Comparative Transcriptomics of Naturally Susceptible and Resistant Trypanosoma cruzi Strains in Response to Benznidazole

Chagas disease (CD) is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi and represents a major public health concern. Currently, treatment options are limited to two medications, Benznidazole and Nifurtimox; both of which have significant side effects and variable efficacy. The emergence of drug-resistant T. cruzi strains, coupled with a limited understanding of resistance mechanisms, further complicates disease control and hinders the development of more effective therapies. To study these resistance mechanisms, we conducted a comparative transcriptomic analysis of two T. cruzi strains: MG (naturally susceptible) and DA (naturally resistant) to Benznidazole. Both strains, classified as TcI, were maintained in RPMI culture medium. The half-maximal effective concentration (EC50) of Benznidazole was determined using the MTT assay on parasites exposed to varying drug concentrations. RNA was extracted and sequenced using RNA-seq, followed by read alignment to the T. cruzi Brazil A4 reference genome. Differential gene expression was analyzed with DESeq2, functional enrichment was assessed using Gene Ontology (GO) analysis, and metabolic pathways were mapped via the KAAS tool. Our results confirmed a stark contrast in drug susceptibility: the EC50 value for Benznidazole was significantly higher in the DA strain (28.92 µg/mL) than in the MG strain (0.8827 µg/mL). Differential gene expression analysis identified 13,725 differentially expressed genes in DA and 13,701 in MG. Specifically, DA exhibited 408 upregulated and 1,515 downregulated genes, while MG had 153 upregulated and 866 downregulated genes (Log2FoldChange = 2 or = -2). GO analysis revealed distinct biological processes associated with resistance and susceptibility. In DA, 53 GO terms were upregulated and 127 were downregulated, whereas MG showed 90 upregulated and 40 downregulated terms. Genes in DA were primarily linked to electron transport and detoxification processes, whereas MG displayed an enrichment of genes related to DNA repair and energy metabolism. Metabolic pathway analysis using KAAS highlighted key differences between the two strains, particularly in three major pathways: the pentose phosphate pathway, glycolysis/gluconeogenesis, and the tricarboxylic acid (TCA) cycle. These findings suggest that T. cruzi's response to Benznidazole involves complex, multifactorial mechanisms that differ between strains. Notably, we identified key candidate genes such as prostaglandin F2a synthase, trypanothione synthase, thioredoxin, and prostaglandin F synthase as potential targets for more effective treatments. Using naturally resistant and susceptible strains, we uncovered distinct transcriptional signatures and metabolic pathways linked to differential drug responses. These findings highlight promising avenues for therapeutic development while emphasizing the need for broader studies across diverse T. cruzi strains and discrete typing units (DTUs).