Glucose-6-phosphate dehydrogenase and Trypanothione reductase interaction protects Leishmania donovani from metalloid mediated oxidative stress.
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, AsIIIand TeIVextraneously, which hint towards metalloid driven structural changes of the interacting proteins. Co-purification patterns and pull-down assays also depicted that metalloids (SbIII, AsIIIand 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 AsIIIand 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.
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India.
Ayan Kumar
AK
Ghosh
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Export Promotion Industrial Park, Hajipur, Vaishali 844101, Bihar, India.
Savita
S
Saini
Department of Microbiology, All India Institute of Medical Sciences, Phulwarisharif, Patna 801505, Bihar, India.
Sushmita
S
Das
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India.
Abhishek
A
Mandal
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India.
Abul Hasan
AH
Sardar
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India.
Md Yousuf
MY
Ansari
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India.
Kumar
K
Abhishek
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India.
Ajay
A
Kumar
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India.
Ruby
R
Singh
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India.
Sudha
S
Verma
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India.
Asif
A
Equbal
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India.
Vahab
V
Ali
Division of Molecular Biology, Bioinformatics and Molecular Biochemistry & Cell Biology, Rajendra Memorial Research Institute of Medical Sciences (I.C.M.R.), Agamkuan, Patna 800007, Bihar, India. Electronic address: drpradeep.das@gmail.com.
Pradeep
P
Das
D000818:Animals; D000965:Antimony / Q000494:pharmacology; D001151:Arsenic / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects; D004351:Drug Resistance / Q000187:drug effects; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007893:Leishmania donovani / Q000187:drug effects* / Q000378:metabolism / Q000472:pathogenicity; D007898:Leishmaniasis, Visceral / Q000188:drug therapy* / Q000201:enzymology / Q000469:parasitology; D058955:Metalloids / Q000633:toxicity; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*; D011487:Protein Conformation / Q000187:drug effects; D017382:Reactive Oxygen Species; D013691:Tellurium / Q000494:pharmacology
D058955:Metalloids; D017382:Reactive Oxygen Species; D000965:Antimony; D005954:Glucosephosphate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D001151:Arsenic; D013691:Tellurium
Apoptosis; Leishmania; Metabolon; Metalloids; NADPH; ROS