title: Up-regulation of cytosolic tryparedoxin in Amp B resistant isolates of Leishmania donovani and its interaction with cytosolic tryparedoxin peroxidase.
abstract: Leishmania is a unicellular protozoan parasite which causes leishmaniasis, a neglected tropical disease. It possess a unique thiol metabolism comprising of several proteins among which, tryparedoxin (cTXN) and tryparedoxin peroxidase (cTXNPx), function in concert as oxidoreductases, utilizing trypanothione as a source of electrons to reduce the hydroperoxides produced by macrophages during infection. This detoxification pathway is unique and essential for the survival of Leishmania. Herein, we report the functional characterization of Leishmania donovani cTXN and its interaction with cTXNPx. The full length recombinant cTXN and cTXNPx proteins were purified in the native state and biochemical analysis showed that the cTXN-cTXNPx coupled system efficiently degraded hydrogen peroxide and tert-butyl hydroperoxide by transferring reducing equivalents from trypanothione. In silico investigation of the potential interaction between cTXN and cTXNPx proteins showed strong interaction of model structures with amino acids Ile109, Thr132, Glu107, Trp70, Trp39, Cys40 and His129 of Ld-cTXN and Thr54, Lys93, Arg128 and Asn152 of Ld-cTXNPx predicted to be involved in interaction. Moreover, co-purification, pull down assay and immunoprecipitation studies confirmed the interaction between Ld-cTXN and Ld-cTXNPx proteins. In addition, for the first time, we demonstrated at the translational level that Ld-cTXN protein is upregulated in Amp B resistant isolates accompanied by enhanced peroxidase activity, as compared to sensitive strains. Thus, our results show that Ld-cTXN and Ld-cTXNPx proteins acts in concert by physical interaction to form a strong peroxide stress detoxification system in Leishmania and their upregulation in Amp B resistant isolates imparts better stress tolerance, and hence fitter pathogens, as compared to sensitive strains.
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna 800007, India.
authors.forename: Shashi S
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 800007, 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 800007, India.
authors.forename: Amir
authors.initials: A
authors.lastname: Zaidi
authors.affiliation: Bioinformatics Centre, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna 800007, India; Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, EPIP Complex, Hajipur 844102, India.
authors.forename: Md Yousuf
authors.initials: MY
authors.lastname: Ansari
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna 800007, 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 800007, India; Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, EPIP Complex, Hajipur 844102, India.
authors.forename: Kuljit
authors.initials: K
authors.lastname: Singh
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna 800007, India.
authors.forename: Bidyut
authors.initials: B
authors.lastname: Purkait
authors.affiliation: Bioinformatics Centre, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna 800007, India.
authors.forename: Ganesh Chandra
authors.initials: GC
authors.lastname: Sahoo
authors.affiliation: Department of Immunology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna 800007, India.
authors.forename: Sanjeeva
authors.initials: S
authors.lastname: Bimal
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna 800007, 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 800007, India; Bioinformatics Centre, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna 800007, India. Electronic address: vahab_ali@yahoo.com.
authors.forename: Vahab
authors.initials: V
authors.lastname: Ali
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology*; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004351:Drug Resistance; D007893:Leishmania donovani / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D010544:Peroxidases / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism*; D011817:Rabbits; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D000981:Antiprotozoal Agents; D015800:Protozoan Proteins; C114110:tryparedoxin; D013879:Thioredoxins; D000666:Amphotericin B; D010544:Peroxidases; C110959:tryparedoxin peroxidase
keywords: Drug resistance; Leishmania donovani; Thiol metabolism; Trypanothione; Tryparedoxin; Tryparedoxin peroxides