Phagocytosis is required for proliferation and pathogenesis of Entamoeba histolytica and erythrophagocytosis is considered to be a marker of invasive amoebiasis. Ca2+ has been found to play a central role in the process of phagocytosis. However, the molecular mechanisms and the signalling mediated by Ca2+ still remain largely unknown. Here we show that Calmodulin-like calcium binding protein EhCaBP3 of E. histolytica is directly involved in disease pathomechanism by its capacity to participate in cytoskeleton dynamics and scission machinery during erythrophagocytosis. Using imaging techniques EhCaBP3 was found in phagocytic cups and newly formed phagosomes along with actin and myosin IB. In vitro studies confirmed that EhCaBP3 directly binds actin, and affected both its polymerization and bundling activity. Moreover, it also binds myosin 1B in the presence of Ca2+. In cells where EhCaBP3 expression was down regulated by antisense RNA, the level of RBC uptake was reduced, myosin IB was found to be absent at the site of pseudopod cup closure and the time taken for phagocytosis increased, suggesting that EhCaBP3 along with myosin 1B mediate the closure of phagocytic cups. Experiments with EhCaBP3 mutant defective in Ca2+ -binding showed that Ca2+ binding is required for phagosome formation. Liposome binding assay revealed that EhCaBP3 recruitment and enrichment to membrane is independent of any cellular protein as it binds directly to phosphatidylserine. Taken together, our results suggest a novel pathway mediating phagocytosis in E. histolytica, and an unusual mechanism of modulation of cytoskeleton dynamics by two calcium binding proteins, EhCaBP1 and EhCaBP3 with mostly non-overlapping functions.

Entamoeba histolytica is the etiological agent of human amoebic colitis and liver abscess, and causes a high level of morbidity and mortality worldwide, particularly in developing countries. There are a number of studies that have shown a crucial role for Ca2+ and its binding protein in amoebic biology. EhCaBP5 is one of the EF hand calcium-binding proteins of E. histolytica. We have determined the crystal structure of EhCaBP5 at 1.9 Å resolution in the Ca2+-bound state, which shows an unconventional mode of Ca2+ binding involving coordination to a closed yet canonical EF-hand motif. Structurally, EhCaBP5 is more similar to the essential light chain of myosin than to Calmodulin despite its somewhat greater sequence identity with Calmodulin. This structure-based analysis suggests that EhCaBP5 could be a light chain of myosin. Surface plasmon resonance studies confirmed this hypothesis, and in particular showed that EhCaBP5 interacts with the IQ motif of myosin 1B in calcium independent manner. It also appears from modelling of the EhCaBP5-IQ motif complex that EhCaBP5 undergoes a structural change in order to bind the IQ motif of myosin. This specific interaction was further confirmed by the observation that EhCaBP5 and myosin 1B are colocalized in E. histolytica during phagocytic cup formation. Immunoprecipitation of EhCaBP5 from total E. histolytica cellular extract also pulls out myosin 1B and this interaction was confirmed to be Ca2+ independent. Confocal imaging of E. histolytica showed that EhCaBP5 and myosin 1B are part of phagosomes. Overexpression of EhCaBP5 increases slight rate (∼20%) of phagosome formation, while suppression reduces the rate drastically (∼55%). Taken together, these experiments indicate that EhCaBP5 is likely to be the light chain of myosin 1B. Interestingly, EhCaBP5 is not present in the phagosome after its formation suggesting EhCaBP5 may be playing a regulatory role.

Plant growth regulators have an important role in various developmental processes during the life cycle of plants. They are involved in abiotic stress responses and tolerance. They have very well-developed capabilities to sense the changes in their external milieu and initiate an appropriate signaling cascade that leads to the activation of plant defense mechanisms. The plant defense system activation causes build-up of plant defense hormones like jasmonic acid (JA) and antioxidant systems like glutathione (GSH). Moreover, calcium (Ca2+) transients are also seen during abiotic stress conditions depicting the role of Ca2+ in alleviating abiotic stress as well. Therefore, these growth regulators tend to control plant growth under varying abiotic stresses by regulating its oxidative defense and detoxification system. This review highlights the role of Jasmonates, Calcium, and glutathione in abiotic stress tolerance and activation of possible novel interlinked signaling cascade between them. Further, phyto-hormone crosstalk with jasmonates, calcium and glutathione under abiotic stress conditions followed by brief insights on omics approaches is also elucidated.

SummaryThe genome of Entamoeba histolytica encodes several calcium binding proteins and those characterized thus far have been shown to participate predominantly in phagocytosis and endocytosis. Our study showed that EhCaBP6 has two EF-hand domains EFI and EFIII; it can bind Ca 2+ in vitro and undergoes conformational transition on binding Ca 2+ suggesting that it can serve as a calcium signal sensor. EhCaBP6 is localized in the nucleus, cytoplasm and plasma membrane and is sensitive to heat stress. Unlike other Ca 2+ binding proteins that have been studied in E. histolytica, EhCaBP6 is found at microtubule ends and at the intercellular bridge with the microtubules during cytokinesis. Furthermore, increased expression of EhCaBP6 was correlated with a significant increase in the number of microtubular structures suggesting that this protein may regulate chromosome segregation and cytokinesis in E. histolytica.

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To investigate the impact of Glutathione (GSH) in mitigating low-temperature stress in Pusa Sheetal cv. of Solanum lycopersicum and imparting low-temperature tolerance by evaluating the different physiological responses. The plant under research was also being studied for its growth and stress tolerance. Low temperatures (LT) stress was applied to seedlings with or without GSH application 12 h before LT stress (prophylactic dose), after 12 h-LT (preemptive dose), and post 12-h recovery (curative dose). Different concentrations of GSH [0, G1 (0.5 mM), G2 (1 mM) and G3 (2 mM)] against LT stress were used. Antioxidant activities, photosynthesis, growth, and stress tolerance indices were quantified. LT stress caused an oxidative burst in S. lycopersicum seedlings of the Pusa Sheetal cv. as indicated by increased peroxidation of lipids and H2O2 concentration. Glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities were enhanced. The best concentration was G2 (1 mM), which resulted in a rise in antioxidant activity. Moreover, a decline in lipid peroxidation and H2O2 levels was also seen. The purpose of this study is to identify the role of GSH in reducing LT stress and to find the best dose concentration. This is the first report to assess the GSH-mediated LT stress tolerance in S. lycopersicum (Pusa Sheetal cv.). Therefore, exogenous GSH application of optimal concentration of GSH to LT stressed S. lycopersicum can be an effective approach for augmenting the plant detoxification system and promoting its growth and development.

GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) is a bifunctional enzyme which catalyzes the conversion of UDP-GlcNAc to ManNAc and ManNAc to ManNAc 6-phosphate, key steps in the sialic acid biosynthesis. Mutations in GNE lead to a neuromuscular disorder, Hereditary Inclusion Body Myopathy (HIBM). A major limitation in understanding the function of GNE is lack of recombinant full length GNE (rGNE) protein for detailed biophysical and structural characterization. In the present study, we have used Dictyostelium discoideum (Dd) as an alternate host for successful expression and secretion of functionally active form of GNE and its mutant proteins. We have generated Dd-AX3 stable cell lines harboring wtGNE or its mutants with Dd specific secretory signal sequence, PsA (prespore antigen). Upon starvation, rGNE was secreted in the medium from secretory vesicles. The rGNE was functionally active with epimerase activity (54±5.2 mU/mg) and kinase activity (66.45±3.48 mU/mg), while both epimerase and kinase activities of mutant GNE were drastically reduced. These activities were found to be statistically significant at p value < 0.05. Our study clearly demonstrates that Dd can be used as an expression host for the production of recombinant and functionally active form of GNE and its mutant proteins that can be used for biophysical characterization and structural determination of GNE to understand the pathomechanism of HIBM.