Up-regulation of cytosolic tryparedoxin in Amp B resistant isolates of Leishmania donovani and its interaction with cytosolic tryparedoxin peroxidase

Biochimie. 2016 Feb;121:312-25. doi: 10.1016/j.biochi.2015.12.017. Epub 2015 Dec 30.

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.

Keywords: Drug resistance; Leishmania donovani; Thiol metabolism; Trypanothione; Tryparedoxin; Tryparedoxin peroxides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amphotericin B / pharmacology*
  • Animals
  • Antiprotozoal Agents / pharmacology
  • Cytosol / metabolism*
  • Drug Resistance
  • Leishmania donovani / drug effects*
  • Leishmania donovani / enzymology*
  • Leishmania donovani / metabolism
  • Male
  • Peroxidases / metabolism*
  • Protozoan Proteins / metabolism*
  • Rabbits
  • Thioredoxins / metabolism*

Substances

  • Antiprotozoal Agents
  • Protozoan Proteins
  • tryparedoxin
  • Thioredoxins
  • Amphotericin B
  • Peroxidases
  • tryparedoxin peroxidase