PROTEASE SECRETION IN PSEUDOMONAS AERUGINOSA

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The broad objective of this investigation is to increase our understanding of the pathogenic mechanisms of Pseudomonas aeruginosa. A property of P. aeruginosa which plays a major role in pathogenesis is its ability to secrete toxic and degradative exoenzymes which cause necrosis at sites of infection. However, little is known about the mechanism of secretion. Among the exoenzymes of P. aeruginosa are two proteases, called elastase and LasA, which have a variety of substrates, but they also can act cooperatively to degrade elastin. The ability of P. aeruginosa to degrade elastin is believed to be a significant factor in its pathogenesis because it is a major protein of connective tissue and a vital component for lung function. Both elastase and LasA protease are initially formed as preproenzymes which are proteolytically processed twice before they are released by the organisms. We have recently provided evidence that proelastase processing occurs by autocatalysis, but the mechanism of LasA processing is unknown. Both elastase and LasA protease, as well as many other exoproteins, are secreted through the outer membrane by a complex set of proteins encoded by the xcp genes. Little is known about the sorting signals which channel exoproteins through this secretion pathway. The objective of this proposal is to understand the mechanisms of processing and secretion of the elastolytic proteases in P. aeruginosa. This proposal combines genetic and biochemical approaches to compare these proteases and their secretion pathways. Much is known about the structure of elastase, but little is known about LasA as a protease. We will determine the structure/function relationships of LasA using genetic, biochemical, and immunological approaches. We will characterize the processing and secretion of elastase and LasA proteins that have been altered by substitutions in their active sites. This will allow us to evaluate the role of autoprocessing and identify the location in the cell where secretion is blocked to better understand the pathway of Secretion. Both elastase and LasA have long amino terminal peptides which are removed during secretion, and may be involved in translocation, folding, and/or the enzymatic activity of the proteases. These possibilities will be evaluated by characterizing the effects of propeptide deletions, amino acid substitutions, and hybrids formed by propeptide domain switching. Little is known about the mechanisms by which secreted proteins interact with the extracellular-translocation machinery. We will identify targeting signals in elastase and LasA, which presumably interact with the Xcp- secretion system, and target these proteins for secretion through the outer membrane. This will be performed through a deletion analysis, site- directed mutagenesis, and a study of the behavior of protein fusions in P. aeruginosa in order to elucidate the sequences in elastase and LasA that contain essential targeting signals for extracellular Secretion. This research provides an excellent model system for understanding protein export in gram-negative bacteria and should result in new information on a significant mechanism of pathogenesis in an important opportunist.