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

ABSTRACT: 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 < 0.05). Furthermore, inhibition of LdeIF2? phosphorylation significantly reduced the infection rate (P < 0.05), emphasizing its deciding role in successful invasion and infection establishment. Notably, our findings suggested the phosphorylation of LdeIF2? under H2O2-induced oxidative stress. Inhibition of H2O2-induced LdeIF2? phosphorylation hampered antioxidant balance by impaired redox homeostasis gene expression, resulting in increased reactive oxygen species accumulation (P < 0.05) and finally leading to decreased parasite viability (P < 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.

SUBMITTER: Abhishek K

PROVIDER: S-EPMC5192082 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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

Abhishek Kumar K Sardar Abul Hasan AH Das Sushmita S Kumar Ashish A Ghosh Ayan Kumar AK Singh Ruby R Saini Savita S Mandal Abhishek A Verma Sudha S Kumar Ajay A Purkait Bidyut B Dikhit Manas Ranjan MR Das Pradeep P

Molecular and cellular biology 20161219 1

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 (eIF2α) phosphorylation by stress-specific eIF2α 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 eIF2α (LdeIF2α). In vit ...[more]

PMID: 27736773

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Project description:The importance of autophagy in parasites with a digenetic life cycle like Leishmania spp. is significant. The parasite survives as promastigotes in the insect gut and as immotile amastigotes in mammals. This study demonstrates increased autophagy in Leishmania parasite during progression of in vitro life cycle and upon exposure to stress stimuli like starvation, oxidative stress, and drugs. Autophagy inhibition during stress exposure increased cell death, indicating the importance of autophagy in cellular defense against adverse conditions. Atg8 protein, a homolog of mammalian autophagy protein LC3 is expressed in Leishmania parasite but its function remains unknown. Overexpression of Atg8 (Atg8-OE) rendered the parasites resistant to stress and capable of infecting macrophages in substantial numbers; however, disruption of the Atg8 gene (?Atg8) resulting in suppression of Atg8 protein expression, increased susceptibility to stress and reduced the capability to cause infection. A critical event in the Leishmania parasite lifecycle is the differentiation of promastigote forms to the disease causing amastigote forms. The failure of ?Atg8 parasites lacking Atg8 protein to differentiate into amastigotes, unlike the Atg8-OE and vector-transfected parasites, clearly indicated Atg8 involvement in a crucial event. The inability of ?Atg8 parasites to infect macrophages in vitro was verified in an in vivo mouse model of leishmaniases where infection could not be induced by the ?Atg8 parasites. Autophagy is known to be involved in the remodeling of damaged organelles. The accumulation of Atg8 around damaged mitochondria suggested increase of autophagy in the vicinity of the organelle. This buildup was prevented when mitochondria generated reactive oxygen species that were quenched, suggesting them as possible signaling molecules for sensing mitochondrial instability. In summary, our study provides new evidences for a crucial role of Atg8 protein in sustaining Leishmania parasite survival during life cycle and stress exposure, differentiation to amastigotes, and their infective abilities.

Project description:BackgroundGene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.Methodology/principal findingsTwo parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.Conclusion/significanceOur study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

Dataset Information

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

Publications

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

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