| A proline-arginine-rich antibacterial peptide (PR-39) from pig neutrophils was isolated and characterized by gel-filtration, reversed-phase high performance liquid chromatography (RP-HPLC), mass spectrometry, and various antibacterial assays. PR-39 was a potent antibiotic mainly against Gram-negative bacteria such as Escherichia coli and salmonellae. PR-39 had postantibiotic effect and inhibited Salmonella typhimurium invasion into rat intestinal epithelial cells. This antibacterial peptide killed bacteria by a nonmembrane pore-forming mechanism.; In a structure-activity relationship study, it was determined that the first 26 amino acids of the NH{dollar}sb2{dollar}-terminus (PR-26) was the antibacterial functional domain of PR-39. The N-terminal first three arginine residues and the central segment containing residues 20 to 26 were essential for the antibacterial activity of PR-26. In addition, PR-39 inhibited the production of superoxide anion (O{dollar}sb2sp-{dollar}) from pig neutrophils in a whole cell assay and human neutrophils in a cell-free assay. Using a cell-free superoxide anion assay and recombinant phagocyte NADPH oxidase components, we demonstrated that PR-39 bound to the Src homology region 3 (SH3) domains of p47{dollar}sp{lcub}phox{rcub},{dollar} one of the cytosol components of phagocyte NADPH oxidase, and blocked the interaction of p47{dollar}sp{lcub}phox{rcub}{dollar} with p22{dollar}sp{lcub}phox{rcub},{dollar} one of the transmembrane protein components of phagocyte NADPH oxidase. This suggested that PR-39 might be a negative regulator for phagocyte NADPH oxidase and have antioxidant activity in host inflammatory responses.; Taken together, these findings show that a multifunctional antibacterial peptide, PR-39, is found in pig neutrophils. The study of PR-39 will generate new information about how the neutrophil regulates its antimicrobial systems in host defense and will provide an excellent candidate for the development of new drugs for infectious diseases. |
