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Currently, there is no approved vaccine for visceral leishmaniasis (VL) caused by L. donovani . The ability to manipulate Leishmania genome by eliminating or introducing genes necessary for parasites’ survival considered as the powerful strategy to generate the live attenuated vaccine. In the present study fructose-1,6-bisphosphatase (LdFBPase) gene deleted L. donovani (Δ fbpase ) was generated using homologous gene replacement strategy. Though LdFBPase gene deletion (Δ fbpase ) does not affect the growth of parasite in the promastigote form but axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Though Δ fbpase L. donovani parasite persisted in BALB/c mice up to 12 weeks but was unable to cause infection, we tested its ability to protect against a virulent L. donovani challenge . Notably, intraperitoneal immunisation with live Δ fbpase parasites displayed the reduction of parasites load in mice spleen and liver post challenge. Moreover, immunised BALB/c mice showed a reversal of T cell anergy and high levels of NO production that result in the killing of the parasite. A significant, correlation was found between parasite clearance and elevated IFNγ, IL12, and IFNγ/IL10 ratio compared to IL10 and TGFβ in immunised and challenged mice. Results suggested the generation of protective Th1 type immune response which induced significant parasite clearance at 12-week, as well as 16 weeks post, challenged immunised mice, signifying sustained immunity. Therefore, we propose that Δ fbpase L. donovani parasites can be a live attenuated vaccine candidate for VL and a good model to understand the correlatives of protection in visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

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 ( Ld G6PDH) and Trypanothione reductase ( Ld TryR) 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 Ld G6PDH and Ld TryR in cytosol. In-silico and in-vitro interaction observed by size exclusion chromatography, co-purification, pull-down assay and spectrofluorimetric analysis revealed Ld G6PDH and Ld TryR physically interact with each other in a NADPH dependent manner. Coupled enzymatic assay displayed that NADPH generation was severely impaired by addition of Sb III , As III and Te IV extraneously, which hint towards metalloid driven structural changes of the interacting proteins. Co-purification patterns and pull-down assays also depicted that metalloids (Sb III , As III and Te IV ) hinder the in-vitro interaction of these two enzymes. Surprisingly, metalloids at sub-lethal concentrations induced the in-vivo interaction of Ld G6PDH and Ld TryR, as analyzed by pull-down assays and fluorescence microscopy signifying protection against metalloid mediated ROS. Inhibition of Ld TryR by thioridazine in Ld G6PDH -/- 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 Ld G6PDH and Ld TryR and showed cross resistivity towards As III and Te IV . Thus, our findings propose the metabolon of Ld G6PDH and Ld TryR as an alternate therapeutic target and provide mechanistic insight about metalloid resistance in Visceral Leishmaniasis. [ABSTRACT FROM AUTHOR]

The antimalarial agents artemisinins inhibit cytomegalovirus (CMV) in vitro and in vivo, but their target(s) has been elusive. Using a biotin-labeled artemisinin, we identified the intermediate filament protein vimentin as an artemisinin target, validated by detailed biochemical and biological assays. We provide insights into the dynamic and unique modulation of vimentin, depending on the stage of human CMV (HCMV) replication. In vitro, HCMV entry and viral progeny are reduced in vimentindeficient fibroblasts, compared with control cells. Similarly, mouse CMV (MCMV) replication in vimentin knockout mice is significantly reduced compared with controls in vivo, confirming the requirement of vimentin for establishment of infection. Early after HCMV infection of human foreskin fibroblasts vimentin level is stable, but as infection proceeds, vimentin is destabilized, concurrent with its phosphorylation and virusinduced calpain activity. Intriguingly, in vimentin-overexpressing cells, HCMV infection is reduced compared with control cells. Binding of artesunate, an artemisinin monomer, to vimentin prevents virus-induced vimentin degradation, decreasing vimentin phosphorylation at Ser-55 and Ser-83 and resisting calpain digestion. In vimentin-deficient fibroblasts, the anti- HCMV activity of artesunate is reduced compared with controls. In summary, an intact and stable vimentin network is important for the initiation of HCMV replication but hinders its completion. Artesunate binding to vimentin early during infection stabilizes it and antagonizes subsequent HCMV-mediated vimentin destabilization, thus suppressing HCMV replication. Our target discovery should enable the identification of vimentin- binding sites and compound moieties for binding. [ABSTRACT FROM AUTHOR]