Transcriptomic analysis of benznidazole-resistant Trypanosoma cruzi clone reveals nitroreductase I-independent resistance mechanisms

The enzyme nitroreductase I (NTRI) has been implicated as the primary gene responsible for resistance to benznidazole (Bz) and nifurtimox in Trypanosoma cruzi. However, Bz-resistant T. cruzi field isolates carrying the wild-type NTR-I enzyme suggest that additional mechanisms independent of this enzyme may contribute to the resistance phenotype. To investigate these alternative mechanisms, in this paper, we pressured a Trypanosoma cruzi clone with a high Bz concentration over several generations to select Bz-resistant clones. Surprisingly, we found a highly drug-resistant clone carrying a wild-type NTRI. However, the knockout of this gene using CRISPR-Cas9 in the sensitive clone showed that NTRI indeed induces resistance to Bz and supports the idea that the resistant one exhibits mechanisms other than NTRI. To explore these new mechanisms, we performed an RNA-seq analysis, which revealed genes involved in metabolic pathways related to oxidative stress, energy metabolism, membrane transporters, DNA repair, and protein synthesis. Our results support the idea that resistance to benznidazole is a multigenic trait. A Deeper understanding of these genes is essential for developing new drugs to treat Chagas disease.