title: Stage-dependent expression and up-regulation of trypanothione synthetase in amphotericin B resistant Leishmania donovani.
abstract: Kinetoplastids differ from other organisms in their ability to conjugate glutathione and spermidine to form trypanothione which is involved in maintaining redox homeostasis and removal of toxic metabolites. It is also involved in drug resistance, antioxidant mechanism, and defense against cellular oxidants. Trypanothione synthetase (TryS) of thiol metabolic pathway is the sole enzyme responsible for the biosynthesis of trypanothione in Leishmania donovani. In this study, TryS gene of L. donovani (LdTryS) was cloned, expressed, and fusion protein purified with affinity column chromatography. The purified protein showed optimum enzymatic activity at pH 8.0-8.5. The TryS amino acids sequences alignment showed that all amino acids involved in catalytic and ligands binding of L. major are conserved in L. donovani. Subcellular localization using digitonin fractionation and immunoblot analysis showed that LdTryS is localized in the cytoplasm. Furthermore, RT-PCR coupled with immunoblot analysis showed that LdTryS is overexpressed in Amp B resistant and stationary phase promastigotes (∼ 2.0-folds) than in sensitive strain and logarithmic phase, respectively, which suggests its involvement in Amp B resistance. Also, H2O2 treatment upto 150 µM for 8 hrs leads to 2-fold increased expression of LdTryS probably to cope up with oxidative stress generated by H2O2. Therefore, this study demonstrates stage- and Amp B sensitivity-dependent expression of LdTryS in L. donovani and involvement of TryS during oxidative stress to help the parasites survival.
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, AgamKuan, Patna, India.
authors.forename: Asif
authors.initials: A
authors.lastname: Equbal
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, AgamKuan, Patna, India.
authors.forename: Shashi Shekhar
authors.initials: SS
authors.lastname: Suman
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, AgamKuan, Patna, India.
authors.forename: Shadab
authors.initials: S
authors.lastname: Anwar
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, AgamKuan, Patna, India.
authors.forename: Krishn Pratap
authors.initials: KP
authors.lastname: Singh
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, AgamKuan, Patna, India.
authors.forename: Amir
authors.initials: A
authors.lastname: Zaidi
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, AgamKuan, Patna, India.
authors.forename: Abul Hasan
authors.initials: AH
authors.lastname: Sardar
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, AgamKuan, Patna, India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, AgamKuan, Patna, India.
authors.forename: Vahab
authors.initials: V
authors.lastname: Ali
mesh_terms: D019732:Amide Synthases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000666:Amphotericin B / Q000494:pharmacology*; D000818:Animals; D003001:Cloning, Molecular; D004351:Drug Resistance / Q000235:genetics*; D004789:Enzyme Activation; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007893:Leishmania donovani / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D007898:Leishmaniasis, Visceral / Q000188:drug therapy / Q000469:parasitology*; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D021261:Parasitic Sensitivity Tests; D055503:Protein Multimerization; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D016415:Sequence Alignment; D015854:Up-Regulation
chemical_list: D017382:Reactive Oxygen Species; D000666:Amphotericin B; D006861:Hydrogen Peroxide; D019732:Amide Synthases; C064407:trypanothione synthetase