Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target.
Aminoacyl tRNA synthetases are central enzymes required for protein synthesis. These enzymes are the known drug targets in bacteria and fungi. Here, we for the first time report the functional characterization of threonyl tRNA synthetase (LdThrRS) of Leishmania donovani, a protozoan parasite, the pr...
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ftdoajarticles:oai:doaj.org/article:eef6c1913d844ec282831c2418a0e817 2023-05-15T15:12:06+02:00 Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target. Sanya Chadha Ramachandran Vijayan Sakshi Gupta Manoj Munde Samudrala Gourinath Rentala Madhubala 2018-06-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0006575 https://doaj.org/article/eef6c1913d844ec282831c2418a0e817 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC6025875?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0006575 https://doaj.org/article/eef6c1913d844ec282831c2418a0e817 PLoS Neglected Tropical Diseases, Vol 12, Iss 6, p e0006575 (2018) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2018 ftdoajarticles https://doi.org/10.1371/journal.pntd.0006575 2022-12-31T09:35:55Z Aminoacyl tRNA synthetases are central enzymes required for protein synthesis. These enzymes are the known drug targets in bacteria and fungi. Here, we for the first time report the functional characterization of threonyl tRNA synthetase (LdThrRS) of Leishmania donovani, a protozoan parasite, the primary causative agent of visceral leishmaniasis.Recombinant LdThrRS (rLdThrRS) was expressed in E. coli and purified. The kinetic parameters for rLdThrRS were determined. The subcellular localization of LdThrRS was done by immunofluorescence analysis. Heterozygous mutants of LdThrRS were generated in Leishmania promastigotes. These genetically manipulated parasites were checked for their proliferation, virulence, aminoacylation activity and sensitivity to the known ThrRS inhibitor, borrelidin. An in silico generated structural model of L. donovani ThrRS was compared to that of human.Recombinant LdThrRS displayed aminoacylation activity, and the protein is possibly localized to both the cytosol and mitochondria. The comparison of the 3D-model of LdThrRS to human ThrRS displayed considerable similarity. Heterozygous parasites showed restrictive growth phenotype and had attenuated infectivity. These heterozygous parasites were more susceptible to inhibition by borrelidin. Several attempts to obtain ThrRS homozygous null mutants were not successful, indicating its essentiality for the Leishmania parasite. Borrelidin showed a strong affinity for LdThrRS (KD: 0.04 μM) and was effective in inhibiting the aminoacylation activity of the rLdThrRS (IC50: 0.06 μM). Borrelidin inhibited the promastigotes (IC50: 21 μM) stage of parasites. Our data shows that LdThrRS is essential for L. donovani survival and is likely to bind with small drug-like molecules with strong affinity, thus making it a potential target for drug discovery efforts. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 12 6 e0006575 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Sanya Chadha Ramachandran Vijayan Sakshi Gupta Manoj Munde Samudrala Gourinath Rentala Madhubala Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Aminoacyl tRNA synthetases are central enzymes required for protein synthesis. These enzymes are the known drug targets in bacteria and fungi. Here, we for the first time report the functional characterization of threonyl tRNA synthetase (LdThrRS) of Leishmania donovani, a protozoan parasite, the primary causative agent of visceral leishmaniasis.Recombinant LdThrRS (rLdThrRS) was expressed in E. coli and purified. The kinetic parameters for rLdThrRS were determined. The subcellular localization of LdThrRS was done by immunofluorescence analysis. Heterozygous mutants of LdThrRS were generated in Leishmania promastigotes. These genetically manipulated parasites were checked for their proliferation, virulence, aminoacylation activity and sensitivity to the known ThrRS inhibitor, borrelidin. An in silico generated structural model of L. donovani ThrRS was compared to that of human.Recombinant LdThrRS displayed aminoacylation activity, and the protein is possibly localized to both the cytosol and mitochondria. The comparison of the 3D-model of LdThrRS to human ThrRS displayed considerable similarity. Heterozygous parasites showed restrictive growth phenotype and had attenuated infectivity. These heterozygous parasites were more susceptible to inhibition by borrelidin. Several attempts to obtain ThrRS homozygous null mutants were not successful, indicating its essentiality for the Leishmania parasite. Borrelidin showed a strong affinity for LdThrRS (KD: 0.04 μM) and was effective in inhibiting the aminoacylation activity of the rLdThrRS (IC50: 0.06 μM). Borrelidin inhibited the promastigotes (IC50: 21 μM) stage of parasites. Our data shows that LdThrRS is essential for L. donovani survival and is likely to bind with small drug-like molecules with strong affinity, thus making it a potential target for drug discovery efforts. |
format |
Article in Journal/Newspaper |
author |
Sanya Chadha Ramachandran Vijayan Sakshi Gupta Manoj Munde Samudrala Gourinath Rentala Madhubala |
author_facet |
Sanya Chadha Ramachandran Vijayan Sakshi Gupta Manoj Munde Samudrala Gourinath Rentala Madhubala |
author_sort |
Sanya Chadha |
title |
Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target. |
title_short |
Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target. |
title_full |
Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target. |
title_fullStr |
Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target. |
title_full_unstemmed |
Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target. |
title_sort |
genetic manipulation of leishmania donovani threonyl trna synthetase facilitates its exploration as a potential therapeutic target. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2018 |
url |
https://doi.org/10.1371/journal.pntd.0006575 https://doaj.org/article/eef6c1913d844ec282831c2418a0e817 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 12, Iss 6, p e0006575 (2018) |
op_relation |
http://europepmc.org/articles/PMC6025875?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0006575 https://doaj.org/article/eef6c1913d844ec282831c2418a0e817 |
op_doi |
https://doi.org/10.1371/journal.pntd.0006575 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
12 |
container_issue |
6 |
container_start_page |
e0006575 |
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1766342841203687424 |