Effects of a Holliday junction-binding peptide on mammalian cells and the inhibition of Salmonella growth inside murine macrophages

Several hexapeptides that inhibit bacterial phage lambda Integrase-mediated site specific recombination were previously identified through screening of combinatorial peptide libraries. The two most potent peptides bind to Holliday junctions, inhibit a number of Holliday junction and branched DNA-processing enzymes and exhibit broad spectrum antibacterial properties in vitro. It was unknown, however, whether these peptides can also inhibit the growth of bacterial pathogens inside mammalian host cells or what effects the peptides may have on the host cells.;Here, I showed that peptide wrwycr was concentrated in murine peritoneal macrophages and J774A.1 macrophage-like cells as well as in HeLa cells. The peptide inhibited the growth of Salmonella enterica serovar Typhimurium inside both J774A.1 cells and peritoneal macrophages, in a concentration-dependent manner. The peptide induces the SOS response in a dose-dependent fashion in intracellular Salmonella, implicating the accumulation of DNA damage as a mechanism of bacterial growth inhibition. This is consistent with, but does not prove that, peptide-dependent inhibition of DNA repair may inhibit the growth of intracellular bacteria. Peptide wrwycr caused low toxicity in the peritoneal macrophages, but greater toxicity in the J774A.1 cells.;Peptide wrwycr also caused concentration-dependent toxicity in HeLa cells, which was exacerbated by co-treatment with DNA-damaging agents. The peptide was at least somewhat synergistic both with etoposide, a topoisomerase II poison, and with hydroxyurea, a ribonucleotide reductase inhibitor, in accumulating apoptosis-independent DNA breaks in HeLa cells. The synergy strongly suggests that DNA repair intermediates are targets of peptide wrwycr. The phosphorylation of histone H2AX is commonly assayed as an indicator of double-strand DNA breaks. Treatment of HeLa cells with wrwycr induced a peptide concentration-dependent phosphorylation of histone H2AX, which was also enhanced with etoposide and hydroxyurea treatments.;Thus, accumulation of DNA breaks appears to be a common mechanism of peptide toxicity in bacterial and mammalian cells. Pending further toxicity studies in animal models, peptide wrwycr may be a promising antibiotic with a novel mechanism of action. Its preferential toxicity in fast-replicating cells and synergy with classical chemotherapeutics suggest that wrwycr may have potential use as a chemotherapeutic, particularly in combination therapies.