Ascorbate Peroxidase, a Key Molecule Regulating Amphotericin B Resistance in Clinical Isolates of Leishmania donovani

Abstract

c 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 AmBresistant strain prevents cells from the deleterious effect of oxidative stress, i.e., mitochondrial dysfunction and cellular death induced by AmB.

Phosphorylation of Translation Initiation Factor 2-Alpha in Leishmania donovani under Stress Is Necessary for Parasite Survival

“…ROS accumulation, decreased viability, and a drastic decrease in the infectivity rate upon the inhibition of stress-induced LdeIF2␣ phosphorylation emphasize the importance of this stressresponsive pathway. Moreover, drug-induced LdeIF2␣ phosphorylation indicates its possible involvement in drug unresponsiveness and resistance, as the above-described drugs follow the same mode of action for parasite clearance (42).…”

“…The nonadherent cells were removed by gentle washing with serum-free medium, and the adherent macrophages were incubated overnight in complete medium. To show the effect of GSK specifically on Leishmania PERK and other ways to null and void the effect of GSK on host PERK, promastigotes grown in regular medium for 4 days were pretreated with GSK2606414 for 3 h, washed with sterile PBS to remove the unreacted GSK (42,48), harvested, and resuspended in PBS; the numbers of flagellated and nonflagellated forms were microscopically estimated; and the parasites were incubated with adherent macrophages at a multiplicity of infection of 10 in RPMI-10% FBS for 6 h at 37°C. Cells were washed three times in PBS to remove unattached parasites and again incubated for 24 h at 37°C in a CO 2 incubator.…”

In Vitro Evaluation of Antileishmanial Activity of Computationally Screened Compounds against Ascorbate Peroxidase To Combat Amphotericin B Drug Resistance

“…Comparative study with Prx. APx is well known to be absent in humans (9,20), but human peroxiredoxin-1 (Prx) (NP_859048) was identified as the closest human peroxidase from BLASTp, sharing 26% query coverage and 33% sequence identity with LdAPx. Primary sequence alignment showed much less sequence conservation between human Prx and LdAPx.…”

“…AmB becomes auto-oxidized inside the cell and produces ROS. It had been reported earlier that ROS was minimized by the overexpression of LdAPx for the survival of the AmB-resistant strain (9). Therefore, we measured the consumption of ROS in AmB-sensitive and -resistant isolates in the presence of AmB (0.125 g/ml) and inhibitors I 1 (14 M), I 2 (42 M), and I 3 (28 M).…”

“…Ascorbate peroxidase (APx [EC 1.11.1.11]) is a unique and attractive therapeutic target of the trypanothione pathway that regulates oxidative stress in the Leishmania parasite, which is devoid of catalase and glutathione (GSH) peroxidases (9). APx is the first hemecontaining class I peroxidase, an integral enzyme of the glutathione-ascorbate cycle that catalyzes the detoxification of hydroperoxide (H 2 O 2 ) through a one-electron transfer mechanism (10) and keeps the parasite free from cell damage.…”