An Antimicrobial Peptide That Targets DNA Repair Intermediates In Vitro Inhibits Salmonella Growth within Murine Macrophages

Abstract

The hexapeptide WRWYCR was previously identified on the basis of its ability to inhibit bacteriophage lambda integrase-mediated recombination by trapping and preventing resolution of the Holliday junction intermediate. This peptide inhibits several unrelated DNA repair enzymes that bind to and process Holliday junctions and branched DNA substrates. WRWYCR and its D stereoisomer, wrwycr, are bactericidal against both Gram-positive and Gram-negative bacteria, causing the accumulation of DNA breaks, chromosome segregation defects, and the filamentation of cells. DNA repair is a novel target of antibiotics. In the present study, we examined the ability of the peptides to inhibit the growth of Salmonella in mammalian cells. J774A.1 macrophage-like cells and murine peritoneal macrophages were infected with Salmonella enterica serovar Typhimurium and grown in the presence or absence of peptide. We found that peptide wrwycr reduced the number of Salmonella cells recovered after 24 h growth in J774A.1 cells by 100 to 1,000 times, depending on the multiplicity of infection. The peptide also inhibited Salmonella growth in peritoneal macrophages, and although higher doses were required, these were not toxic to the host cells. The apparent lower level of potency of the peptide paralleled the lower level of replication of Salmonella and the lower level of permeation of the peptide in the peritoneal macrophages than in the J774.1 cells. Treatment with peptide wrwycr elicited the SOS response in a significant fraction of the intracellular bacteria, as would be expected if the mechanism of bacterial killing was the same in pure culture and in host cells. These results represent a proof of principle of the antimicrobial activities of compounds that target DNA repair.The hexapeptide WRWYCR was previously identified on the basis of its ability to inhibit bacteriophage lambda integrase (Int)-mediated site-specific recombination by accumulating the Holliday junction (HJ) intermediates during the reaction (4). Holliday junctions are also central intermediates in homologous recombination-dependent DNA repair, which often but not exclusively involves RecA-dependent strand invasion. The double-strand breaks that are generated by oxidative damage, irradiation, or interstrand cross-links or nicks that have been converted to double-strand breaks during replication require repair of the break by use of the homology on the sister chromosome (14,15,28). During the repair process, chromosome dimers may arise. Such dimeric chromosomes cannot be properly segregated to daughter cells, unless they are converted to monomers by the Int-related bacterial site-specific recombinases XerC and XerD, which also generate Holliday junction intermediates during recombination at dif sites (3). These processes are extremely important, if not essential; for example, up to 50% of recA mutant cells are nonviable, and the remainder are hypersensitive to any DNA damage (14). Although mutations in XerC and/or XerD are not lethal, they are detrimental to cell growth, ...

Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus

“…1. The ability to induce the SOS response has been shown previously for the hexapeptide WRWYCR, which exerts its broad bactericidal activity through stalling of bacterial replication forks (Su et al, 2010).…”

“…Other studies have shown that binding of AMPs to DNA can induce the SOS response (Gunderson & Segall, 2006;Su et al, 2010). To analyse the effect of FL9 on the SOS response, we examined the expression of a LexA-controlled gene, recA, using a S. aureus recA : : lacZ fusion and an agar diffusion assay (Nielsen et al, 2010).…”

Escherichia coli enterobactin synthesis and uptake mutants are hypersensitive to an antimicrobial peptide that limits the availability of iron in addition to blocking Holliday junction resolution

“…Because mammalian host defences such as gastric acid and the oxidative burst in macrophages induce DNA damage and thus DNA repair in bacterial invaders, use of wrwycr in antimicrobial therapy may increase the efficacy of these natural defence systems. Recently, wrwycr was shown to inhibit the growth of Salmonella enterica serovar Typhimurium inside macrophages (Su et al, 2010) and significantly enhance acid-induced killing of Shiga toxin-producing strains of Escherichia coli associated with haemolytic uraemia syndrome (Lino et al, 2011).…”

A novel antimicrobial peptide significantly enhances acid-induced killing of Shiga toxin-producing Escherichia coli O157 and non-O157 serotypes

“…The peptide WRWYCR, with the same sequence but composed of Lamino acids, interacts with HJs with a K d of 14 nM, inhibits several HJ resolution enzymes with an IC 50 of 5-64 nM and is also bactericidal against non-pathogenic E. coli, although it is less antimicrobial than wrwycr (Gunderson & Segall, 2006;Kepple et al, 2005Kepple et al, , 2008. The peptide wrwycr has also been shown to inhibit Salmonella enterica growing inside murine macrophages (Su et al, 2010) and to have antimicrobial activity against non-pathogenic E. coli (Gunderson & Segall, 2006). Importantly, wrwycr was found to have no toxic effect on J774A.1 macrophage-like cells and murine peritoneal macrophages in the study by Su et al (2010).…”