UC San Diego

Efflux pumps are a detoxification mechanism widely distributed in biological systems. Their ability to extrude a broad range of pharmaceutical agents intended to treat infectious diseases makes them a formidable obstacle to effective treatment. Overuse, misuse and abuse of antibiotics throughout much of the world have led to the emergence of bacterial strains against which current therapies are becoming increasingly ineffective. The MefA pump is encoded by the mefA gene and confers resistance to 14- and 15-membered macrolide antibiotics (e.g. erythromycin, azithromycin) in Streptococcus pyogenes (Group A Streptococcus) and Streptococcus pneumoniae. A compound that blocks, or inhibits the action of this efflux pump could restore sensitivity of these bacteria to this important family of antibiotics. In this study, attention is turned toward secondary metabolites of marine origin as sources of novel bioactive compounds. An assay was developed to screen both crude extracts and pure compound samples for their ability to restore sensitivity of a mefA(+) strain of GAS to erythromycin. Parallel screens were performed using a serotype matched mefA(-) strain to control for independent antimicrobial activity. Several extracts were identified that reversed MefA- mediated erythromycin resistance, and these were fractionated using HPLC and subjected to various analytical techniques including mass spectrometry, UV spectroscopy and NMR spectroscopy to isolate the bioactive compounds and determine their chemical structures. Identification of a specific MefA inhibitor can serve as a lead point for rational design of novel antimicrobial agents to combat bacterial resistance mediated by drug efflux pumps. Additional molecular studies involve targeted mutagenesis and heterologous expression of mefA to determine its contribution to GAS host cell interactions, invasiveness and intracellular survival