The broad objective of this investigation is to increase our understanding of the pathogenic mechanisms of Pseudomonas aeruginosa, an opportunistic pathogen which causes a variety of severe and lethal infections in humans. P. aeruginosa is notable for its ability to secrete into its environment a number of toxic and degradative enzymes, a property which has been shown to play a dominant role in the organism's pathogenic processes. Little is known about the pathway of secretion of these enzymes in P. aeruginosa. The proposed study will examine the synthesis, activation, and secretin of two proteases in P. aeruginosa. This bacterium secretes two distinct proteases called elastase and alkaline protease, and these extracellular enzymes are responsible for much of the necrosis and general invasiveness seen at sites of infection. We have recently shown that the gene lasA encodes a 31 Kd protein which activates the elastolytic potential of the precursor, pro-elastase. We will investigate the nature of this activation step employing genetic and biochemical techniques. The structural gene for elastase (lasB) will be cloned, characterized, and sequenced. Processing of elastase precursors will be determined at the molecular level, and intermedicates in the secretion pathway will be characterized. Mutations known to block the secretion of a number of extracellular enzymes, including elastase, will be characterized for their role in the processing of elastase intermediates. Gene fusions will be used to compare the regulation of lasA and lasB in P. aeruginosa. lasB mutants will be constructed by in vitro manipulation of cloned DNA followed by gene replacement and then characterized for protease production. lasA protein also has a secondary role in the secretion of elastase which will be investigated and defined. We will also clone and characterize the structural gene for alkaline protease (alpA), a secreted enzyme with a pathway of secretion which is apparently distinct from that of elastase. alpA mutants will be constructed by gene replacement and characterized. The effect of lasA and lasB mutations on alkaline protease secretion will be examined.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Tennessee Health Science Center
Schools of Medicine
United States
Zip Code
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

Showing the most recent 10 out of 18 publications