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 enzyme⁴³ (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.