Role of the NaHCO3 Transporter MpsABC in the NaHCO3-β-Lactam-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing concern due to their intrinsic resistance to most standard-of-care β-lactam antibiotics. Recent studies of clinical isolates have documented a novel phenotype, termed NaHCO₃ responsiveness, in which a substantial proportion of MRSA strains exhibit enhanced susceptibility to β-lactams such as cefazolin and oxacillin in the presence of NaHCO₃. A bicarbonate transporter, MpsAB (membrane potential-generating system), was recently found in S. aureus, where it plays a role in concentrating NaHCO₃ for anaplerotic pathways. Here, we investigated the role of MpsAB in mediating the NaHCO₃ responsiveness phenotype. Radiolabeled NaH¹⁴CO₃ uptake profiling revealed significantly higher accumulation in NaHCO₃-responsive vs nonresponsive MRSA strains when grown in ambient air. In contrast, under 5% CO₂ conditions, NaHCO₃-responsive (but not nonresponsive) strains exhibited repressed uptake. Oxacillin MICs were measured in four prototype strains and their mpsABC deletion mutants in the presence of NaHCO₃ supplementation under 5% CO₂ conditions. NaHCO₃-mediated reductions in oxacillin MICs were observed in the responsive parental strains but not in mpsABC deletion mutants. No significant impact on oxacillin MICs was observed in the nonresponsive strains under the same conditions. Transcriptional and translational studies were carried out using both quantitative reverse transcription-PCR (qRT-PCR) and mpsA-green fluorescent protein (GFP) fusion constructs; these investigations showed that mpsA expression and translation were significantly upregulated during mid-exponential-phase growth in oxacillin-NaHCO₃-supplemented medium in responsive versus nonresponsive strains. Taken together, these data show that the NaHCO₃ transporter MpsABC is a key contributor to the NaHCO₃-β-lactam responsiveness phenotype in MRSA. IMPORTANCE MRSA infections are increasingly difficult to treat, due in part to their resistance to most β-lactam antibiotics. A novel and relatively common phenotype, termed NaHCO₃ responsiveness, has been identified in which MRSA strains show increased susceptibility in vitro and in vivo to β-lactams in the presence of NaHCO₃. A recently described S. aureus NaHCO₃ transporter, MpsAB, is involved in intracellular NaHCO₃ concentration for anaplerotic pathways. We investigated the role of MpsAB in mediating the NaHCO₃ responsiveness phenotype in four prototype MRSA strains (two responsive and two nonresponsive). We demonstrated that MpsABC is an important contributor to the NaHCO₃-β-lactam responsiveness phenotype. Our study adds to the growing body of well-defined characteristics of this novel phenotype, which could potentially translate to alternative targets for MRSA treatment using β-lactams.

Keywords: CO2; MRSA; MpsABC; bicarbonate transporter; membrane potential-generating system ABC; methicillin-resistant Staphylococcus aureus; sodium bicarbonate; β-lactams.

FIG 1

(A) Uptake of NaH¹⁴CO₃ by bicarbonate-responsive strains (S. aureus JE2 and MRSA 11/11; red lines and symbols) and nonresponsive strains (S. aureus BMC1001 and COL; blue lines and symbols). Bacterial cultures grown in ambient air until mid-exponential phase were washed and adjusted to the same OD. Fluorocitrate was added to the cells and incubated for 30 min before the addition of NaH¹⁴CO₃ (50 μCi). Aliquots of cell suspensions were collected at the indicated time points, and the H¹⁴CO₃ uptake was determined by the ¹⁴C accumulation in cells, as measured by liquid scintillation counting. Each value is the mean and SD from three independent biological replicates. (B) The P values represent the significant differences in the AUC for the uptake of each strain, analyzed using one-way ANOVA followed by Tukey’s multiple-comparison test. The H¹⁴CO₃ uptake in the responsive strains JE2 and MRSA 11/11 was significantly higher than that in the nonresponsive strains BMC1001 and COL.

FIG 2

Uptake of NaH¹⁴CO₃ by strain sets grown in ambient air (A; solid lines and symbols) and 5% CO₂ conditions (CO₂; dashed lines and symbols). The bicarbonate-responsive strains (A) JE2 and (B) MRSA 11/11 showed significantly higher H¹⁴CO₃ uptake when grown in ambient air than in CO₂. **, P = 0.0092; ***, P = 0.0003. The H¹⁴CO₃ uptake for cells grown in ambient air and 5% CO₂ was not significantly different in the nonresponsive strains (C) BMC1001 (ns, not significant [P = 0.7847]) and (D) COL (P = 0.6652). Each point represents the mean and SD from three independent biological replicates. Statistical significance between the uptake in ambient air and CO₂ was calculated using Student’s t test from the AUC values for each strain set.

FIG 3

Expression of mpsA in NaHCO₃-responsive (JE2 and MRSA 11/11) and NaHCO₃-nonresponsive (BMC1001 and COL) strains. Gene expression data were obtained by qRT-PCR of RNA from (A) mid-exponential-phase (3 h) and (B) early-stationary-phase (6 h) strains grown in CA-MHB–Tris with (+) or without (−) NaHCO₃ and 1/2 MIC of oxacillin. NaCl (2%) was included in growth media in which oxacillin was also included. For each strain, mpsA expression was normalized to the value obtained in CA-MHB–Tris (−), with this value set to 1.0. Data are means and SD from three independent biological replicates. Statistical comparisons were determined by Student’s t test. ns, not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

FIG 4

Translational efficiency of mpsA promoter sequences for all the prototype strains in the presence of NaHCO₃ and oxacillin. Translational efficiency was assessed by flow cytometry using strains harboring the promoter-GFP fusions (Fig. S4) for JE2, MRSA 11/11, BMC1001, and COL promoter regions. Transcriptional (txn) constructs harbor the promoter region of mpsA with the sarA RBS fused to gfp (left), while translational (tln) constructs harbor the promoter region of mpsA with the mpsA RBS fused to gfp (right). Cells were grown in CA-MHB–Tris, 1/2 MIC of oxacillin (16 μg/mL for JE2 and MRSA 11/11, 64 μg/mL for BMC1001, and 128 μg/mL for COL), and 2% NaCl for 3 h before being assessed for MFI via flow cytometry. NaHCO₃ (44 mM) was added to all the media (+). Statistical significance of the MFI between the responsive and nonresponsive strains was determined by Student’s t test. **, P = 0.0044; ***, P = 0.0003. Data are the results of four independent biological replicates for each strain/condition.

FIG 5

Differences in MFI of strains JE2, MRSA 11/11, BMC1001, and COL grown with and without NaHCO₃ in the presence of 1/2 MIC of oxacillin for (A) transcriptional (txn) and (B) translational (tln) constructs. The constructs and conditions used are indicated in the legend to Fig. 4. Each bar shows the difference (increase/decrease) between the MFIs of the strain grown with and without NaHCO₃ (baseline levels), and the statistical significance between them was calculated using one-way ANOVA followed by Tukey’s multiple-comparison test. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are the results from four independent biological replicates for each strain/condition.