Identification and Characterization of a Novel Major Facilitator Superfamily (MFS) Efflux Pump SA09310 Mediating Tetracycline Resistance inStaphylococcus aureus

Abstract

AbstractDrug efflux systems have recently been recognized as an important mechanism of multidrug resistance in bacteria. Here, we described the identification and characterization of a novel chromosomally encoded multidrug efflux pump (SA09310) inStaphylococcus aureus. SA09310 is a 43-kDa protein with 12 transmembrane helices. The conserved amino acid sequence motifs of the major facilitator superfamily (MFS) were identified in the protein SA09310, which indicated SA09310 belonged to MFS transporters. Expression of thesa09310gene was induced by different types of antibiotics, including aminoglycoside, tetracycline, macrolides, and chloramphenicol. Thesa09310gene knockout mutant (Δsa09310) was constructed, and its susceptibility to 30 different antibiotics was evaluated. Mutant△sa09310exhibited increased sensitivity to tetracycline and doxycycline, with 64-fold and 8-fold decreased MICs, respectively. The mechanism of SA09310 mediating tetracycline resistance was demonstrated by its ability to extrude intracellular tetracycline from within the cells into the environment. The efflux activity of SA09310 was further confirmed by EtBr accumulation and efflux assays. In addition, the efflux activity of SA09310 was observed to be blocked by the known efflux pump inhibitor carbonyl cyanide-chlorophenylhydrazone (CCCP), which provided direct evidence that suggested the H+-dependent activity of SA09310 efflux pump. The conservation of SA09310 homologs in Staphylococcus indicated the universal function of these SA09310-like protein clusters. In conclusion, the function-unknown protein SA09310 has been identified and characterized as a tetracycline efflux pump, thereby mediating tetracycline resistance inS. aureus.