Queuosine tRNA modification regulates translational adaptation and virulence of Leishmania mexicana

The complex life cycle of the human parasite Leishmania mexicana requires rapid translational adaptation for survival in two distinct environments: the insect vector and the mammalian host. These protists lack conventional transcriptional control due to their unusual genome organization. Consequently, tRNA modifications may represent an additional mechanism for post-transcriptional regulation of gene expression. One such modification is queuosine (Q), which is incorporated at the anticodon wobble position 34 of specific tRNAs. Here, we demonstrate that Q-tRNA levels increase substantially during Leishmania differentiation from the insect stage to the mammalian-infective stage, implying an important role for virulence. Hence, we generated mutant cells lacking the enzyme responsible for Q incorporation, tRNA-guanine transglycosylase (TGT), which exhibited substantial changes in the proteome during differentiation in vitro. Specifically, downregulated proteins were enriched in NAU codons, whereas upregulated proteins predominantly contained NAC codons. Although LmxTGT knockout parasites exhibited normal growth and differentiation in vitro, they demonstrated impaired survival within macrophages and reduced pathogenicity in mice, highlighting the role of the Q-tRNAs under stress conditions. To our knowledge, we present here the first direct evidence that queuosine tRNA modification controls the infectivity of Leishmania via codon-biased translation. To date, gene expression regulation in Leishmania and other trypanosomatids has been attributed mostly to RNA stability and processing; however, our findings demonstrate that tRNA modifications also play a key regulatory role. Specifically, the Q-tRNA modification provides a novel layer of gene expression regulation, maintaining translational balance and supporting the parasite's ability to adapt to changing environments, and contributing to Leishmania virulence.