Genetic Validation of<italic toggle="yes"> Leishmania donovani</italic> Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

ABSTRACT Leishmania donovani 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 synth...

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Autores principales: Sanya Chadha, N. Arjunreddy Mallampudi, Debendra K. Mohapatra, Rentala Madhubala
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Publicado: American Society for Microbiology 2017
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spelling oai:doaj.org-article:7101a03993594215a882b8c8217e29982021-11-15T15:22:04ZGenetic Validation of<italic toggle="yes"> Leishmania donovani</italic> Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity10.1128/mSphereDirect.00340-172379-5042https://doaj.org/article/7101a03993594215a882b8c8217e29982017-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphereDirect.00340-17https://doaj.org/toc/2379-5042ABSTRACT Leishmania donovani 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 L. donovani lysyl-tRNA synthetase (LdLysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. LdLysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LdLysRS-1. Recombinant LdLysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LdLysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LdLysRS-1 appears to be an essential gene, as a chromosomal knockout of LdLysRS-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 [IC50], 4.19 µM) and intracellular amastigotes (IC50, 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 LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LdLysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE 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. L. donovani 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 (LdLysRS) are present in this parasite. LdLysRS-1 is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 is closer to bacteria. Here, we have characterized LdLysRS-1 of L. donovani. LdLysRS-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 LdLysRS-1 as a potential drug target.Sanya ChadhaN. Arjunreddy MallampudiDebendra K. MohapatraRentala MadhubalaAmerican Society for MicrobiologyarticleLeishmania donovanilysyl-tRNA synthetasedrug targetsgenetic validationMicrobiologyQR1-502ENmSphere, Vol 2, Iss 4 (2017)
institution DOAJ
collection DOAJ
language EN
topic Leishmania donovani
lysyl-tRNA synthetase
drug targets
genetic validation
Microbiology
QR1-502
spellingShingle Leishmania donovani
lysyl-tRNA synthetase
drug targets
genetic validation
Microbiology
QR1-502
Sanya Chadha
N. Arjunreddy Mallampudi
Debendra K. Mohapatra
Rentala Madhubala
Genetic Validation of<italic toggle="yes"> Leishmania donovani</italic> Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity
description ABSTRACT Leishmania donovani 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 L. donovani lysyl-tRNA synthetase (LdLysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. LdLysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LdLysRS-1. Recombinant LdLysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LdLysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LdLysRS-1 appears to be an essential gene, as a chromosomal knockout of LdLysRS-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 [IC50], 4.19 µM) and intracellular amastigotes (IC50, 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 LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LdLysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE 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. L. donovani 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 (LdLysRS) are present in this parasite. LdLysRS-1 is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 is closer to bacteria. Here, we have characterized LdLysRS-1 of L. donovani. LdLysRS-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 LdLysRS-1 as a potential drug target.
format article
author Sanya Chadha
N. Arjunreddy Mallampudi
Debendra K. Mohapatra
Rentala Madhubala
author_facet Sanya Chadha
N. Arjunreddy Mallampudi
Debendra K. Mohapatra
Rentala Madhubala
author_sort Sanya Chadha
title Genetic Validation of<italic toggle="yes"> Leishmania donovani</italic> Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity
title_short Genetic Validation of<italic toggle="yes"> Leishmania donovani</italic> Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity
title_full Genetic Validation of<italic toggle="yes"> Leishmania donovani</italic> Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity
title_fullStr Genetic Validation of<italic toggle="yes"> Leishmania donovani</italic> Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity
title_full_unstemmed Genetic Validation of<italic toggle="yes"> Leishmania donovani</italic> Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity
title_sort genetic validation of<italic toggle="yes"> leishmania donovani</italic> lysyl-trna synthetase shows that it is indispensable for parasite growth and infectivity
publisher American Society for Microbiology
publishDate 2017
url https://doaj.org/article/7101a03993594215a882b8c8217e2998
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