Role of dithiol glutaredoxin 1 protein of Leishmania donovani in Amphotericin B resistance and redox homeostasis.

Visceral leishmaniasis, caused by Leishmania, remains a global health concern with limited treatment options. Although the polyene drug Amphotericin-B has been considered effective, the emergence of drug resistance compromises its efficacy. Managing redox metabolism is crucial for protozoan parasites wherein low molecular weight thiols are crucial for preserving a reduced intracellular environment for the normal functioning of metabolic processes. Glutaredoxin, a small ubiquitous redox protein belonging to the thioredoxin-fold superfamily, is an important component of the trypanothione-based redox metabolism of Leishmania; indispensable for the maintenance of cellular redox homeostasis. The L.donovani dithiol glutaredoxin 1 (LdGrx1) protein was expressed, purified, and biochemically characterized. Kinetic studies revealed that the LdGrx1 protein catalyzes the reduction of disulfide substrates. Digitonin fractionation and confocal microscopy analysis demonstrated cytosolic localization of LdGrx1 protein. The role of LdGrx1 protein in Amphotericin-B drug resistance was investigated by comparing three different strains of L. donovani- Amp-B sensitive, clinical isolate, and lab-generated Amp-B resistant parasites. The lab-generated parasite mimicked the pattern of resistant clinical isolate exhibiting higher Amp-B IC50 value, antioxidant activities, and thiol levels with subsequently lower ROS content, indicating more efficient thiol-based antioxidative defense than the sensitive parasites. In both Amp-B-resistant parasites, the LdGrx1 expression was found to be upregulated at transcriptional and translational levels, suggesting a possible association between LdGrx1 protein and Amp-B resistance. Thus, our results unveil the role of LdGrx1 in oxidative stress management, parasite survival under drug pressure, and it restores protein function through the reduction of mixed-disulfide substrates.