Mitomycin C potentiates metronidazole activity in resistant Trichomonas vaginalis through suppression of thioredoxin...

Metronidazole (MTZ) remains the first-line therapy for trichomoniasis; however, increasing resistance in Trichomonas vaginalis threatens treatment efficacy. Because iron availability enhances MTZ sensitivity, we investigated whether MTZ-induced parasite death involves ferroptosis-related mechanisms and explored alternative pathways contributing to drug resistance. Exposure of metronidazole-sensitive and -resistant isolates to ferroptosis inducers reduced parasite viability, yet neither ferroptosis inhibition nor iron chelation restored survival, and lipid peroxidation was not detected. These findings indicate that MTZ-associated cytotoxicity does not proceed via canonical ferroptotic pathways in T. vaginalis. Instead, resistant parasites exhibited elevated expression of thioredoxin-associated transcripts, consistent with enhanced redox defense capacity. Functional suppression of thioredoxin reductase (TrxR) activity by mitomycin C (MMC) significantly enhanced MTZ-mediated killing and produced a synergistic antiparasitic effect in resistant isolates. Although selective TrxR inhibition alone was insufficient to fully reproduce this phenotype, these findings implicate thioredoxin-dependent redox regulation as a contributor to MTZ susceptibility. Combination treatment was accompanied by selective metabolic reprogramming, including upregulation of pentose phosphate pathway genes linked to NADPH generation. Collectively, our results demonstrate that MTZ induces a non-ferroptotic form of cell death and identify thioredoxin-dependent redox buffering as a resistance-associated vulnerability. Targeting redox homeostasis through rational combination therapy may represent a mechanistically guided strategy to overcome metronidazole resistance in trichomoniasis.