Comparative transcriptomics of naturally susceptible and resistant Trypanosoma cruzi strains in response to Benznidazole

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, remains a major public health challenge due to limited treatment options, Benznidazole and Nifurtimox; which are associated with adverse effects and variable efficacy. The emergence of drug-resistant in T. cruzi strains, along with limited knowledge of the molecular mechanisms underlying resistance, hampers the development of more effective therapies. To explore these mechanisms, we performed a comparative transcriptomic analysis of two T. cruzi TcI strains: MG (naturally susceptible) and DA (naturally resistant) to Benznidazole. Parasites were cultured in LIT medium, and IC50 values were determined using the MTT assay. RNA was extracted and sequenced (RNA-seq), with reads aligned to a reference genome. Differential gene expressions were analyzed with DESeq2, functional enrichment through Gene Ontology (GO), and metabolic pathways were mapped via KAAS. The IC50 for Benznidazole in DA (28.92 μg/mL; 111.13 μM) was substantially higher than in MG (0.88 μg/mL; 3.39 μM), confirming differential susceptibility. DA showed 408 upregulated and 1515 downregulated genes, while MG had 153 upregulated and 866 downregulated (Log₂FoldChange ≥ 2 or ≤ -2). GO analysis indicated divergent biological processes between strains: DA exhibited enrichment in electron transport and detoxification, while MG was enriched in DNA repair and energy metabolism. Metabolic mapping revealed significant differences in the pentose phosphate pathway, glycolysis/gluconeogenesis, and the tricarboxylic acid (TCA) cycle. Key genes potentially involved in resistance like prostaglandin F2α synthase, trypanothione synthase, thioredoxin, and prostaglandin F synthase were identified as candidate therapeutic targets. These findings suggest that Benznidazole resistance in T. cruzi involves multifactorial, strain-specific responses at the transcriptomic and metabolic levels. By analyzing naturally resistant and susceptible TcI strains of T. cruzi under identical experimental conditions, this study reveals strain-specific transcriptomic adaptations that have not been previously characterized in naturally resistant and susceptible populations. These findings expand our current understanding of intrinsic Benznidazole resistance in T. cruzi, moving beyond purely experimental models. Specifically, they highlight novel metabolic and redox pathways that could serve as therapeutic targets effective against diverse T. cruzi strains and Discrete Typing Units (DTUs).