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.