular structures, the mechanisms of their chaperone-assisted folding, pathways of secretion in P. aeruginosa, and clinical relevance. This proposal combines molecular, biochemical and biophysical approaches to compare the processing and secretion of these two important proteases. The investigators will analyze the structures of LasA and LasB exoproteins by genetic and crystallographic methods. They will determine the mechanisms of propeptide (chaperone)-mediated folding and secretion of LasA and LasB proteins. They will determine the mechanism by which proLasA and proLasB interact with the type II secretion apparatus for release to the environment. They will also determine the clinical relevance of LasA and LasB in pathogenesis, and test the efficacy of LasA as a specific anti-staphylococcal therapeutic treatment. This research will result in new information on a significant mechanism of pathogenesis in an important opportunistic organism. It also provides a model system for understanding a major pathway of protein export in gram-negative bacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI026187-09A1
Application #
2901877
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Taylor, Christopher E,
Project Start
1988-04-01
Project End
2004-06-30
Budget Start
1999-07-15
Budget End
2000-06-30
Support Year
9
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Barequet, Irina S; Bourla, Nirit; Pessach, Yuval N et al. (2012) Staphylolysin is an effective therapeutic agent for Staphylococcus aureus experimental keratitis. Graefes Arch Clin Exp Ophthalmol 250:223-9
Barequet, Irina S; Habot-Wilner, Zohar; Mann, Oran et al. (2009) Evaluation of Pseudomonas aeruginosa staphylolysin (LasA protease) in the treatment of methicillin-resistant Staphylococcus aureus endophthalmitis in a rat model. Graefes Arch Clin Exp Ophthalmol 247:913-7
Grande, Kerian K; Gustin, Jean K; Kessler, Efrat et al. (2007) Identification of critical residues in the propeptide of LasA protease of Pseudomonas aeruginosa involved in the formation of a stable mature protease. J Bacteriol 189:3960-8
Makal, Umit; Wood, Lynn; Ohman, Dennis E et al. (2006) Polyurethane biocidal polymeric surface modifiers. Biomaterials 27:1316-26
Kessler, Efrat; Safrin, Mary; Blumberg, Shmaryahu et al. (2004) A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction. Anal Biochem 328:225-32
Barequet, Irina S; Ben Simon, Guy J; Safrin, Mary et al. (2004) Pseudomonas aeruginosa LasA protease in treatment of experimental staphylococcal keratitis. Antimicrob Agents Chemother 48:1681-7
McIver, Kevin S; Kessler, Efrat; Ohman, Dennis E (2004) Identification of residues in the Pseudomonas aeruginosa elastase propeptide required for chaperone and secretion activities. Microbiology 150:3969-77
Cahan, R; Axelrad, I; Safrin, M et al. (2001) A secreted aminopeptidase of Pseudomonas aeruginosa. Identification, primary structure, and relationship to other aminopeptidases. J Biol Chem 276:43645-52
Malhotra, S; Silo-Suh, L A; Mathee, K et al. (2000) Proteome analysis of the effect of mucoid conversion on global protein expression in Pseudomonas aeruginosa strain PAO1 shows induction of the disulfide bond isomerase, dsbA. J Bacteriol 182:6999-7006
Kessler, E; Safrin, M; Gustin, J K et al. (1998) Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides. J Biol Chem 273:30225-31

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