Identification and Characterization of a Novel Major Facilitator Superfamily Efflux Pump, SA09310, Mediating Tetracycline Resistance in Staphylococcus aureus

Abstract

Drug 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) in Staphylococcus 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 that SA09310 belonged to the MFS transporters. Expression of the sa09310 gene was induced by different types of antibiotics, including aminoglycoside, tetracycline, macrolides, and chloramphenicol. An sa09310 gene knockout mutant (Δsa09310) was constructed, and its susceptibility to 30 different antibiotics was evaluated. The Δsa09310 mutant exhibited increased sensitivity to tetracycline and doxycycline, with 64-fold- and 8-fold-decreased MICs, respectively. The mechanism of SA09310 mediation of 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 ethidium bromide (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 the 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 mediating tetracycline resistance in S. aureus.

Keywords: Staphylococcus aureus; antibiotic resistance.

FIG 1

Transmembrane topology and phylogenetic analysis of the SA09310 protein. (A) The transmembrane topology of the SA09310 protein was created using the TOPO2 online tool based on the predicted transmembrane structure by TMHMM 2.0. The amino acids of SA09310 highlighted in orange are the conserved motif (motif A) of MFS transporters between the TMS2 and TMS3 helices. The amino acids of SA09310 highlighted in purple are another conserved motif of MFS transporters located in the fifth α-helix. (B) The phylogenetic tree of SA09310 was constructed based on the sequence alignment between SA09310 and proteins from 14 groups of classified MFS transporters using ClustalW, as implemented in the CLC Main Workbench. The resulting tree was calculated using the neighbor-joining method and Jukes-Cantor protein distance model. The abbreviations for the classified groups are defined in Materials and Methods, and SA09310 is highlighted with a star.

FIG 2

Expression of the sa09310 gene with the stimulation of different types of antibiotics. (A) Bacterial cells from the mid-exponential phase were treated with 100 μg/mL of different antibiotics. After induction for 1 h, the mRNA levels of the sa09310 gene were determined by RT-qPCR with the 16S rRNA gene as the internal control. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.05 relative to the level without antibiotic induction. NS, not significant. (B) The expression of the sa09310 gene was induced by tetracycline in a concentration-dependent manner. S. aureus cells were induced with different concentrations of tetracycline (0, 1, 10, 25, or 100 μg/mL), and the mRNA levels of the sa09310 gene were checked by RT-qPCR.

FIG 3

Tetracycline (Tet) and doxycycline (Doxy) MIC assay. MICs of Tet and Doxy were tested with S. aureus WT, sa09310 gene knockout mutant (Δsa09310), and Δsa09310 repaired (Δsa09310_com) strains using the Etest strip on TSA plates according to CLSI guidelines (39). The MICs of Tet and Doxy for each strain were read from the plate and presented as histograms on the right side.

FIG 4

Intracellular tetracycline, EtBr accumulation, and efflux assays. (A) The S. aureus WT and the Δsa09310 mutant were treated with a final concentration of 5 μg/mL of tetracycline. At the indicated time points (0, 0.5, 1, 1.5, 2, 3, and 4 h) after treatment, the bacterial cells were harvested and resuspended in lysis buffer by adjusting the OD₆₀₀ value to 1.0 (5 × 10⁸ CFU/mL). The tetracycline concentration of the cell lysate was quantified by ELISA. Concentrations from suspensions of the WT and Δsa09310 groups during the indicated period were compared and analyzed. **, P ≤ 0.05 by paired t test. (B) EtBr accumulation assay. S. aureus WT, Δsa09310, and sa09310 overexpression strains were treated with 4 μg/mL of EtBr. The fluorescence of each strain was measured in a 96-well plate by a Bioreader and normalized to the OD₆₀₀. The dotted line indicates the time point at which EtBr was added. Data are presented as the means of three independent assays. ****, P ≤ 0.0001 by paired t test. (C) EtBr accumulation efflux assay. S. aureus cells were treated with EtBr as described above for 40 min. Then, the extracellular EtBr was removed by centrifugation and resuspension of the cells in fresh PBS, and the fluorescence of each strain was measured. After 20 min, the efflux pump inhibitor carbonyl cyanide chlorophenylhydrazone (CCCP) at a final concentration of 100 μM was added to each well, and the fluorescence was read for an additional 40 min. The dotted line indicates the time point when CCCP was added. All data are presented as the mean of three independent assays. **, P ≤ 0.05 by paired t test.

FIG 5

(A) Survival curves of G. mellonella larvae infected with the S. aureus USA300 WT at doses ranging from 1 × 10⁴ to 1 × 10⁷ per larva. Larvae were incubated at 37°C, and their viability was assessed over 6 days. (B) Clearance of the S. aureus WT and Δsa09310 mutant by tetracycline in the larval infection model. Larvae were infected with an optimal dose of 1 × 10⁵ CFU/larva of the WT or Δsa09310 mutant. After 2 h of infection, 10 μg of tetracycline was injected into each larva (tetracycline-treated group). PBS was administered as the control for antibiotic treatment. Five live larvae were randomly selected from each group and were homogenized. The bacterial burden of each selected larva at 12 and 24 h after infection was determined by serial dilution and plating assays. ***, P ≤ 0.001. ns, not significant.

FIG 6

Conservation of SA09310 efflux pump in staphylococci. (A) Conserved genomic context of sa09310 and its homologous genes in different Staphylococcus species. (B) Heat map of percentage identities between the SA09310 protein and its analogous proteins from other Staphylococcus species.