Shafer and colleagues have found that human lysosomal cathepsin G is particularly antimicrobial against volutionary diverse pathogens such as Capnocytophaga sputigena, Escherichia coli, Neisseri gonorrhoeae, Pseudomonas aeruginosa, and Staphylococcus aureus. These investigators have studied the structure-function relationship of cathepsin G through the use of synthetic peptides that span the entire protein and have found that this protein has multiple antimicrobial domains that display """"""""target-specific"""""""" killing activity. However, one of these peptides, spanning residues 117-136 (CG 117-136) in the full-length protein, display the broad-spectrum antibacterial characteristic of the intact cathepsin G protein. Therefore, its activity may reflect the bactericidal capacity of the full-length protein. It is most active against S. aureus, a pathogen that often displays resistance to multiple antibiotics. This group has shown that the capacity of CG 117-136 to exert antibacterial action is independent of stereochemistry at the a carbon, suggesting that killing does not require recognition of a target with a chiral center but rather is mediated by a membrane-damaging mechanism. Shafer and colleagues now seek to ascertain the mechanism by which peptide CG 117-136 exerts broad-spectrum antimicrobial action in vitro. Using peptide synthesis procedures, these investigators will chemically modify CG 117-136 in order to identify regions essential for antimicrobial action (AIM 1). They will increase the cationic or hydrophobic properties of CG 117-136, which should enhance its capacity to interact with negatively charged microbial surface structures or promote insertion into the cytoplasmic membrane. Specifically chemical modifications will include substitution of neutral amino acids with arginine, extending the peptide with additional arginine groups, exchanging certain amino acids with more nonpolar residues, and attaching isoprenoid or fatty acid moieties to the peptide backbone. With such peptide variants Shafer and colleagues will conduct genetic and molecular experiments to help determine its mechanism of killing (AIM 2). Specifically, they will utilize Tn551 mutagenesis to identify mutants that express enhanced resistance to CG 117-136. Finally, using a rat infection model, this group will examine the capacity of CG 117-136 to exert antibacterial action in vivo and to reduce the incidence of abscesses in infected wounds (AIM 3). It is anticipated that these studies will help in the design and testing of new antimicrobial agents that can be used in the treatment of infections, particularly in those individuals with impaired host defenses or when the infecting pathogen display resistance to clinically useful antibiotics.
