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.
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