Genetic Validation of Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity.

is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of lysyl-tRNA synthetase (LysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the genome database. LysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LysRS-1. Recombinant LysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LysRS-1 appears to be an essential gene, as a chromosomal knockout of LysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC], 4.19 µM) and intracellular amastigotes (IC, 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using LysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LysRS. Our data provide a framework for the development of a new class of drugs against this parasite. Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the proper construction of peptide chains. These enzymes provide raw materials for protein translation and also ensure fidelity of translation. is a protozoan parasite that causes visceral leishmaniasis. It is a continuously proliferating parasite that depends heavily on efficient protein translation. Lysyl-tRNA synthetase is one of the aaRSs which charges lysine to its cognate tRNA. Two different coding sequences for lysyl-tRNA synthetases (LysRS) are present in this parasite. LysRS-1 is closer to apicomplexans and eukaryotes, whereas LysRS-2 is closer to bacteria. Here, we have characterized LysRS-1 of . LysRS-1 appears to be an essential gene, as the chromosomal null mutants did not survive. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. This study also provides a platform to explore LysRS-1 as a potential drug target.