title: Universal minicircle sequence binding protein of Leishmania donovani regulates pathogenicity by controlling expression of cytochrome-b.
abstract: BACKGROUND
  Leishmania contains a concatenated mitochondrial DNA, kDNA. Universal minicircle sequence binding protein (UMSBP), a mitochondrial protein, initiates kDNA replication by binding with a conserved universal minicircle sequence (UMS) of kDNA. Here, we describe first time in L. donovani the regulation of DNA binding activity of UMSBP and the role of UMSBP in virulence.
  
  
  METHODS
  Insilco and EMSA study were performed to show UMS-binding activity of UMSBP. Tryparedoxin(TXN)-tryparedoxin peroxidase(TXNPx) assay as well as co-overexpression of cytochrome-b5 reductase-like protein (CBRL) and tryparedoxin in L. donovani were done to know the regulation of DNA binding activity of UMSBP. Knockout and episomal-expression constructs of UMSBP were transfected in L. donovani. The cell viability assay and immunofluorescence study to know the status of kDNA were performed. Macrophages were infected with transfected parasites. mRNA level of cytochrome b, activity of complex-III, intracellular ATP level of both transfected promastigotes and amastigotes as well as ROS concentration and the level of apoptosis of transfected promastigotes were measured. Level of oxidative phosphorylation of both transfected and un-transfected amastigotes were compared. Burden of transfected amastigotes in both macrophages and BALB/c mice were measured.
  
  
  RESULTS
  L. donovani UMSBP is capable of binding with UMS, regulated by redox through mitochondrial enzymes, TXN, TXNPx and CBRL. Depletion of UMSBP (LdU(-/-)) caused kDNA loss, which decreased cytochrome-b expression [component of complex-III of electron transport chain (ETC)] and leads to the disruption of complex-III activity, decreased ATP generation, increased ROS level and promastigotes exhibited apoptosis like death. Interestingly, single knockout of UMSBP (LdU(-/+)) has no effect on promastigotes survival. However, single knockout in intracellular amastigotes demonstrate loss of mRNA level of cytochrome-b, disruption in the activity of complex-III and reduced production of ATP in amastigotes than wild type. This process interfere with the oxidative-phosphorylation and thereby completely inhibit the intracellular proliferation of LdU(-/+) amastigotes in human macrophages and in BALB/c mice. Amastigotes proliferation was restored as wild type after episomal expression of LdUMSBP in LdU(-/+) parasites (LdU(-/+)AB).
  
  
  CONCLUSIONS
  The LdUMSBP regulates leishmanial mitochondrial respiration and pathogenesis. So, LdUMSBP may be an attractive target for rational drug designing and LdU(-/+) parasites could be considered as a live attenuated vaccine candidate against visceral leishmaniasis.
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Ruby
authors.initials: R
authors.lastname: Singh
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Bidyut
authors.initials: B
authors.lastname: Purkait
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Kumar
authors.initials: K
authors.lastname: Abhishek
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Hajipur, Vaishali, Bihar 844101 India.
authors.forename: Savita
authors.initials: S
authors.lastname: Saini
authors.affiliation: Department of Microbiology, All India Institute of Medical Sciences (AIIMS), Patna, 801105 India.
authors.forename: Sushmita
authors.initials: S
authors.lastname: Das
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Sudha
authors.initials: S
authors.lastname: Verma
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Mandal
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Ayan Kr
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Yousuf
authors.initials: Y
authors.lastname: Ansari
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Ashish
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Abul H
authors.initials: AH
authors.lastname: Sardar
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Ajay
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry, Bose Institute, Kolkata, 700009 India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Parrack
authors.affiliation: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Indian Council of Medical Research (ICMR), Agamkuan, Patna, Bihar 800007 India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
keywords: Apoptosis; CBRL; ETC; Leishmania donovani; Oxidative phosphorylation; UMSBP; kDNA