Multidrug resistance in Staphylococcus aureus due to overexpression of a novel multidrug and toxin extrusion (MATE) transport protein

Abstract

Efflux is an important mechanism of multidrug resistance (MDR) in bacteria. The multidrug and toxin extrusion (MATE) family is the most recently described group of MDR efflux proteins, none of which have previously been identified in Staphylococcus aureus. Two independently derived S. aureus mutants having efflux-related MDR phenotypes were studied using microarray technology and a marked overexpression of an open reading frame (ORF; mepA) encoding a protein homologous with MATE family proteins was observed in both. There was concomitant overexpression of ORFs in close proximity to mepA (approximately 100 bp) encoding a MarR-type regulator (mepR, upstream of mepA) and a protein of unknown function (mepB, downstream). Experiments in which mepA was overexpressed or disrupted revealed that the encoded protein has a broad substrate profile that includes several monovalent and divalent biocides and the fluoroquinolone antimicrobial agents norfloxacin and ciprofloxacin. The function of MepB is obscure, it does not contribute to the MDR phenotype conferred by MepA. MepR overexpression reversed the MDR phenotypes of both mutants by repressing mepA transcription. All three ORFs are preferentially transcribed as a single mepRAB unit, suggesting that the three genes form an operon.

FIG. 1.

Selected nucleotide sequences of the mepRAB locus. (A) Terminal portion of mepB and flanking DNA. The mepB stop codon is indicated, followed by inverted repeats (A and A', B and B', C and C') that may function as a mepRAB transcription terminator (arrows). (B) Promoter regions of mepR and mepB. Transcription start sites (+1), ribosome binding sites (RBS), and start codons (SC) are shown in boldface type and highlighted with a grey background. Possible −35 and −10 motifs are shown in boldface type and italicized. Two transcription start sites were identified for mepB (+1₁ and + 1₂).

FIG. 2.

MepA-mediated efflux of EtBr. Bars: 1, SA-K1758-P; 2, SA-K1758-P with reserpine (20 μg/ml); 3, SA-K1758-A; 4, SA-K1758-A with reserpine. Results are expressed as percent reduction in fluorescence (means ± standard deviations), which correlates with EtBr efflux, over 5 min.

FIG. 3.

(A) Effect of overexpression of mepR on mepA expression. Lane 1, SA-K2068-P; lane 2, SA-K2068-R; lane 3, SA-K1748-P; lane 4, SA-K1748-R. The negative effect of MepR on mepA expression is clearly evident. (B) Expression of mepRAB. Lane 1, S. aureus NCTC 8325-4; lane 2, SA-K2068; lane 3, SA-K1712; lane 4, SA-K1748.