Up-regulation of silent information regulator 2 (Sir2) is associated with amphotericin B resistance in clinical isolates of Leishmania donovani
Authors: Ruby Singh
Kirti Wasnik
Abul H Sardar
Pradeep Das
Dharmendra Singh
Manas Dikhit
Ayan K Ghosh
Sushmita Das
Bidyut Purkait
Mark Paine
Ashish Kumar
Journal: Journal of Antimicrobial Chemotherapy (2015)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: AmB resistance; MDR1; ROS; Sir2; apoptosis
Affiliations: Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India., Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Hajipur, Bihar, India., Department of Microbiology, All India Institute of Medical Sciences, Patna, India., Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK., Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India .

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. 

Retracted: No

PubPeer comments:
 Concern about Figures 1h and 5a: * Blue boxes: Four lanes in the PARP1 panel in Figure 1h look like four lanes in the MDR1 panel in Figure 5a, albeit in mirror image. * Red boxes: Four lanes in the A-tub panel in Figure 1h look like four lanes in the A-tub panel in Figure 5a, albeit in mirror image. ![file] , 
 Concern about Figure 6B (image made a bit lighter to bring out details): * Pink boxes: Unexpected similarities between lanes 2 and 6 * Yellow boxes: Unexpected similarities between lanes 3 and 7 Legend states:  Lane 1, marker; lane 2, R; lane 3, R + AmB; lane 4, sirtinol+R+AmB; lane 5, Sir22 + R+AmB; lane 6, S; lane 7, OE S+AmB; and lane 8, S+AmB.  ![file] , 
 Concern about Figure 6C. * Boxes of the same color highlight unexpected similarities between panels. ![file] ,