Using Inverse PCR and Sequencing to Identify Genes in Pseudomonas aeruginosa that Contribute to Susceptibility to the Antimicrobial Peptide DASamp2

As modern medicine moves further into the 21st century, our available pharmacopoeia of antimicrobial agents is currently threatened by the rate of increased multi-drug resistance in pathogenic microorganisms. Increased pathogen resistance to conventional antibiotics is making treating infections more difficult, and increasing mortality rates in infected patients. Due to this dilemma, new alternatives to traditional antibiotics are currently being sought. Antimicrobial peptides are one potential source of new antibiotics. DASamp2 is a promising new antimicrobial peptide, developed by Dr. Wang at UNMC, that is experimentally shown to be effective against both Gram positive and Gram negative bacteria including Pseudomonas aeruginosa. In an effort to determine the target of DASamp2, and to determine the ease at which the bacterium P. aeruginosa can develop resistance to this compound, the Rowen lab previously isolated mutant P. aeruginosa strains with showed increased resistance to DASamp2. I determined the mutation in one such mutant (RMB1) was located in the gene mexT by using inverse PCR and sequencing. The gene, mexT, is believed to encode a transcriptional regulator involved in the the production of efflux pumps (pumps that move drugs and other chemicals out of the cells). However, the regulation of the production of efflux pumps in P. aeruginosa is complex and poorly understood. Our observation that the mutation of mexT in the RMB1 caused a large increase in resistance to DASamp2 (~8 fold) suggests that these efflux pumps also play a significant role in determining the susceptibility of bacterial cells to AMPs such as DASamp2