The long term objective of this research is to prepare therapeutically useful inhibitors of the proteinases that degrade the structural macromolecules of the cornea during bacterial keratitis, a relatively common condition accompanying corneal infection in man. The first step in this process of inhibitor design is to identify the proteinase present during infection and to describe the time course of their appearance in a rabbit model of Pseudomonas keratitis. This will be accomplished by the technique of enzymography to separate based on molecular mass and isoelectric point those proteinases present in corneal homogenates subsequent to bacterial infection. In addition, newly synthesized bacterial and host proteinases will be identified by metabolic radiolabelling with sulfate of methionine followed by separation of the proteins by 2-dimensional electrophoresis or immunoprecipitation with specific antibodies. Preliminary work indicates that only one bacterial proteinase, the Pseudomonas alkaline metalloproteinase is present in the Pseudomonas- infected, ulcerating rabbit cornea, suggesting that this proteinase may play an important role in the ulcerative process. We propose to investigate the inhibitor specificity of this proteinase using synthetic metalloproteinase inhibitors we have previously designed and tested for mammalian extracellular matrix degrading metalloproteinases. Data obtained from these studies will provide a rational basis for the design of inhibitors of higher potency. In addition, we propose to study the substrate specificity of the alkaline proteinase in order to further define the enzyme's active site. Finally, we propose to continue word initiated previously to clone and sequence the structural gene for Pseudomonas alkaline proteinase. Thus, the specific aims of this proposal include: (1) identification of bacterial and host proteinases present in the rabbit cornea at various stages after infection with Pseudomonas aeruginosa; (2) characterize alkaline proteinase with respect to substrate specificity and gene sequence; (3) evaluate peptide-based, metal chelating metalloproteinase inhibitors for potency with alkaline proteinase.
| Feltzer, R E; Gray, R D; Dean, W L et al. (2000) Alkaline proteinase inhibitor of Pseudomonas aeruginosa. Interaction of native and N-terminally truncated inhibitor proteins with Pseudomonas metalloproteinases. J Biol Chem 275:21002-9 |
