Proteome changes associated with Leishmania donovani promastigote adaptation to oxidative and nitrosative stresses

Sardar, Abul Hasan; Kumar, Sudeep; Kumar, Ashish; Purkait, Bidyut; Das, Sushmita; Sen, Abhik; Kumar, Manish; Sinha, Kislay Kumar; Singh, Dharmendra; Equbal, Asif; Ali, Vahab; Das, Pradeep

doi:10.1016/j.jprot.2013.01.011

Cited by 54 publications

(54 citation statements)

References 62 publications

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“…We analyzed intracellular ROS generation in promastigotes using 2=,7=-dichlorodihydrofluorescein diacetate (H 2 DCFDA) dye. The ROS level for L. donovani promastigotes exposed to the higher dose of 70 M SAG (SAG 70 ) was found to be ϳ1.2-fold higher (P Ͻ 0.05) than that for promastigotes exposed to the lower dose (SAG 40 ). Similarly, for amphotericin B treatment, promastigotes subjected to the higher dose of Amp B (Amp B 0.06 ) showed ϳ1.3-fold-higher (P Ͻ 0.05) ROS accumulation than did promastigotes subjected to the lower dose of Amp B (Amp B 0.03 ).…”

Section: Resultsmentioning

confidence: 93%

“…As results from previous experiments showed that drug-generated ROS are also capable of inducing LdeIF2␣ phosphorylation, we estimated parasite viability under drug pressure in the presence of GSK2606414. The percent viabilities of promastigotes after 48 h of exposure to SAG 40 or SAG 70 were 71% and 59%, respectively, whereas in the presence of GSK, viabilities were 62% and 46%, respectively. Similarly, with exposure to amphotericin B, viz., Amp B 0.03 or Amp B 0.06 , the percent viabilities were 76% and 52%, respectively, which were further decreased significantly in the presence of GSK2606414 to 61% for treatment with Amp B 0.03 plus GSK and to 39% for treatment with Amp B 0.06 plus GSK (Fig.…”

Section: Resultsmentioning

confidence: 94%

“…(E) Percent cell viability of L. donovani promastigotes subjected to oxidative stress generated due to H 2 O 2 treatment or in the presence of GSK2606414 and under H 2 O 2 stress along with GSK2606414 after 4 h of treatment. (F) Percent viability at 48 h of promastigotes exposed to SAG 40 …”

Section: Resultsmentioning

confidence: 99%

“…Overall, we conclude that stress-induced phosphorylation of LdeIF2␣ is a crucial event in the life cycle of L. donovani through (i) transformation of the motile promastigote form into the nonmotile amastigote form; (ii) decreased global translation and upregulation of stress-responsive genes during transformation or in the amastigote form, which indicates the adaptive response of parasites to conserve energy and divert it toward the minimum essential pathways required for survival, exemplified by thiol redox metabolism (27,40); and (iii) use of the pentose phosphate pathway (41) for coping with oxidative stress and energy generation (37), respectively, while converting the more active form into the low-metabolic amastigote form. 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.…”

Section: Donovani Promastigotes (Uns) (B) or With Promastigotes Preinmentioning

confidence: 89%

“…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). The treatment time for deducing LD 50 s was kept well below 48 h to ensure that parasite viability is not significantly affected upon treatment.…”

Section: Methodsmentioning

confidence: 99%

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

Abhishek

Sardar

Das

et al. 2017

Molecular and Cellular Biology

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The transformation of Leishmania donovani from a promastigote to an amastigote during mammalian host infection displays the immense adaptability of the parasite to survival under stress. Induction of translation initiation factor 2-alpha (eIF2␣) phosphorylation by stress-specific eIF2␣ kinases is the basic stress-perceiving signal in eukaryotes to counter stress. Here, we demonstrate that elevated temperature and acidic pH induce the phosphorylation of Leishmania donovani eIF2␣ (LdeIF2␣). In vitro inhibition experiments suggest that interference of LdeIF2␣ phosphorylation under conditions of elevated temperature and acidic pH debilitates parasite differentiation and reduces parasite viability (P Ͻ 0.05). Furthermore, inhibition of LdeIF2␣ phosphorylation significantly reduced the infection rate (P Ͻ 0.05), emphasizing its deciding role in successful invasion and infection establishment. Notably, our findings suggested the phosphorylation of LdeIF2␣ under H 2 O 2 -induced oxidative stress. Inhibition of H 2 O 2 -induced LdeIF2␣ phosphorylation hampered antioxidant balance by impaired redox homeostasis gene expression, resulting in increased reactive oxygen species accumulation (P Ͻ 0.05) and finally leading to decreased parasite viability (P Ͻ 0.05). Interestingly, exposure to sodium antimony glucamate and amphotericin B induces LdeIF2␣ phosphorylation, indicating its possible contribution to protection against antileishmanial drugs in common use. Overall, the results strongly suggest that stress-induced LdeIF2␣ phosphorylation is a necessary event for the parasite life cycle under stressed conditions for survival.

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Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Donovani Promastigotes (Uns) (B) or With Promastigotes Preinmentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

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

Abhishek

Sardar

Das

et al. 2017

Molecular and Cellular Biology

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Oxidant activated soluble adenylate cyclase of Leishmania donovani regulates the cAMP–PKA signaling axis for its intra‐macrophage survival during infection

Kumar

Das²,

Sen

et al. 2021

J of Cellular Biochemistry

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Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulates antioxidant gene expression and phosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by~1.54 ± 0.35, and~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclize ATP to enhance the cAMP basal level that regulates antioxidant gene expression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression was upregulated by~5.7 ± 0.35,~2.57 ± 0.56,~4.7 ± 0.36, and 2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and 25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KD was found less Biotechnology, Ministry of Science and Technology, Grant/Award Number: DBT/ JRF/16/AL/986 infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.

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Proteomic Analysis of Posttranslational Modifications Using iTRAQ in Leishmania

Zilberstein¹

2014

Methods in Molecular Biology

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iTRAQ is a high coverage quantitative proteomics technique identifies and quantitates abundance changes of multiple (up to eight) distinct protein samples. To date, one iTRAQ-MS/MS assay can identify up to quarter of cells proteome. Each of the eight tags covalently binds to the N-terminus as well as arginine and lysine side chains of peptides, enabling labeling of the entire peptide population in each sample. Following tagging, the various protein samples are mixed and subjected to LC-MS/MS analysis. In the first round identical peptides from the different protein populations focus in a single pick. Subsequently, sequence of each peptide is determined. The tags whose m/z is similar to that of natural amino acids are used to determine relative abundance. To date, iTRAQ enabled identification of almost 2,000 Leishmania proteins. Here, we provide protocols for protein abundance changes and for phosphoproteomics analysis in Leishmania parasites.

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Proteome changes associated with Leishmania donovani promastigote adaptation to oxidative and nitrosative stresses

Cited by 54 publications

References 62 publications

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

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

Oxidant activated soluble adenylate cyclase of Leishmania donovani regulates the cAMP–PKA signaling axis for its intra‐macrophage survival during infection

Proteomic Analysis of Posttranslational Modifications Using iTRAQ in Leishmania

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