Our use of antibiotics is outpacing the rate at which bacterial pathogens become resistant to the currently available drugs. Meanwhile, the development of new antibiotics is being abandoned by large pharmaceutical companies to small biotech concerns and academic labs. While studying site-specific recombination, we have identified several hexapeptides that inhibit recombination reactions and bind to Holliday junctions, preventing their resolution. We have discovered that these hexapeptides have antimicrobial activity in both gram+ and gram- bacteria, although they are 4-8 fold more potent in gram+ bacteria (including methicillin resistant Staph aureus). Depending on the organism, MICs range from 8-64 mu/g/ml. The antimicrobial activity is associated with chromosome partitioning defects, filamentation, and induction of DNA breaks. Based on confocal microscopy with fluorescently labeled peptides, we have found that the peptides penetrate both gram+ and gram- bacteria. Despite the fact that they are also able to penetrate eukaryotic cells, we have not detected cytotoxic effects in HeLa cells at doses up to 250 mu/g/ml. Our short term goals are to understand the basis of the mechanism of action of these peptides in bacterial cells, to test their antimicrobial activity against intracellular pathogens, and their ability to cure bacterial infections in animal model systems. In addition, we will investigate their activities in different subcellular compartments of eukaryotic cells with an eye towards maximizing their specificity against bacteria and minimizing cytotoxic side effects. Since the peptides may not be optimal for pharmacological use, we are also identifying non-peptide lead compounds that mimic the activity of the peptides.
The specific aims of this study are to: 1) Identify and characterize nonpeptide small molecules that trap Holliday junctions. 2 & 3) Investigate the bacterial targets of the Holliday-junction trapping compounds using a combination of biochemical and genetic approaches. 4) Investigate effects on bacterial membranes. 5) Investigate the effects of Holliday-junction trapping compounds on eukaryotic cells. As part of this specific aim, we will also determine whether the peptides kill intracellular bacteria. 6) Investigate the effect of the peptides on Salmonella and Staph infections in mice in two well-studied infectious disease model systems.
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