tRNA lysidinylation is essential for the minimal translation system in the Plasmodium falciparum apicoplast

For decades, researchers have sought to define minimal translation systems to uncover fundamental principles of life and advance biotechnology. tRNAs, essential components of this machinery, decode mRNA codons into amino acids. The apicoplast of malaria parasites contains 25 tRNA isotypes in its organellar genome-the lowest number found in known translation systems. Efficient translation in such minimal systems depends heavily on post-transcriptional tRNA modifications. One such modification, lysidine at the wobble position (C34) of tRNA_CAU, distinguishes between methionine (AUG) and isoleucine (AUA) codons. tRNA isoleucine lysidine synthetase (TilS) produces lysidine, which is nearly ubiquitous in bacteria and essential for cellular viability. Here, we report a TilS ortholog (PfTilS) targeted to the apicoplast of Plasmodium falciparum. We demonstrate that PfTilS activity is essential for parasite survival and apicoplast function, likely due to its role in protein translation. This study is the first to characterize TilS in an endosymbiotic organelle, contributing to research on eukaryotic organelles and minimal translational systems. Moreover, the absence of lysidine in humans highlights a potential target for antimalarial strategies.