Glucose-6-phosphate dehydrogenase and Trypanothione reductase interaction protects Leishmania donovani from metalloid mediated oxidative stress

Abstract

Exploration of metabolons as viable drug target is rare in kinetoplastid biology. Here we present a novel protein-protein interaction among Glucose-6-phosphate dehydrogenase (LdG6PDH) and Trypanothione reductase (LdTryR) of Leishmania donovani displaying interconnection between central glucose metabolism and thiol metabolism of this parasite. Digitonin fractionation patterns observed through immunoblotting indicated localisation of both LdG6PDH and LdTryR in cytosol. In-silico and in-vitro interaction observed by size exclusion chromatography, co-purification, pull-down assay and spectrofluorimetric analysis revealed LdG6PDH and LdTryR physically interact with each other in a NADPH dependent manner. Coupled enzymatic assay displayed that NADPH generation was severely impaired by addition of SbIII, AsIII and TeIV extraneously, which hint towards metalloid driven structural changes of the interacting proteins. Co-purification patterns and pull-down assays also depicted that metalloids (SbIII, AsIII and TeIV) hinder the in-vitro interaction of these two enzymes. Surprisingly, metalloids at sub-lethal concentrations induced the in-vivo interaction of LdG6PDH and LdTryR, as analyzed by pull-down assays and fluorescence microscopy signifying protection against metalloid mediated ROS. Inhibition of LdTryR by thioridazine in LdG6PDH-/- parasites resulted in metalloid induced apoptotic death of the parasites due to abrupt fall in reduced thiol content, disrupted NADPH/NADP+ homeostasis and lethal oxidative stress. Interestingly, clinical isolates of L.donovani resistant to SAG exhibited enhanced interaction between LdG6PDH and LdTryR and showed cross resistivity towards AsIII and TeIV. Thus, our findings propose the metabolon of LdG6PDH and LdTryR as an alternate therapeutic target and provide mechanistic insight about metalloid resistance in Visceral Leishmaniasis.