Silva, H. G.; De Freitas Nascimento, J.; Braga, M. S.; Silber, A. M.; Waldor, M.; da Cunha, J. P.; Kimura, S.

Trypanosoma cruzi, the etiological agent of Chagas disease, infects millions of people in the Americas. This parasite undergoes drastic changes in its morphology and metabolism between infective and noninfective forms through global remodeling of its proteome. Chemical modification of tRNA (tRNA modification) contributes to the control of protein expression by modulating the codon decoding process. However, knowledge of tRNA modification profiles, the enzymes that create modifications and their regulation in different cellular conditions is largely restricted to relatively few model organisms. Here, we profile tRNA modifications in both infective and noninfective forms of T. cruzi to probe their dynamic changes. Genome mining of tRNA modifying enzymes identified 66 putative tRNA modifying enzymes in T. cruzi, each responsible for at least one of fifty-seven modifications. tRNA sequencing detected reverse transcription-derived signatures at 182 sites in T. cruzi tRNAs that are likely derived from 18 tRNA modifications. tRNA modifications and tRNA modification enzymes are differentially modulated across the life stages of T. cruzi. We found that hydroxywybutosine (OHyW) at position 37 on tRNAPhe had a reduced level in the infective form (metacyclic trypomastigote) and the associated modification enzyme Tyw1a exhibited reduced expression in this stage. Knockout of Tyw1a increased the differentiation from epimastigote (noninfective form) to metacyclic trypomastigote, suggesting that OHyW37 modification levels control the rate of metacyclogenesis. Overall, our findings suggest that the global regulation of tRNA modifications in the life stages of T. cruzi plays a critical role in the differentiation of this parasite.