Transcriptomic Signatures of Trichomonas vaginalis Isolates That Exhibit Low, Intermediate, and High In Vitro Resistance to Metronidazole

As part of efforts to identify genes associated with Trichomonas vaginalis resistance to 5-nitroimidazole drugs, thirty cryopreserved T. vaginalis isolates were revived and grown using Diamond’s TYM medium. Minimum lethal concentrations (MLCs) for metronidazole (MTZ), tinidazole (TDZ), and secnidazole (SEC) were determined using a drug susceptibility assay. Transcriptome profiling was performed for 15 MTZ-sensitive (MTZ-S, MLC < 50 µg/mL) and 15 MTZ-resistant (MTZ-R, MLC ≥ 50 µg/mL) isolates using next-generation RNA sequencing. Bioinformatics analyses identified differentially expressed genes (DEGs). Among the MTZ-R isolates, six exhibited low MLCs of 50 µg/mL, five had intermediate MLCs between 100 and 200 µg/mL, and four had high MLCs ≥ 400 µg/mL. Differential gene expression analysis identified 28, 140, and 73 significantly altered genes in low-, intermediate-, and high-level MTZ resistance groups, respectively, with predominantly upregulated expression patterns. The SEC-resistant (SEC-R) isolates exhibited 136 differentially expressed genes, whereas the TDZ-resistant (TDZ-R) isolates showed minimal transcriptional changes. Focused analyses of iron transport pathways revealed reduced expression of ZIP-family iron import genes, particularly TvZIP4 (TVAG_273550), the strongest predictor of resistance in elastic-net modeling (AUC = 0.795). Resistant isolates also demonstrated coordinated upregulation of iron–sulfur cluster assembly and hydrogenosomal protein-import pathways. Weighted gene co-expression network analysis (WGCNA) identified multiple resistance-associated transcriptional modules correlated with MTZ and SEC MLCs. A comparative transcriptomic–proteomic analysis revealed concordant upregulation of iron–sulfur cluster machinery but discordant regulation of hydrogenosomal cargo proteins, likely supporting a post-transcriptional restriction model. These findings provide a broader mechanistic framework for understanding 5-nitroimidazole resistance in T. vaginalis and identifying candidate biomarkers and pathways that may support future therapeutic and diagnostic development.