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 1,515 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). • Natural resistance in T. cruzi involves complex, multigenic adaptations. • Resistant strain upregulates genes in redox, DNA repair, and energy metabolism. • Susceptible strain shows limited gene response, linked to higher drug susceptibility. • Distinct metabolic rewiring supports parasite survival under treatment. • Transcriptomics reveal strain-specific drug response in T. cruzi.