Description
Our laboratory has previously isolated and characterized peptides that trap Holliday Junction (HJ) structures and prevent their resolution to products. The peptides were shown to induce DNA damage and chromosome segregation defects in Gram-positive and Gram-negative bacteria. Subsequently, we screened small molecule libraries and identified compounds with similar activities as the peptides. The small molecules were evaluated first for their ability to accumulate HJ in phage lambda site-specific recombination pathways in vitro, and second for their potential to inhibit bacterial growth. Small molecule 1609-10 (SM10) has greater antimicrobial activity than the most potent peptide (wrwycr), as judged by the minimal inhibitory concentration. In investigating the mechanism of action of wrwycr and the SM10, we found that both induce several envelope stress responses, based on the activity of cpxP::lacZ, rpoH::lacZ and spy::lacZ reporter fusions. E. coli mutants deficient in the 2-component sensor kinase CpxA or in the envelope stress sigma factor RpoE are hypersensitive to SM10. Both the peptide wrwycr and the SM10 led to depolarization of the membrane, although on different time scales. While testing a potential link between membrane damage and DNA damage, we observed that overexpression of OmpC and OmpF (known inducers of envelope stress), but not overexpression of the cytoplasmic proteins PurE or FolD, causes DNA damage and induces the SOS response. In addition to envelope perturbations, SM10 and wrwycr also induce DNA damage, including double strand breaks, in E. coli. Aerobically, wrwycr, SM10, and OmpC/OmpF overexpression also increase formation of reactive oxygen species (ROS), which may be the primary cause of DNA damage in all cases. Using EPR, we found that peptide wrwycr significantly increased intracellular free iron levels, which may contribute to the formation of hydroxyl radicals. Surprisingly, only about 33% of the DNA damage is dependent on aerobic growth. Interestingly the lethality of SM10 and the over-expression of OmpC were similar under anaerobic and aerobic conditions. The antimicrobial activity of peptide wrwycr and the SM10 is partly due to their effects on the cell envelope, thus making them likely to have broad-spectrum antibiotic capabilities.
