title: Ascorbate peroxidase, a key molecule regulating amphotericin B resistance in clinical isolates of Leishmania donovani.
abstract: Amphotericin B (AmB), a polyene macrolide, is now a first-line treatment of visceral leishmaniasis cases refractory to antimonials in India. AmB relapse cases and the emergence of secondary resistance have now been reported. To understand the mechanism of AmB, differentially expressed genes in AmB resistance strains were identified by a DNA microarray and real-time reverse transcriptase PCR (RT-PCR) approach. Of the many genes functionally overexpressed in the presence of AmB, the ascorbate peroxidase gene from a resistant Leishmania donovani strain (LdAPx gene) was selected because the gene is present only in Leishmania, not in humans. Apoptosis-like cell death after exposure to AmB was investigated in a wild-type (WT) strain in which the LdAPx gene was overexpressed and in AmB-sensitive and -resistant strains. A higher percentage of apoptosis-like cell death after AmB treatment was noticed in the sensitive strain than in both the resistant isolate and the strain sensitive to LdAPx overexpression. This event is preceded by AmB-induced formation of reactive oxygen species and elevation of the cytosolic calcium level. Enhanced cytosolic calcium was found to be responsible for depolarization of the mitochondrial membrane potential and the release of cytochrome c (Cyt c) into the cytosol. The redox behavior of Cyt c showed that it has a role in the regulation of apoptosis-like cell death by activating metacaspase- and caspase-like proteins and causing concomitant nuclear alterations, as determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and DNA fragmentation in the resistant strain. The present study suggests that constitutive overexpression of LdAPx in the L. donovani AmB-resistant strain prevents cells from the deleterious effect of oxidative stress, i.e., mitochondrial dysfunction and cellular death induced by AmB.
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, India.
authors.forename: Ashish
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Microbiology, All India Institute of Medical Sciences, Patna, Bihar, India.
authors.forename: Sushmita
authors.initials: S
authors.lastname: Das
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, India.
authors.forename: Bidyut
authors.initials: B
authors.lastname: Purkait
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, India.
authors.forename: Abul Hasan
authors.initials: AH
authors.lastname: Sardar
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, India.
authors.forename: Ayan Kumar
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: Department of Biomedical Informatics, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, India.
authors.forename: Manas Ranjan
authors.initials: MR
authors.lastname: Dikhit
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, India.
authors.forename: Kumar
authors.initials: K
authors.lastname: Abhishek
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, India drpradeep.das@gmail.com.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology*; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D060387:Ascorbate Peroxidases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D007893:Leishmania donovani / Q000187:drug effects*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D015800:Protozoan Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000981:Antiprotozoal Agents; D015800:Protozoan Proteins; D000666:Amphotericin B; D045304:Cytochromes c; D060387:Ascorbate Peroxidases
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