Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired reduced folate transporter

Abstract

Described antimicrobial resistance mechanisms enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. Here we describe a mechanism of sulfamethoxazole resistance that requires a host metabolite for activity. Using a combination of in vitro evolution and metabolic rescue experiments, we identify an energy-coupling factor (ECF) transporter S component gene (thfT) that enables Group A Streptococcus to acquire extracellular reduced folate compounds. ThfT likely expands the substrate specificity of an endogenous ECF transporter to acquire reduced folate compounds directly from the host, thereby bypassing the inhibition of folate biosynthesis by sulfamethoxazole. As such, ThfT is a functional equivalent of eukaryotic folate uptake pathways that confers very high levels of resistance to sulfamethoxazole, yet remains undetectable when Group A Streptococcus is grown in the absence of reduced folates. Our study highlights the need to understand how antibiotic susceptibility of pathogens might function during infections to identify additional mechanisms of resistance and reduce ineffective antibiotic use and treatment failures, which in turn further contribute to the spread of antimicrobial resistance genes amongst bacterial pathogens.

Fig. 1. Overview of folate metabolism in bacteria and mammals and targets of SMX and TMP.

Orange arrows are specific to bacteria. Blue arrows are pathways specific to mammals. Black arrows are common to both bacteria and mammals. Mammalian dihydrofolate reductase (mDHFR) is able to reduce folic acid to DHF, while bacterial dihydrofolate reductase enzymes are unable to perform this reaction. Mammalian exogenous folate uptake pathways can accommodate multiple reduced folate substrates (RFC and hFR) or folic acid in addition to reduced folates (PCFT). RFC is the major transporter in systemic tissues while hFR and PCFT are tissue-specific. RFC reduced folate carrier, hFR human folate receptor, PCFT proton-coupled folate transporter, GTP guanosine triphosphate, DHP dihydropteroate, DHF dihydrofolate, THF tetrahydrofolate, PABA para-aminobenzoic acid, dUMP deoxyuridine monophosphate, dTMP deoxythymidine monophosphate, TMP trimethoprim, SMX sulfamethoxazole, DHFR dihydrofolate reductase (mammalian). Created with BioRender.com.

Fig. 2. A DfrG-positive GAS strain can evolve SXT resistance due to upregulation of an ECF transporter S component gene (thfT).

a Schematic of in vitro evolution procedure using SXT gradient plates. An overnight culture of TB08 on HBA was used to inoculate a SXT gradient plate (0–50 μg/ml) and incubated for 24 h at 37 °C. Growth was then harvested from the the highest antibiotic concentration (i.e. where growth started to be inhibited) and used to seed successive rounds of evolution. Created with BioRender.com. b MICs to SXT on MHF agar from bioMerieux (MHF-Bm; blue circles) and Oxoid (MHF-Ox; red squares) following in vitro evolution of GAS strain TB08. Results from three independent experiments are shown. Values are expressed as a ratio of the initial MIC as determined by Etest following each round of evolution. c Genomic context of thfT (purple arrow) as determined by tblastX pairwise blast which highlights evidence of horizontal gene transfer of thfT (purple) and shared origin with that in Streptococcus dysgalactiae subsp equisimilis. Percent identity is by shading as indicated to the right. d Relative expression of thfT in TB08 and TB08-2-14 measured by RT-qPCR. Values are normalised to average expression of TB08 ME samples (dashed line). ME, mid-exponential phase (OD₆₀₀ = 0.4–0.6); St, stationary phase (OD₆₀₀ = 1.2–1.4). Data from three biological replicates are presented as mean values ± SD. Differences assessed using a one-tailed, unpaired Student’s t-test. Source data for panels b and d are provided as a Source Data file.

Fig. 3. ThfT confers SXT resistance in the presence of exogenous THF.

a Dose-response curves for the impact of exogenous THF on SXT resistance of M6^JRS4, TB08 and TB08-2-14. MICs are expressed as a percentage the maximum resolution of the Etest assay (32 μg/ml). Data from three biological replicates on MHF-Bm agar are presented as mean values ± SD. EC₅₀ values were calculated using Graphpad Prism software. b Representative Etest assays on MHF-Bm agar for data presented in a. c Schematic showing predicted effect of increased ThfT expression in TB08-2-14 on competition with other ECF S components for ECF transport modules and the effect on SXT MICs. Under conditions of low ThfT expression and low exogenous THF, competition between THF-loaded ThfT and S components loaded with other compounds limits THF import, resulting in low SXT MICs (top left). This competition is overcome by increased ThfT expression (top right) or increased extracellular THF, which results in high SXT MICs. Source data for panel a is provided as a Source Data file.

Fig. 4. ThfT is an SMX resistance protein that allows GAS strains to bypass the folate synthesis pathway in the presence of exogenous THF.

a Heterologous expression of T71 and A71 variants of thfT in M6^JRS4. The constructs with a constitutive P23 promoter are indicated with a blue arrow and thfT is indicated with a red arrow. SMX, TMP and SXT MICs were determined by Etest. Exogenous THF was supplied at a concentration of 50 ng/cm². Results are median values of three independent experiments determined with Etest strips on MHF-Bm agar. Statistical analysis of groups is in Supplementary Table 4. b Model of ThfT mediated SMX-resistance. ThfT is able to acquire extracellular THF which then feeds directly into the folate cycle (blue box), which bypasses the inhibitory effect of SMX on folate synthesis (green box). As THF is recycled back to DHF during the folate cycle, only small amounts of exogenous THF are required to support growth in the presence of SMX. GTP, guanosine triphosphate; DHP, dihydropteroate; DHF, dihydrofolate; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate. Created with BioRender.com. c Single gene complementation of thfT in the same genetic locus as in TB08 is sufficient for high-level SMX resistance in GAS strain NS5347. SMX, TMP and SXT MICs were determined by Etest. Exogenous THF was supplied at a concentration of 50 ng/cm². Results are median values of three independent experiments determined with Etest strips on MHF-Bm agar. Statistical analysis of groups is in Supplementary Table 5. d Ability of THF to confer ThfT-mediated SMX resistance as measured by broth microdilution. Data from three biological replicates are presented as mean values ± SEM. Differences assessed using a two-tailed, unpaired Student’s t-test. Source data for panels a, c and d are provided as a Source Data file.

Fig. 5. ThfT enables SMX-resistance by uptake of multiple one-carbon folate cycle intermediates from host cells.

a Ability of NS5347 encoding thfT variants to utilise multiple one carbon THF intermediate compounds. SMX MICs (μg/ml) were determined by Etest in the presence of 50 ng/cm² of each compound on MHF-Bm agar. Results are median values from three independent experiments. Statistical analysis of groups is in Supplementary Table 7. 5,10-me⁺THF, 5,10-methenyl-THF. b Model of ThfT mediated SMX resistance in the presence of one-carbon folate cycle intermediate compounds. Compounds utilised by ThfT to bypass the inhibitory action of SMX are highlighted in red and compounds which could not be utilised by ThfT are highlighted in green. 5-formimino-THF (grey) was not available for testing. Solid black arrows indicate pathways that are likely functional in GAS based on the presence of predicted enzymes in the core GAS genome, while pathways indicated with dashed grey arrows indicate the absence of corresponding enzyme (Supplementary Fig. 8). Created with BioRender.com. GTP guanosine triphosphate, DHP dihydropteroate, DHF dihydrofolate, dUMP deoxyuridine monophosphate, dTMP deoxythymidine monophosphate, 5,10-me-THF 5,10-methylene-THF. c Cartoon schematic of experiment to test uptake of cellular folates by ThfT. SMX MICs were by broth microdilution across a 2-fold serial dilution of SMX (2500 to 5 μg/ml, left to right) in the presence of a 2-fold serial dilution of epithelial cell lysates (1 × 10⁵ to 1.6 × 10³ cells/well, top to bottom). After overnight incubation, MIC values were determined by examining GAS growth (orange wells) for each epithelial cell lysate concentration. d Ability of epithelial cell lysates to confer ThfT-mediated SMX resistance using procedure in c. Data from three biological replicates are presented as mean values ± SEM. Differences assessed using a one-tailed, paired Student’s t-test. Source data for panels a and d are provided as a Source Data file.