The DegP (HtrA) protease is essential for virulence in several Gram- negative pathogens: S.typhimurium, B. abortus, Y. enterocolitica, E. coli and P. aeruginosa. This important virulence factor was recently identified in S. pyogenes and S. aureus suggesting that inhibitors of DegP function may represent true broad-spectrum antiinfectives. The DegP protease is a multifunctional protein essential for the removal of misfolded and aggregated proteins in the periplasm of Gram-negative bacteria. In vitro, DegP has shown weak protease activity on casein and several other non- native substrates. We have identified the major pilin subunit of the Pap pilus, PapA, as a native DegP substrate and demonstrate binding and proteolysis of this substrate in vitro. This is the only known substrate that relates the proteolytic function of this important protein to pathogenesis. With this finding the DegP recognition and cleavage sites in PapA will be mapped and two HTS assays developed based on cleavage of a fluorescently tagged peptide substrate or whole PapA protein, respectively. We will validate the Gram-positive DegP as a virulence factor and develop this target as well. There is ample biological precedent for the efficacy of protease inhibitors as therapeutic agents. PROPOSED COMMERCIAL APPLICATIONS: The experiments outlined in this proposal will validate and develop DegP as an anti-infective target. The assays and procedures developed will allow HTS and rational drug design to be initiated in Phase II. This technology should allow a new class of antibiotics to be developed to combat antibiotic-resistant gram-negative pathogens.