Understanding The Mechanism Of Action Of Anti-Microbial Peptides Using A Novel Dna-Based Strategy And Enantiomers.

Antibiotic resistance is a key problem to healthcare worldwide and the rate of development of new classes of antibiotics continues to lag. Anti-Microbial Peptides (AMPs) are a promising class of therapeutic agents, since they are effective against broad spectrum of bacteria and fungi. Moreover, AMPs not only act on bacterial membrane, but they also have various intracellular targets, which makes it harder for bacteria to develop resistance against an AMP. It is important to understand the detailed mechanism of action of AMPs in order to develop them into novel drugs. The classical method of studying their mechanisms of action by treatment of bacteria with chemically synthesized AMPs has a major pitfall. This method cannot differentiate between the distinctive effects of AMPs on bacterial membranes and on intracellular targets in the cytoplasm. In order to study the effect of AMP at a subcellular location, we have developed an innovative, DNAbased regulatable system in E. coli. In this system, NCR247 (an antimicrobial peptide produced by plants) is expressed from within the bacteria and is localized to a subcellular location. Our results indicate that NCR247 is bactericidal when expressed as a fusion protein to an outer membrane protein and is presented outside the cell. However, NCR247 is bacteriostatic when it is expressed within the cytoplasm. Our data shows that NCR247 exhibits distinctive physiological effects when expressed in different subcellular, bacterial compartments. We are currently extending this strategy to study the mechanism of action of other AMPs. Since NCR247 is capable of exhibiting pleiotropic effects on bacteria, we wish to understand which of these effects are due to peptide’s interaction with bacterial membrane and which are due to stereo-specific peptide-protein interactions. Using Denantiomer of NCR247 we have shown that peptides effect on membrane permeability and lethality are chirally independent, but other physiological effects are due to peptide-protein interaction. We are currently investigating the protein partners of NCR peptide in bacteria.