Discussion

Benznidazole (BNZ) is the front-line drug for Chagas disease. However, treatment failures are widely reported. To assess mechanisms of action and resistance, we sequenced the genomes of T. cruzi Y strain and three BNZ-resistant clones derived from a single drug-selected population. The genomes (35.5 Mb) contain 8,289 predicted protein-coding genes, 5119 where function can be inferred. Surprisingly, we identified a total of 26,495 point mutations in the drug-resistant clones. In coding regions, 49% of these were non-synonymous, many linked to radical amino acid changes. Analysis of copy number also revealed widespread DNA amplifications. Mutations were identified in genes of all functional categories, including DNA repair. This was associated with increased susceptibility to DNA alkylating and/or interstrand cross-linking agents. Resistance to benznidazole could be partially explained by stop-codon generating mutations in the drug-activating nitroreductase, a phenomenon which conferred widespread cross-resistance to other nitroheterocyclic compounds. Unexpectedly, we found that each clone was also resistant to posaconazole (6-22 fold), a drug which has been proposed for use against T. cruzi infections, in combination with benznidazole. Our findings therefore identify the highly mutagenic activity of benznidazole metabolites in T. cruzi, demonstrate how these might promote the development of multi-drug resistance, and indicate the need for vigilance when benznidazole is used in combination therapy.