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.

Intraspecies differences in natural susceptibility to amphotericine B of clinical isolates of Leishmania subgenus Viannia

“…(L . ) donovani species in the Bihar region of India [ 15 , 29 , 53 , 54 ]. Alterations in the sterol composition of the membrane [ 53 ] and alterations in the expression levels of enzymes of the thiol cascade, such as trypanothione reductase [ 55 ] and tripareduccin [ 15 ], have been reported for these isolates, among other mechanisms [ 54 ].…”

“…donovani species in the Bihar region of India [ 15 , 29 , 53 , 54 ]. Alterations in the sterol composition of the membrane [ 53 ] and alterations in the expression levels of enzymes of the thiol cascade, such as trypanothione reductase [ 55 ] and tripareduccin [ 15 ], have been reported for these isolates, among other mechanisms [ 54 ]. Changes have also been reported in the levels of other enzymes related to the control of reactive oxygen species, such as ascorbate peroxidase [ 56 ], cysteine synthetase [ 53 ] and cysteine proteinase B [ 54 ].…”

Deciphering the interplay between cysteine synthase and thiol cascade proteins in modulating Amphotericin B resistance and survival of Leishmania donovani under oxidative stress

“…Previously, we reported up-regulation of LdTryS expression in Amp B resistant L. donovani clinical isolates at both transcriptional and translational levels [8], [20]. In T. brucei , dsRNAi studies of TryS resulted in declined T(SH) 2 and glutathionylspermidine (Gsp) level, growth arrest, impaired antioxidant capacity and infectivity [21].…”

ATrypanosoma bruceiORFeome-based Gain-of-Function Library identifies genes that promote survival during melarsoprol treatment

“…Despite the established relationship between melarsoprol and trypanothione, the specific mode of melarsoprol cell killing remains unknown (24). Because the biosynthetic and redox utilization pathways contain enzymes unique to Trypanosomatids, they have been broadly explored as drug targets against American trypanosomes and Leishmania species (15,(28)(29)(30)(31)(32).…”