Abstract
BackgroundLeishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator) from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator) related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2) of L. donovani.MethodologyRecombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors.ConclusionOur data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis.
Highlights
Acetylation and deacetylation of proteins have recently emerged as a major post-translational modification [1]
Our data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity
Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning
Summary
Acetylation and deacetylation of proteins have recently emerged as a major post-translational modification [1]. The Silent Information Regulator (SIR2), the founding member of the family of sirtuins, was originally described as a regulator of transcriptional silencing of mating-type loci, telomeres and ribosomal DNA [4], and involved in the lifespan extension of yeast [5]. Since their discovery, SIR2-like genes, known as sirtuins, have been studied in most organisms, including plants, bacteria, and animals, where they play a vital role in promoting an organism’s health and survival [6]. We for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2) of L. donovani
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