Metabolic reconfiguration of the central glucose metabolism: a crucial strategy ofLeishmania donovanifor its survival during oxidative stress

Abstract

Understanding the mechanism that allows the intracellular protozoan parasite Leishmania donovani (Ld) to respond to reactive oxygen species (ROS) is of increasing therapeutic importance because of the continuing resistance toward antileishmanial drugs and for determining the illusive survival strategy of these parasites. A shift in primary carbon metabolism is the fastest response to oxidative stress. A 14 CO 2 evolution study, expression of glucose transporters together with consumption assays, indicated a shift in metabolic flux of the parasites from glycolysis toward pentose phosphate pathway (PPP) when exposed to different oxidants in vitro/ex vivo. Changes in gene expression, protein levels, and enzyme activities all pointed to a metabolic reconfiguration of the central glucose metabolism in response to oxidants. Generation of glucose-6-phosphate dehydrogenase (G6PDH) (∼5-fold) and transaldolase (TAL) (∼4.2-fold) overexpressing Ld cells reaffirmed that lethal doses of ROS were counterbalanced by effective manipulation of NADPH:NADP + ratio and stringent maintenance of reduced thiol content. The extent of protein carbonylation and accumulation of lipid peroxidized products were also found to be less in overexpressed cell lines. Interestingly, the LD 50 of sodium antimony gluconate (SAG), amphotericin-B (AmB), and miltefosine were significantly high toward overexpressing parasites. Consequently, this study illustrates that Ld strategizes a metabolic reconfiguration for replenishment of NADPH pool to encounter oxidative

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

“…7C). This study is incongruent with the study of Ghosh et al, who observed significant depletion of GSH upon treatment with oxidants (29).…”

“…Thereafter, the cells were centrifuged at 10,000 ϫ g for 15 min at 4°C, and supernatant was collected to measure the fluorescence intensity using a spectrofluorometer (LS55; PerkinElmer) with excitation/ emission (ex/em) ϭ 503/529 nm in relative fluorescence units (RFU). The reagent blank was prepared with 0.4 mM H 2 DCFDA in lysis buffer as published earlier (29).…”

“…The parasites (2 ϫ 10 6 cells) treated with inhibitors for 4 h were subsequently washed with PBS (pH 7.2), and afterward AmB (0.125 g/ml) was added. After 24 h, these cells were pelleted and lysed, cell lysate was incubated with monochlorobimane dye (2 mM) for 1 h at 37°C, and the GSH level was monitored at ex/em ϭ 395/480 nm with a fluorescence plate reader as reported earlier (29).…”

Effects of nitro-heterocyclic derivatives against Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes

“…One hypothesis is that the compounds of the BSF group could act as a NO donor, in a cellular stress situation, as with cells infected with L. L. infantum in our experiment. The mechanism NO exerts on cytotoxicity activity is not well understood, but it may be by inhibition of mitochondrial respiration, glycolysis, peroxidation of membrane lipids, inactivation of peroxidases and arginases (Fonseca-Silva et al, 2015;Ghosh et al, 2015;da Silva et al, 2015;Giudice et al,, 2007;Grandoni and Ascendi, 2004;Vickers et al, 2004).…”

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

“…For the generation of axenic amastigotes, late-stationary-phase promastigotes were inoculated in M199 medium supplemented with 20% serum in 25-cm 2 ventilated flasks and grown at 37°C at pH 5.5 with 5% CO 2 for an average of 5 days for promastigote-to-amastigote differentiation in a cell-free culture; maintenance of axenic amastigotes was carried out as described previously (43). For in vitro generation of oxidative stress, H 2 O 2 (200 M) (Merck GmbH, Darmstadt, Germany) was added to the promastigote culture, suspended in fresh medium, and incubated at 25°C inside a basic oxygen demand (BOD) incubator (41). Due to the short half-life of H 2 O 2 , the treatment time was limited to 4 h. SAG and Amp B treatments were given for 8 h at two different doses, viz., 40 M (SAG 40 ) and 70 M (SAG 70 ) for SAG and 0.060 M (Amp B 0.06 ) and 0.030 M (Amp B 0.03 ) for amphotericin B, based on the 50% lethal doses (LD 50 s) of the drugs, as described previously (44).…”