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The target of rapamycin (TOR) is a central hub kinase that promotes growth and development in all eukaryote cells. TOR induces protein synthesis through the phosphorylation of the S6 kinase (S6K), which, in turn, phosphorylates ribosomal S6 protein (RPS6) increasing this anabolic process. Therefore, S6K and RPS6 phosphorylation are generally used as readouts of TOR activity. Protein phosphorylation levels are measured by a western blot (WB) technique using an antibody against one specific phosphosite in cell extracts. However, at the tissue/cell-specific level, there is a huge gap in plants due to the lack of alternative techniques for the evaluation of TOR activity as there are for other organisms such as mammals. Here, we describe an in vivo protocol to detect S6K phosphorylation in tissues/cells of model photosynthetic organisms such as Arabidopsis thaliana and Chlamydomonas reinhardtii . Our proposed method consists of the immunolocalization of a phosphorylated target of TOR kinase using a fluorescent secondary antibody by confocal microscopy. The protocol involves four main steps: tissue/cell fixation, permeabilization, and incubation with primary and secondary antibodies. It is an easy technique that allows handling different samples at the same time. In addition, different ultrastructural cell markers can also be used, such as for nucleus and cell wall detection, allowing a detailed analysis of cell morphology. To our knowledge, this is the first protocol to detect TOR activity in situ in photosynthetic organisms; we consider that it will pave the research on the TOR kinase, opening new possibilities to better understand its complex signaling. Key features • The protocol is an easy and non-destructive method to detect S6K phosphorylation at the cellular level for plants and algae. • First method for in situ immunolocalization of target proteins of TOR kinase in photosynthetic organisms., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Metformin, a safe biguanide derivative with antiproliferative properties, has shown antiparasitic efficacy against the Echinococcus larval stage. Hence, we assessed the efficacy of a dose of 250 mg kg -1 day -1 in experimental models of advanced CE, at 6 and 12 months post-infection with oral and intraperitoneal administration, respectively. At this high dose, metformin reached intracystic concentrations between 0.7 and 1.7 mM and triggered Eg-TOR inhibition through AMPK activation by AMP-independent and -dependent mechanisms, which are dependent on drug dose. Cystic metformin uptake was controlled by increased expression of organic cation transporters in the presence of the drug. In both experimental models, metformin reduced the weight of parasite cysts, altered the ultrastructural integrity of their germinal layers, and reduced the intracystic availability of glucose, limiting the cellular carbon and energy charge and the proliferative capacity of metacestodes. This glucose depletion in the parasite was associated with a slight increase in cystic uptake of 2-deoxiglucose and the transcriptional induction of GLUT genes in metacestodes. In this context, drastic glycogen consumption led to increased lactate production and altered intermediary metabolism in treated metacestodes. Specifically, the fraction of reducing soluble sugars decreased twofold, and the levels of non-reducing soluble sugars, such as sucrose and trehalose, were modified in both cystic fluid and germinal cells. Taken together, our findings highlight the relevance of metformin as a promising candidate for CE treatment and warrant further research to improve the therapeutic conditions of this chronic zoonosis in humans., Competing Interests: The authors declare no conflict of interest.

In an attempt to find new anti-echinococcal drugs, resveratrol (Rsv) effectiveness against the larval stages of Echinococcus granulosus and E. multilocularis was evaluated. The in vitro effect of Rsv on parasites was assessed via optical and electron microscopy, RT-qPCR and immunohistochemistry. In vivo efficacy was evaluated in murine models of cystic (CE) and alveolar echinococcosis (AE). The impact of infection and drug treatment on the mouse bone marrow hematopoietic stem cell (HSC) population and its differentiation into dendritic cells (BMDCs) was investigated via flow cytometry and RT-qPCR. In vitro treatment with Rsv reduced E. granulosus metacestode and protoscolex viability in a concentration-dependent manner, caused ultrastructural damage, increased autophagy gene transcription, and raised Eg-Atg8 expression while suppressing Eg-TOR. However, the intraperitoneal administration of Rsv was not only ineffective, but also promoted parasite development in mice with CE and AE. In the early infection model of AE treated with Rsv, an expansion of HSCs was observed followed by their differentiation towards BMCDs. The latter showed an anti-inflammatory phenotype and reduced LPS-stimulated activation compared to control BMDCs. We suggest that Rsv ineffectiveness could have been caused by the low intracystic concentration achieved in vivo and the drug's hormetic effect, with opposite anti-parasitic and immunomodulatory responses in different doses.

Background: The Echinococcus granulosus sensu lato species complex causes cystic echinococcosis, a zoonotic disease of medical importance. Parasite-derived small extracellular vesicles (sEVs) are involved in the interaction with hosts intervening in signal transduction related to parasite proliferation and disease pathogenesis. Although the characteristics of sEVs from E. granulosus protoscoleces and their interaction with host dendritic cells (DCs) have been described, the effect of sEVs recovered during parasite pharmacological treatment on the immune response remains unexplored., Methods: Here, we isolated and characterized sEVs from control and drug-treated protoscoleces by ultracentrifugation, transmission electron microscopy, dynamic light scattering, and proteomic analysis. In addition, we evaluated the cytokine response profile induced in murine bone marrow-derived dendritic cells (BMDCs) by qPCR., Results: The isolated sEVs, with conventional size between 50 and 200 nm, regardless of drug treatment, showed more than 500 cargo proteins and, importantly, 20 known antigens and 70 potential antigenic proteins, and several integral-transmembrane and soluble proteins mainly associated with signal transduction, immunomodulation, scaffolding factors, extracellular matrix-anchoring, and lipid transport. The identity and abundance of proteins in the sEV-cargo from metformin- and albendazole sulfoxide (ABZSO)-treated parasites were determined by proteomic analysis, detecting 107 and eight exclusive proteins, respectively, which include proteins related to the mechanisms of drug action. We also determined that the interaction of murine BMDCs with sEVs derived from control parasites and those treated with ABZSO and metformin increased the expression of pro-inflammatory cytokines such as IL-12 compared to control cells. Additionally, protoscolex-derived vesicles from metformin treatments induced the production of IL-6, TNF-α, and IL-10. However, the expression of IL-23 and TGF-β was downregulated., Conclusions: We demonstrated that sEV-cargo derived from drug-treated E. granulosus protoscoleces have immunomodulatory functions, as they enhance DC activation towards a type 1 pro-inflammatory profile against the parasite, and therefore support the proposal of a new approach for the prevention and treatment of secondary echinococcosis., (© 2023. The Author(s).)

Alveolar echinococcosis (AE) is a severe disease caused by Echinococcus multilocularis. Its chemotherapeutic treatment is based on benzimidazoles, which are rarely curative and cause several adverse effects. Therefore, it is necessary to develop alternative and safer chemotherapeutic strategies against AE. It has previously been shown that metformin (Met) exhibits considerable in vivo activity on an early-infection model of AE when administered at 50 mg kg−1 day−1 for 8 weeks. Here, the challenge is heightened by a 2-fold increase in parasite inoculum or by starting the treatment 6 weeks post-infection. In both cases, only the combination of Met (100 mg kg−1 day−1) together with a sub-optimal dose of albendazole (ABZ) (5 mg kg−1 day−1) led to a significant reduction in parasite weight compared to the untreated group. Coincidentally, drug combination showed the highest level of damage in E. multilocularis metacestodes. Likewise, Met alone or combined with ABZ led to a decrease in parasite glucose availability, which was evidenced as a lower intracystic glucose concentration. Therefore, the results demonstrate that combination therapy with Met and ABZ offers an alternative to improve the efficacy and reduce the toxicity of the high-dose ABZ monotherapy currently employed.

Immune evasion is a hallmark of persistent echinococcal infection, comprising modulation of innate immune cells and antigen-specific T cell responses. However, recognition of Echinococcus granulosus by dendritic cells (DCs) is a key determinant of the host's response to this parasite. Given that mTOR signaling pathway has been described as a regulator linking metabolism and immune function in DCs, we reported for the first time in these cells, global translation levels, antigen uptake, phenotype, cytokine transcriptional levels, and splenocyte priming activity upon recognition of the hydatid fluid (HF) and the highly glycosylated laminar layer (LL). We found that LL induced a slight up-regulation of CD86 and MHC II in DCs and also stimulated the production of IL-6 and TNF-α. By contrast, HF did not increase the expression of any co-stimulatory molecules, but also down-modulated CD40 and stimulated the expression of the anti-inflammatory cytokine IL-10. Both parasitic antigens promoted protein synthesis through mTOR activation. The use of rapamycin decreased the expression of the cytokines tested, empowered the down-modulation of CD40 and also reduced splenocyte proliferation. Finally, we showed that E. granulosus antigens increase the amounts of LC3-positive structures in DCs which play critical roles in the presentation of these antigens to T cells., (© 2021. The Author(s).)

Alveolar echinococcosis (AE) is a severe disease caused by the larval stage of the tapeworm Echinococcus multilocularis Current chemotherapeutic treatment options based on benzimidazoles are of limited effectiveness, which underlines the need to find new antiechinococcosis drugs. Metformin is an antihyperglycemic and antiproliferative agent that shows activity against the related parasite Echinococcus granulosus Hence, we assessed the in vitro and in vivo effects of the drug on E. multilocularis Metformin exerted significant dose-dependent killing effects on in vitro cultured parasite stem cells and protoscoleces and significantly reduced the dedifferentiation of protoscoleces into metacestodes. Likewise, oral administration of metformin (50 mg/kg of body weight/day for 8 weeks) was effective in achieving a significant reduction of parasite weight in a secondary murine AE model. Our results revealed mitochondrial membrane depolarization, activation of Em-AMPK, suppression of Em-TOR, and overexpression of Em-Atg8 in the germinal layer of metformin-treated metacestode vesicles. The opposite effects on the level of active Em-TOR in response to exogenous insulin and rapamycin suggest that Em-TOR is part of the parasite's insulin signaling pathway. Finally, the presence of the key lysosomal pathway components, through which metformin reportedly acts, was confirmed in the parasite by in silico assays. Taken together, these results introduce metformin as a promising candidate for AE treatment. Although our study highlights the importance of those direct mechanisms by which metformin reduces parasite viability, it does not necessarily preclude any additional systemic effects of the drug that might reduce parasite growth in vivo ., (Copyright © 2020 American Society for Microbiology.)

Cystic echinococcosis (CE) is a worldwide zoonosis caused by the Echinococcus granulosus larval stage. The currently available therapy for this disease is based on benzimidazoles, which are rarely curative and cause several adverse effects. Therefore, new treatment options are needed. Octreotide (Oct) is a somatostatin analogue which exhibits anti-proliferative and anti-secretory effects over several cancer cell lines expressing somatostatin receptors. Here, we assessed the in vitro pharmacological effect of Oct against the E. granulosus larval stage. The drug caused a significant dose-dependent decrease in the viability of both protoscoleces and metacestodes. SEM and TEM analysis showed ultrastructural damage in both larval forms under drug treatment. Based on this, we investigated the possible presence of an Oct binding receptor in the parasite. The putative somatostatin/allatostatin-like receptor (Eg-s/ast) conserves the characteristic topology and signature sequences of the prototype somatostatin receptor common to vertebrates and is expressed in both metacestodes and protoscoleces. Moreover, Oct treated-parasites showed the presence of autophagic structures and a significant increase in transcriptional expression of autophagy key genes such as Eg-atg6, Eg-atg8, Eg-atg12 and Eg-atg16. In addition, by in toto immunolocalization assays, an increase in the punctate pattern and Eg-Atg8 protein expression was detected in Oct-treated metacestodes. Subsequently, the combination of Oct and Met had an additive effect on the viability of both larval forms. Our results provide additional evidence for the participation of PI3K/AKT/TOR/autophagy pathway in the Echinococcus survival and suggest the concomitant use of these drugs as potential therapeutic agents in treating of CE., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The secretion of extracellular vesicles (EVs) in helminth parasites is a constitutive mechanism that promotes survival by improving their colonization and adaptation in the host tissue. In the present study, we analyzed the production of EVs from supernatants of cultures of Echinococcus granulosus protoscoleces and metacestodes and their interaction with dendritic cells, which have the ability to efficiently uptake and process microbial antigens, activating T lymphocytes. To experimentally increase the release of EVs, we used loperamide, a calcium channel blocker that increases the cytosolic calcium level in protoscoleces and EV secretion. An exosome-like enriched EV fraction isolated from the parasite culture medium was characterized by dynamic light scattering, transmission electron microscopy, proteomic analysis and immunoblot. This allowed identifying many proteins including: small EV markers such as TSG101, SDCBP, ALIX, tetraspanins and 14-3-3 proteins; proteins involved in vesicle-related transport; orthologs of mammalian proteins involved in the immune response, such as basigin, Bp29 and maspardin; and parasite antigens such as antigen 5, P29 and endophilin-1, which are of special interest due to their role in the parasite-host relationship. Finally, studies on the EVs-host cell interaction demonstrated that E. granulosus exosome-like vesicles were internalized by murine dendritic cells, inducing their maturation with increase of CD86 and with a slight down-regulation in the expression of MHCII molecules. These data suggest that E. granulosus EVs could interfere with the antigen presentation pathway of murine dendritic cells inducing immunoregulation in the host. Further studies are needed to better understand the role of these vesicles in parasite survival and as diagnostic markers and new vaccines., Competing Interests: The authors have declared that no competing interests exist.

Cystic echinococcosis is a neglected parasitic disease caused by the larval stage of Echinococcus granulosus for which an effective treatment is not yet available. Since autophagy constitutes a homeostatic mechanism during stress, either inhibition or activation of its activity might be detrimental for survival of the parasite. Amongst the critical molecules that regulate autophagy, TOR, AMPK and sirtuins are the best characterized ones. Previously, we have identified the autophagic machinery, the occurrence of TORC1-controlled events, and the correlation between autophagy and the activation of the unfolded protein response in E. granulosus larval stage. In addition, we have demonstrated that the parasite is susceptible to metformin (Met), a drug that indirectly activates Eg-AMPK and induces energy stress. In this work, we demonstrate that Met induces autophagy in the E. granulosus larval stage. Electron microscopy analysis revealed the presence of autophagic structures in Met-treated protoscoleces. In accordance with these findings, the autophagic marker Eg-Atg8 as well as the transcriptional expression of Eg-atg6, Eg-atg8, Eg-atg12 and Eg-atg16 genes were significantly up-regulated in Met-treated parasites. The induction of the autophagic process was concomitant with Eg-foxO over-expression and its nuclear localization, which could be correlated with the transcriptional regulation of this pathway. On the other hand, the expression of Eg-AKT and Eg-Sirts suggests a possible participation of these conserved proteins in the regulation of Eg-FoxO. Therefore, through pharmacological activation of the AMPK-FoxO signaling pathway, Met could play a role in the death of the parasite contributing to the demonstrated anti-echinococcal effects of this drug. The understanding of the regulatory mechanisms of this pathway in E. granulosus represents a solid basis for choosing appropriate targets for new chemotherapeutic agents., (Copyright © 2018. Published by Elsevier B.V.)

Cystic echinococcosis (CE) is a worldwide parasitic zoonosis caused by the larval stage of Echinococcus granulosus. Current chemotherapy against this disease is based on the administration of benzimidazoles (BZMs). However, BZM treatment has a low cure rate and causes several side effects. Therefore, new treatment options are needed. The antidiabetic drug glibenclamide (Glb) is a second-generation sulfonylurea receptor inhibitor that has been shown to be active against protozoan parasites. Hence, we assessed the in vitro and in vivo pharmacological effects of Glb against the larval stage of E. granulosus. The in vitro activity was concentration dependent on both protoscoleces and metacestodes. Moreover, Glb combined with the minimum effective concentration of albendazole sulfoxide (ABZSO) was demonstrated to have a greater effect on metacestodes in comparison with each drug alone. Likewise, there was a reduction in the cyst weight after oral administration of Glb to infected mice (5 mg/kg of body weight administered daily for a period of 8 weeks). However, in contrast to in vitro assays, no differences in effectiveness were found between Glb + albendazole (ABZ) combined treatment and Glb monotherapy. Our results also revealed mitochondrial membrane depolarization and an increase in intracellular Ca2+ levels in Glb-treated protoscoleces. In addition, the intracystic drug accumulation and our bioinformatic analysis using the available E. granulosus genome suggest the presence of genes encoding sulfonylurea transporters in the parasite. Our data clearly demonstrated an anti-echinococcal effect of Glb on E. granulosus larval stage. Further studies are needed in order to thoroughly investigate the mechanism involved in the therapeutic response of the parasite to this sulfonylurea.

Cystic echinococcosis (CE) is a worldwide distributed helminthic zoonosis caused by Echinococcus granulosus. Benzimidazole derivatives are currently the only drugs for chemotherapeutic treatment of CE. However, their low efficacy and the adverse effects encourage the search for new therapeutic targets. We evaluated the in vitro efficacy of Bortezomib (Bz), a proteasome inhibitor, in the larval stage of the parasite. After 96 h, Bz showed potent deleterious effects at a concentration of 5 μM and 0.5 μM in protoscoleces and metacestodes, respectively (P < 0.05). After 48 h of exposure to this drug, it was triggered a mRNA overexpression of chaperones (Eg-grp78 and Eg-calnexin) and of Eg-ire2/Eg-xbp1 (the conserved UPR pathway branch) in protoscoleces. No changes were detected in the transcriptional expression of chaperones in Bz-treated metacestodes, thus allowing ER stress to be evident and viability to highly decrease in comparison with protoscoleces. We also found that Bz treatment activated the autophagic process in both larval forms. These facts were evidenced by the increase in the amount of transcripts of the autophagy related genes (Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16) together with the increase in Eg-Atg8-II detected by western blot and by in toto immunofluorescence labeling. It was further confirmed by direct observation of autophagic structures by electronic microscopy. Finally, in order to determine the impact of autophagy induction on Echinococcus cell viability, we evaluated the efficacy of Bz in combination with rapamycin and a synergistic cytotoxic effect on protoscolex viability was observed when both drugs were used together. In conclusion, our findings demonstrated that Bz induced endoplasmic reticulum stress, autophagy and subsequent death allowing to identify unstudied parasite-host pathways that could provide a new insight for control of parasitic diseases.

Metformin (Met) is an anti-hyperglycemic and potential anti-cancer agent which may exert its anti-proliferative effects via the induction of energetic stress. In this study we investigated the in vitro and in vivo efficacy of Met against the larval stage of Echinococcus granulosus. Metformin showed significant dose- and time-dependent killing effects on in vitro cultured protoscoleces and metacestodes. Notably, the combination of Met together with the minimum effective concentration of ABZSO had a synergistic effect after days 3 and 12 on metacestodes and protoscoleces, respectively. Oral administration of Met (50 mg/kg/day) in E. granulosus-infected mice was highly effective in reducing the weight and number of parasite cysts, yet its combination with the lowest recommended dose of ABZ (5 mg/kg/day) was even more effective. Coincidentally, intracystic Met accumulation was higher in animals treated with both drugs compared to those administered Met alone. Furthermore, the safe plant-derived drug Met exhibited remarkable chemopreventive properties against secondary hydatidosis in mice. In conclusion, based on our experimental data, Met emerges as a promising anti-echinococcal drug as it has proven to efficiently inhibit the development and growth of the E. granulosus larval stage and its combination with ABZ may improve the current anti-parasitic therapy.

Calcineurin (CaN) is a Ca(2+)-calmodulin activated serine-threonine protein phosphatase that couples the local or global calcium signals, thus controlling important cellular functions in physiological and developmental processes. The aim of this study was to characterize CaN in Echinococcus granulosus (Eg-CaN), a human cestode parasite of clinical importance, both functionally and molecularly. We found that the catalytic subunit isoforms have predicted sequences of 613 and 557 amino acids and are substantially similar to those of the human counterpart, except for the C-terminal end. We also found that the regulatory subunit consists of 169 amino acids which are 87% identical to the human ortholog. We cloned a cDNA encoding for one of the two catalytic subunit isoforms of CaN (Eg-can-A1) as well as the only copy of the Eg-can-B gene, both constitutively transcribed in all Echinococcus larval stages and responsible for generating a functionally active heterodimer. Eg-CaN native enzyme has phosphatase activity, which is enhanced by Ca(2+)/Ni(2+) and reduced by cyclosporine A and Ca(2+) chelators. Participation of Eg-CaN in exocytosis was demonstrated using the FM4-64 probe and Eg-CaN-A was immunolocalized in the cytoplasm of tegumental cells, suckers and excretory bladder of protoscoleces. We also showed that the Eg-can-B transcripts were down-regulated in response to low Ca(2+) intracellular level, in agreement with decreased enzyme activity. Confocal microscopy revealed a striking pattern of Eg-CaN-A in discrete fluorescent spots in the protoscolex posterior bladder and vesicularized protoscoleces beginning the vesicular differentiation. In contrast, Eg-CaN-A was undetectable during the pre-microcyst closing stage while a high DDX-like RNA helicase expression was evidenced. Finally, we identified and analyzed the expression of CaN-related endogenous regulators., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Metformin (Met) is a biguanide anti-hyperglycemic agent, which also exerts antiproliferative effects on cancer cells. This drug inhibits the complex I of the mitochondrial electron transport chain inducing a fall in the cell energy charge and leading 5'-AMP-activated protein kinase (AMPK) activation. AMPK is a highly conserved heterotrimeric complex that coordinates metabolic and growth pathways in order to maintain energy homeostasis and cell survival, mainly under nutritional stress conditions, in a Liver Kinase B1 (LKB1)-dependent manner. This work describes for the first time, the in vitro anti-echinococcal effect of Met on Echinococcus granulosus larval stages, as well as the molecular characterization of AMPK (Eg-AMPK) in this parasite of clinical importance. The drug exerted a dose-dependent effect on the viability of both larval stages. Based on this, we proceeded with the identification of the genes encoding for the different subunits of Eg-AMPK. We cloned one gene coding for the catalytic subunit (Eg-ampkɑ) and two genes coding for the regulatory subunits (Eg-ampkβ and Eg-ampkγ), all of them constitutively transcribed in E. granulosus protoscoleces and metacestodes. Their deduced amino acid sequences show all the conserved functional domains, including key amino acids involved in catalytic activity and protein-protein interactions. In protoscoleces, the drug induced the activation of AMPK (Eg-AMPKɑ-P176), possibly as a consequence of cellular energy charge depletion evidenced by assays with the fluorescent indicator JC-1. Met also led to carbohydrate starvation, it increased glucogenolysis and homolactic fermentation, and decreased transcription of intermediary metabolism genes. By in toto immunolocalization assays, we detected Eg-AMPKɑ-P176 expression, both in the nucleus and the cytoplasm of cells as in the larval tegument, the posterior bladder and the calcareous corpuscles of control and Met-treated protoscoleces. Interestingly, expression of Eg-AMPKɑ was observed in the developmental structures during the de-differentiation process from protoscoleces to microcysts. Therefore, the Eg-AMPK expression during the asexual development of E. granulosus, as well as the in vitro synergic therapeutic effects observed in presence of Met plus albendazole sulfoxide (ABZSO), suggest the importance of carrying out chemoprophylactic and clinical efficacy studies combining Met with conventional anti-echinococcal agents to test the potential use of this drug in hydatidosis therapy.

Cystic echinococcosis is a zoonotic infection caused by the larval stage of the cestode Echinococcus granulosus. Chemotherapy currently employs benzimidazoles; however, 40% of cases do not respond favorably. With regard to these difficulties, novel therapeutic tools are needed to optimize treatment in humans. The aim of this work was to explore the in vitro and in vivo effects of tamoxifen (TAM) against E. granulosus. In addition, possible mechanisms for the susceptibility of TAM are discussed in relation to calcium homeostasis, P-glycoprotein inhibition, and antagonist effects on a putative steroid receptor. After 24 h of treatment, TAM, at a low micromolar concentration range (10 to 50 μM), inhibited the survival of E. granulosus protoscoleces and metacestodes. Moreover, we demonstrated the chemotherapeutic and chemopreventive pharmacological effects of the drug. At a dose rate of 20 mg/kg of body weight, TAM induced protection against the infection in mice. In the clinical efficacy studies, a reduction in cyst weight was observed after the administration of 20 mg/kg in mice with cysts developed during 3 or 6 months, compared to that of those collected from control mice. Since the collateral effects of high TAM doses have been largely documented in clinical trials, the use of low doses of this drug as a short-term therapy may be a novel alternative approach for human cystic echinococcosis treatment., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Autophagy is a fundamental catabolic pathway conserved from yeast to mammals, but which remains unknown in parasite cestodes. In this work, the pharmacological induction of autophagy was cellularly and molecularly analysed in the larval stages of Echinococcus granulosus. Metacestode sensitivity to rapamycin and TORC1 expression in protoscoleces and metacestodes were shown. Ultrastructural studies showed that treated parasites had an isolation membrane, autophagosomes and autolysosomes, all of which evidenced the autophagic flux. Genes coding for key autophagy-related proteins were also identified in the Echinococcus genome. These genes were involved in autophagosome formation and transcriptional over-expression of Eg-atg5, Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16 and Eg-atg18 was shown in presence of rapamycin or arsenic trioxide. Thus, Echinococcus autophagy could be regulated by non-transcriptional inhibition through TOR and by transcription-dependent up-regulation via FoxO-like transcription factors and/or TFEB proteins. An increase in the punctate pattern and Eg-Atg8 polypeptide level in the tegument, parenchyma cells and excretory system of protoscoleces and in vesicularised parasites was detected after rapamycin treatment. This suggests the occurrence of basal autophagy in the larval stages and during vesicular development. In arsenic-treated protoscoleces, high Eg-Atg8 polypeptide levels within the free cytoplasmic matrix of calcareous corpuscles were observed, thus verifying the occurrence of autophagic events. These experiments also confirmed that the calcareous corpuscles are sites of arsenic trioxide accumulation. The detection of the autophagic machinery in this parasite represents a basic starting point to unravel the role of autophagy under both physiological and stress conditions which will allow identification of new strategies for drug discovery against neglected parasitic diseases caused by cestodes., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

P-glycoprotein (Pgp) is an ATP-dependent transporter involved in the efflux of a wide variety of lipophilic substrates, such as toxins and xenobiotics, out of cells. Pgp expression level is associated with the ineffective therapeutic treatment of cancer cells and microbial pathogens which gives it high clinical importance. Research on these transporters in helminths is limited. This work describes for the first time the Echinococcus granulosus Pgp (Eg-Pgp) expression, in a model cestode parasite and an important human pathogen. Based on calcein efflux assays in the presence of common Pgp modulators, we demonstrated the occurrence of active Eg-Pgp in protoscoleces and metacestodes. Eg-Pgp, which showed a molecular mass of ~130 kDa in western blots, is localized in the suckers and the tegument of control protoscoleces as well as in the subtegument or all parenchymatous cells of protoscoleces treated with Pgp-interfering agents. We also identified five genes encoding Pgp which are constitutively expressed in protoscoleces and metacestodes. We showed that the Eg-pgp1 and Eg-pgp2 transcripts were up-regulated in response to in vitro drug treatment with amiodarone and loperamide, in agreement with the increased polypeptide levels. Finally, in vitro treatment of protoscoleces and metacestodes with trifluoperazine and loperamide was lethal to the parasites. This indicates that both drugs as well as cyclosporine A negatively modulate the E. granulosus Pgp efflux activity, favoring the retention of these drugs in the larval tissue. These events could be associated with the reduction in protoscolex and metacestode viability., (© 2013.)

In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The net synthesis of sucrose (Suc) is catalysed by the sequential action of Suc-phosphate synthase (SPS) and Suc-phosphate phosphatase (SPP). SPS and SPP from Anabaena sp. PCC 7120 (7120-SPS and 7120-SPP) define minimal catalytic units. Bidomainal SPSs, where both units are fused, occur in plants and cyanobacteria, but they display only SPS activity. Using recombinant proteins that have fused 7120-SPS and 7120-SPP, we demonstrated that they are bifunctional chimeras and that the arrangement 7120-SPS/SPP is the most efficient to catalyse the sequential reactions to yield Suc. Moreover, we present the first evidence of a bidomainal SPS present in the cyanobacterium Synechococcus elongatus PCC 7942 with both, SPS and SPP activity., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Cystic echinococcosis is a chronic, complex, and neglected disease. Novel therapeutical tools are needed to optimize human treatment. A number of compounds have been investigated, either using in vitro cultured parasites and/or applying in vivo rodent models. Although some of these compounds showed promising activities in vitro, and to some extent also in the rodent models, they have not been translated into clinical applications. Membrane enzyme activities in culture supernatants of treated protoscoleces with calcium modulator drugs and anthelmintic drugs were measured and provided an indication of compound efficacy. This work describes for the first time the detection of alkaline phosphatase, gamma-glutamyl-transpeptidase and acetylcholinesterase activities in supernatants of in vitro treated Echinococcus granulosus protoscoleces. Marked differences on the enzymatic activities in supernatants from drug treated cultures were detected. We demonstrated that those genes that show the highest degree of conservation when compared to orthologs, are constitutively and highly expressed in protoscoleces and metacestodes. Due to high sensibility and the lack of activity in supernatants of intact protoscoleces, gamma-glutamyl-transpeptidase is proposed as the ideal viability marker during in vitro pharmacological studies against E. granulosus protoscoleces., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

It has been reported that higher plants and cyanobacteria synthesize sucrose (Suc) by a similar sequential action of sucrose-phosphate synthase (SPS) and sucrose-phosphate phosphatase (SPP). In the genome of the marine unicellular cyanobacterium Synechococcus sp. PCC 7002 there is a sequence that was not annotated as a putative SPP encoding gene (sppA), although the sequence was available. In this study, we functionally characterize the sppA gene of that strain and demonstrate that it is cotranscribed with spsA, the SPS encoding gene. This is the first report on the coordination of Suc synthesis gene expression in an oxygenic-photosynthetic organism., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Cell cultures of parasitic helminths are an invaluable tool for investigations of basic biological processes, as well as for development of improved chemotherapeutic agents and molecular interactions between host and parasite. We carried out a simple and efficient methodology to isolate Echinococcus granulosus germinal cells which were maintained for at least 4 months while cultivated in the presence of reducing agents and hormones. Microscopic analysis of the primary cell culture revealed the presence of cells with similar Echinococcus germinal cell morphology and behaviour. Population doubling time was estimated at 48 h, showing a rapid division rate. To discard possible host contamination, the specificity of the primary culture was tested by nested PCR, analyzing mdh gene expression and obtaining only one product with the expected size. We also studied the expression of specific E. granulosus proteins in primary cell culture. The novel and systematized method described here constitutes a powerful tool for investigations in cystic echinococcosis on biochemical and biological aspects related to the life cycle of the parasite and mechanisms of host-parasite interactions. This method also constitutes a powerful tool for the design of more efficient therapeutic alternatives., (Copyright (c) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

FK506 (tacrolimus) and polyketide macrolides such as rapamycin and its derivates bind to FK506-binding proteins (FKBPs). These proteins display a peptidyl-prolyl rotamase function that is believed to catalyze protein folding and they are well-validated anti-proliferative drug targets in certain pathogenic microorganisms, and their functions have been characterized in parasitic protozoa. However, much less is known in helminths and trials with rapalogs on cestoda have not yet been reported. Due to a growing need for new treatment options for human cystic echinococcosis, the in vitro efficacy of rapalogs in Echinococcus granulosus was investigated. We determined the effect of ramapycin, FK506 and everolimus against this cestode, demonstrating their protoscolicidal ability. Also, we observed synergic scolicidal actions during combined therapy with rapalogs plus cyclosporine A, proposing dual administration of drugs to improve pharmacological effects in vivo. We have identified an E. granulosus (Eg)-fkb1 gene that encodes Eg-FKBP, an archetypal protein of the FKBP family, which includes all residues implicated in the binding of pharmacological ligands, in the enzymatic activity and in interactions with possible target proteins. Levels of Eg-fkb1 mRNA are over-expressed by acid but not rapalog treatment. We also described the presence of receptor-operated calcium channels in the larval stage, suggesting that exogenous ligands may dissociate the interaction of Eg-FKBP from these intracellular channels, enhancing the activity of the Ca(2+) release and interfering with their normal regulatory functions. As rapamycin sensitivity is the major criterion used to detect targets of rapamycin kinase, we identified and analyzed in silico critical residues of putative homologs in the Echinococcus genome. These preliminary results will allow us to continue subsequent studies that could reveal the precise intracellular functions of Eg-FKBP, providing greater knowledge for further identification of downstream target proteins, a promising target for chemotherapy of cystic echinococcosis., ((c) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The problem of chemotherapeutic treatments for human echinococcosis has not been completely solved. The benzimidazole-methylcarbamates (BZD), broad-spectrum antihelminthic agents, such as mebendazole and albendazole are the only drugs licensed for treatment of hydatid cysts. These drugs bind directly to beta-tubulin causing the disruption of microtubule-based processes in helminths. However, the molecular bases of their multiple biological activities are poorly understood. Recently, the effect of halogenated derivative flubendazole (FLBZ), against E. granulosus larvae has been conclusively demonstrated. The comparative effectiveness of FLBZ, among other BZDs, was shown by means of vitality tests and time of appearance of morphological damage of larvae. In the present study, we examined biochemical and molecular changes on protoscoleces treated with FLBZ. We show that FLBZ induces: 1) an increase in cytosolic free calcium, 2) a decrease in tubulin transcripts, 3) a reduction of mMDH expression and 4) a significant decrease in glycogen levels. These results are consistent with the existence of multiple targets for FLBZ, such as calcium signaling and energy metabolism, and contribute to the understanding of the pharmaceutical properties of FLBZ.

In the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120), it has been shown that spsB and susA, the genes coding for proteins related to sucrose synthesis and cleavage, respectively, exhibit converse expression regarding the nitrogen source. In the nitrogen-fixing filament, spsB expression is mostly localized to the heterocysts and susA is only expressed in vegetative cells. The aim of this work was to investigate the participation of NtcA, a global nitrogen regulator that operates in cyanobacteria, in the regulation of sucrose metabolism genes in Anabaena sp. PCC 7120. The induction of spsB expression observed in the filaments upon combined-nitrogen depletion was abolished in an NtcA-deficient mutant. In vitro experiments showed that NtcA binds specifically but with different affinities to two sites in the spsB promoter region. When susA expression was analyzed after a combined-nitrogen starvation, the levels of mRNA, polypeptide and activity increased in the mutant in comparison with the wild-type strain. Also, NtcA interacted with one site in the promoter region of susA. We conclude that sucrose metabolism is coordinated at the transcriptional level with nitrogen metabolism, suggesting a global metabolism regulating role for NtcA.

Higher plants and cyanobacteria metabolize sucrose (Suc) by a similar set of enzymes. Suc synthase (SuS, UDP-glucose: D: -fructose 2-alpha-D: -glucosyl transferase, EC 2.4.1.13) catalyses the synthesis and cleavage of Suc, and in higher plants, it plays an important role in polysaccharides biosynthesis and carbon allocation. In this work, we have studied the functional relationship between SuS and the metabolism of polysaccharides in filamentous nitrogen-fixing cyanobacteria. We show that the nitrogen and carbon sources and light regulate the expression of the SuS encoding gene (susA), in a similar way that they regulate the accumulation of polysaccharides. Furthermore, glycogen content in an Anabaena sp. mutant strain with an insertion inactivation of susA was lower than in the wild type strain under diazotrophic conditions, while both glycogen and polysaccharides levels were higher in a mutant strain constitutively overexpressing susA. We also show that there are soluble and membrane-bound forms of SuS in Anabaena. Taken together, these results strongly suggest that SuS is involved in the Suc to polysaccharides conversion according to nutritional and environmental signals in filamentous nitrogen-fixing cyanobacteria.

Nitrogen (N) available to plants mostly originates from N(2) fixation carried out by prokaryotes. Certain cyanobacterial species contribute to this energetically expensive process related to carbon (C) metabolism. Several filamentous strains differentiate heterocysts, specialized N(2)-fixing cells. To understand how C and N metabolism are regulated in photodiazotrophically grown organisms, we investigated the role of sucrose (Suc) biosynthesis in N(2) fixation in Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120). The presence of two Suc-phosphate synthases (SPS), SPS-A and SPS-B, directly involved in Suc synthesis with different glucosyl donor specificity, seems to be important in the N(2)-fixing filament. Measurement of enzyme activity and polypeptide levels plus reverse transcription-polymerase chain reaction experiments showed that total SPS expression is greater in cells grown in N(2) versus combined N conditions. Only SPS-B, however, was seen to be active in the heterocyst, as confirmed by analysis of green fluorescent protein reporters. SPS-B gene expression is likely controlled at the transcriptional initiation level, probably in relation to a global N regulator. Metabolic control analysis indicated that the metabolism of glycogen and Suc is likely interconnected in N(2)-fixing filaments. These findings suggest that N(2) fixation may be spatially compatible with Suc synthesis and support the role of the disaccharide as an intermediate in the reduced C flux in heterocyst-forming cyanobacteria.

Culture amplification in colon adenocarcinoma cell line (CaCo-2) combined with enzyme immunoassay (Pathfinder ELISA) was developed as a supplementary tool for rotavirus diagnosis. One hundred and thirty stools in which results by polyacrylamide gel electrophoresis (PAGE) were in agreement with those obtained by ELISA were amplified in the CaCo-2 cell line. After the first passage 100% specimens were revealed as positive by ELISA. This result was confirmed by PAGE and direct electron microscopy (EM) which increased the rates of rotavirus detection up to 100% after the third and fifth cell passages, respectively. All of the amplified negative stools were confirmed as negative. Among discordant results, three of the eight specimens positive by ELISA but negative by PAGE were confirmed as true positive after the third cell passage. False positive ELISA results could be discarded when the samples were culture amplified and retested by the same ELISA. Using the CaCo-2 amplification-ELISA as supplementary assay, sensitivity and specificity were 1.000 and 0.953 for ELISA and 0.917 and 1.000 for PAGE, respectively. The combined CaCo-2 cell line amplification-immunoassay method proved to be suitable both to evaluate increase in sensitivity of newly developed rotavirus assays and for rotaviral amplification before antigen assays.

In this paper we determined the prevalence of mycoplasma contamination in 17 cell lines. Eighty per cent of the laboratories that currently use cell culture techniques participated in this study. Hoechst 33258 dye was used to detect mycoplasma contamination. The relationship between culture maintenance conditions and the presence of mycoplasma were analyzed, considering the use of antibiotics in the culture media, fetal calf serum (FCS) quality, culture media processing, use of disponsable labware, type of laminar flow cabinet, quantity of operators, and cell culture system. Thirty-five per cent of the analyzed cell lines showed mycoplasma contamination. Those lines belonged to 2 of the 8 surveyed laboratories. When confronting the working conditions versus mycoplasma contamination, 66% of the laboratories that employ non-certified FCS or reuse their labware, show mycoplasma contamination. Mycoplasma presence was found in 50% of the laboratories that use closed culture system, or more than one operator. Laboratories that process their culture media or that include antibiotic in the growing media, show a 40% contamination. The results obtained help to establish working conditions necessary to avoid introducing or spreading the microorganism.