Characterization of Mechanism of Action of DASamP2 against Pseudomonas aeruginosa by Isolation of Mutants

Human bacterial pathogens are evolving resistance to antibiotics currently used for therapy. There is a need to develop new strategies to combat resistant microbes such as Staphylococcus aureus and Pseudomonas aeruginosa which exhibit resistance to many drugs available today. Antimicrobial peptides (AMP) are a diverse group of molecules that possess antimicrobial properties and are being studied for use as antibiotics. They are naturally produced by organisms as a defense mechanism against pathogens. The mechanism of action of these antimicrobial agents is not well known. Recently a new antimicrobial peptide(DASamP2) designed by Dr. Wang(University of Nebraska Medical Center) was observed to be effective against both S. aureus and P. aeruginosa and holds great promise as a potential antibiotic agent. The goal of my project is to determine the mechanism of action and potential targets of DASamP2 by isolating mutants of P.aeruginosa with altered resistance to DASamP2. We have performed transposon mutagenesis of a sensitive wild type P. aeruginosa strain and screened for increased resistance to DASamP2. Out of the 3600 mutants screened, we isolated 10 mutants that showed increased resistance to the DASamp2 AMP. We are performing Inverse Polymerase Chain Reaction (iPCR) to identify the gene mutated by transposon insertion. Location of the transposon and the gene disrupted by its insertion will then be determined by sequencing. Characterizing the genes will help in elucidating the target and mechanism of action of DASamp2