Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target

PLoS Negl Trop Dis. 2018 Jun 13;12(6):e0006575. doi: 10.1371/journal.pntd.0006575. eCollection 2018 Jun.

Abstract

Background: 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.

Methodology: 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.

Conclusions: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Drug Delivery Systems
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Fatty Alcohols / pharmacology
  • Gene Expression
  • Humans
  • Leishmania donovani / drug effects
  • Leishmania donovani / enzymology*
  • Leishmania donovani / genetics
  • Leishmania donovani / pathogenicity
  • Leishmaniasis, Visceral / parasitology*
  • Organisms, Genetically Modified
  • Phylogeny
  • Protein Domains
  • Protein Transport
  • Protozoan Proteins / antagonists & inhibitors
  • Protozoan Proteins / genetics
  • Protozoan Proteins / isolation & purification
  • Protozoan Proteins / metabolism
  • Recombinant Proteins
  • Sequence Deletion
  • Threonine-tRNA Ligase / antagonists & inhibitors
  • Threonine-tRNA Ligase / genetics*
  • Threonine-tRNA Ligase / isolation & purification
  • Threonine-tRNA Ligase / metabolism

Substances

  • Fatty Alcohols
  • Protozoan Proteins
  • Recombinant Proteins
  • borrelidin
  • Threonine-tRNA Ligase

Grants and funding

This work was supported by the Department of Biotechnology, Ministry of Science and Technology (Grant No. BT/PR6742/BRB/10/1152/2012; www.dbtindia.nic.in/), Department of Science and Technology, Government of India (Grant No. SB/SO/HS/009/2013; www.dst.gov.in/), and JC Bose (SR/S2/JCB-72/2010; http://serb.gov.in/jcbn.php) grant sanctioned to RM. RM is a BSR-University Grants Commission faculty. SC is supported by Council for Scientific and Industrial Research (CSIR), India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.