Human and mouse neutrophils contain neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR 3). The capacity of these serine proteinases, especially NE, to kill bacteria in vitro and to cleave extracellular matrix (ECM) proteins leading to tissue damage is well established, but whether these enzymes kill bacteria in vivo and have specific bacterial target molecules remains unknown. Also, the potential role of the ECM degradation products generated by these proteinases in host defense against bacteria has not been explored. Using mice deficient in NE and CG, we have demonstrated that NE, but not CG, reduces mortality from Gram negative bacterial infections. To date, the antibacterial role of PR 3 has not been clarified in vivo. We propose to generate mice deficient in PR 3 by gene targeting and subject them to bacterial lung infections to determine the relative importance of PR 3 in host defense against bacteria in the lung. Our preliminary data show that the outer membrane protein (Omp) A represents a critical target for NE-mediated killing of E. coli. We hypothesize that Omps represent essential targets of NE to kill other Gram negative bacteria. Recently, we have found that NE kills Pseudomonas aeruginosa in vitro, and degrades its major Omp F, which is unrelated to Omp A. We propose to determine the importance of Omp F in NE-mediated killing of Pseudomonas aeruginosa in isolated neutrophils and in vivo models of acute and chronic pulmonary infections. Wild type Pseudomonas aeruginosa and isogenic strains deficient in Omp F will be used in these studies. We have observed that elastin peptides from NE-digested human lung elastin are bactericidal for Klebsiella pneumoniae in vitro. These data constitute the first evidence of antibacterial role of ECM derived peptides and reveal a novel role for ECM. We will isolate these elastin fragment(s) and investigate their bactericidal activity in vitro and in vivo. These studies will advance our knowledge of the functional properties of lung serine proteinases and ECM peptides. Also, characterization of NE-degraded elastin may provide novel antimicrobial peptides in humans.
