title: Up-regulation of silent information regulator 2 (Sir2) is associated with amphotericin B resistance in clinical isolates of Leishmania donovani.
abstract: OBJECTIVE
  Silent information regulator 2 (Sir2) is involved in parasite survival and apoptosis. Here, we aimed to explore the involvement of Sir2 in amphotericin B (AmB) resistance mechanism in Leishmania donovani.
  
  
  METHODS
  The expression levels of Sir2, MDR1 and NAD(+) biosynthetic pathway enzymes in AmB-resistant and -susceptible parasites were measured and total intracellular NAD(+)/NADH ratios were compared. Overexpression and knockout constructs of Sir2 were transfected in AmB-resistant and -susceptible parasites. Both resistant and susceptible parasites were inhibited with sirtinol for 4 h. The deacetylase activity of Sir2, the expression level of MDR1, the rate of AmB efflux, concentrations of reactive oxygen species (ROS) and levels of apoptosis were examined in WT, inhibited and transfected parasites, and the AmB susceptibility of the respective parasites was measured by determining the LD50 of AmB.
  
  
  RESULTS
  Levels of mRNA, protein and NAD(+)-dependent deacetylase activity of Sir2 were elevated in resistant versus susceptible parasites. Inhibition and/or deletion of Sir2 allele showed a decreased mRNA level of MDR1, lower drug efflux, increased ROS concentration, apoptosis-like phenomenon and decreased LD50 of AmB in resistant parasites. In contrast, Sir2 overexpression in susceptible parasites reversed drug susceptibility producing a resistant phenotype. This was associated with increased LD50 of AmB along with increased expression levels of MDR1, drug efflux and reduced concentrations of ROS, corresponding to decreased apoptosis of resistant to WT sensitive.
  
  
  CONCLUSIONS
  Sir2 plays a critical role in AmB resistance by regulating MDR1, ROS concentration and apoptosis-like phenomena and may be a new resistance marker for visceral leishmaniasis.
authors: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India.
authors: Bidyut
authors: B
authors: Purkait
authors: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India.
authors: Ruby
authors: R
authors: Singh
authors: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Hajipur, Bihar, India.
authors: Kirti
authors: K
authors: Wasnik
authors: Department of Microbiology, All India Institute of Medical Sciences, Patna, India.
authors: Sushmita
authors: S
authors: Das
authors: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India.
authors: Ashish
authors: A
authors: Kumar
authors: Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK.
authors: Mark
authors: M
authors: Paine
authors: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India.
authors: Manas
authors: M
authors: Dikhit
authors: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India.
authors: Dharmendra
authors: D
authors: Singh
authors: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India.
authors: Abul H
authors: AH
authors: Sardar
authors: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India.
authors: Ayan K
authors: AK
authors: Ghosh
authors: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India drpradeep.das@gmail.com.
authors: Pradeep
authors: P
authors: Das
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000666:Amphotericin B / Q000494:pharmacology*; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D017209:Apoptosis; D004351:Drug Resistance; D015870:Gene Expression; D020869:Gene Expression Profiling; D055786:Gene Knockout Techniques; D007893:Leishmania donovani / Q000187:drug effects* / Q000235:genetics*; D017382:Reactive Oxygen Species; D037761:Sirtuins / Q000096:biosynthesis*; D015854:Up-Regulation
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000981:Antiprotozoal Agents; D017382:Reactive Oxygen Species; D000666:Amphotericin B; D037761:Sirtuins
keywords: AmB resistance; MDR1; ROS; Sir2; apoptosis; visceral leishmaniasis