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.