Bidyut Purkait1, Ruby Singh1, Kirti Wasnik2, Sushmita Das3, Ashish Kumar1, Mark Paine4, Manas Dikhit1, Dharmendra Singh1, Abul H Sardar1, Ayan K Ghosh1, Pradeep Das5. 1. Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Agamkuan, Patna, 800007 Bihar, India. 2. Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Hajipur, Bihar, India. 3. Department of Microbiology, All India Institute of Medical Sciences, Patna, India. 4. Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK. 5. 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.
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.
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: Edubiel A Alpizar-Sosa; Nur Raihana Binti Ithnin; Wenbin Wei; Andrew W Pountain; Stefan K Weidt; Anne M Donachie; Ryan Ritchie; Emily A Dickie; Richard J S Burchmore; Paul W Denny; Michael P Barrett Journal: PLoS Negl Trop Dis Date: 2022-09-28
Authors: Alberto Rastrojo; Raquel García-Hernández; Paola Vargas; Esther Camacho; Laura Corvo; Hideo Imamura; Jean-Claude Dujardin; Santiago Castanys; Begoña Aguado; Francisco Gamarro; Jose M Requena Journal: Int J Parasitol Drugs Drug Resist Date: 2018-04-13 Impact factor: 4.077