Background: Asparagine synthetase A catalyzes the synthesis of asparagine from aspartate in the presence of ammonia as a nitrogen donor. Results: L. donovani ASNA enzyme is of bacterial origin and is both ammonia-and glutamine-dependent. Conclusion: LdASNA is essential for survival of the Leishmania parasite. Significance: The absence of LdASNA homologs from humans validates Leishmania ASNA as a novel drug target.
Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Currently available drugs manifest severe side effects. Hence, there is a need to identify new drug targets and drugs. Aminoacyl-tRNA synthetases, required for protein synthesis, are known drug targets for bacterial and fungal pathogens. The aim of the present study was to obtain essentiality data for leucyl-tRNA synthetase (LRS) by gene replacement. Gene replacement studies indicate that this enzyme plays an essential role in the viability of this pathogenic organism and appears to be indispensable for its survival The heterozygous mutant parasites demonstrated a growth deficit and reduced infectivity in mouse macrophages compared to the wild-type cells. We also report that recombinant LRS displayed aminoacylation activity and that the protein localized to both the cytosol and the mitochondrion. A broad-spectrum antifungal, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), was found to inhibit parasite growth in both the promastigote and amastigote stages as well as in BALB/c mice. This compound exhibited low toxicity to mammalian cells. AN2690 was effective in inhibiting the aminoacylation activity of the recombinant LRS. We provide preliminary chemical validation ofLRS as a drug target by showing that AN2690 is an inhibitor both of LRS and of cell growth.
SummaryRecently, we reported molecular characterization, localization and functional importance of Arabino-1, 4-lactone oxidase (ALO) enzyme from Leishmania donovani that catalyses the last step in ascorbate biosynthesis pathway. Vitamin C (L-ascorbic acid) is implicated in several crucial physiological processes. To elucidate the biological role of D-arabinono-γ-lactone oxidase in Leishmania, we made L. donovani ALO null mutant (ΔALO) by double targeted gene replacement. This mutant lacked ALO activity, showed transient growth defect and reduced ascorbate levels. ΔALO grown in ascorbate depleted media further enhanced growth defect with no detectable levels of ascorbate, implying that parasites have the ability to scavenge ascorbate. ΔALO mutants showed reduced survival in mouse macrophages and are impaired in their infectivity in vivo. Furthermore, the ΔALO mutant induced production of pro-inflammatory cytokines gamma interferon (IFN-gamma), interleukin-12 (IL-12) and tumour necrosis factor-α (TNF-α) by infected mouse macrophages. These mutants were susceptible to oxidative stresses in vitro as revealed by the decreased survival inside macrophages by increased production of reactive oxygen or nitrogen species. Complementation of the ΔALO mutants restored the phenotypic effects in these parasites. Our description of ALO null mutant parasite that triggers proinflammatory host responses provides a novel platform for targeting ALO in anti-parasitic strategies.
The synthesis of selenocysteine, the 21st amino acid, occurs on its transfer RNA (tRNA), tRNA Sec . tRNA Sec is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNA Sec and selenophosphate to form Sec-tRNA Sec in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from Leishmania donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ⌬selA deletion in Escherichia coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins, which were only expressed in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed the same sensitivity toward the wild-type and null mutants suggesting its effect is not through binding to selenoproteins. The three-dimensional structural comparison indicates that human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.