Modulatory actions of Echinococcus granulosus antigen B on macrophage inflammatory activation.

Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1ß, IL-6, IL-12p40, IFN-ß) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid's effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages.

Echinococcus granulosus cyst fluid inhibits the type I interferon response by promoting ROS in macrophages.

In cystic echinococcosis (CE), Echinococcus granulosus cystic fluid (EgCF) could impede macrophage-mediated immunity. However, whether EgCF is implicated in the type I interferon response remains to be established. Here, we revealed that EgCF reduced 2'3'-cGAMP-induced IFN-ß production in macrophages by inhibiting the cGAS-STING-IRF3 signaling. EgCF also increased the intracellular reactive oxygen species (ROS) levels. Administration of the ROS inhibitor N-acetylcysteine (NAC) restored the cGAS-STING-IRF3 signaling, which, in turn, upregulated IFN-ß expression. The findings disclose that EgCF could increase macrophage ROS levels, thereby blocking cGAS-STING-IRF3 signaling and repressing the IFN-I response.

Therapeutic effect of Echinococcus granulosus cyst fluid on bacterial sepsis in mice.

BACKGROUND: The primary pathophysiological process of sepsis is to stimulate a massive release of inflammatory mediators to trigger systemic inflammatory response syndrome (SIRS), the major cause of multi-organ dysfunction and death. Like other helminths, Echinococcus granulosus induces host immunomodulation. We sought to determine whether E. granulosus cyst fluid (EgCF) displays a therapeutic effect on sepsis-induced inflammation and tissue damage in a mouse model. METHODS: The anti-inflammatory effects of EgCF were determined by in vitro culture with bone marrow-derived macrophages (BMDMs) and in vivo treatment of BALB/C mice with cecal ligation and puncture (CLP)-induced sepsis. The macrophage phenotypes were determined by flow cytometry, and the levels of cytokines in cell supernatants or in sera of mice were measured (ELISA). The therapeutic effect of EgCF on sepsis was evaluated by observing the survival rates of mice for 72 h after CLP, and the pathological injury to the liver, kidney, and lung was measured under a microscope. The expression of TLR-2/MyD88 in tissues was measured by western blot to determine whether TLR-2/MyD88 is involved in the sepsis-induced inflammatory signaling pathway. RESULTS: In vitro culture with BMDMs showed that EgCF promoted macrophage polarization to M2 type and inhibited lipopolysaccharide (LPS)-induced M1 macrophages. EgCF treatment provided significant therapeutic effects on CLP-induced sepsis in mice, with increased survival rates and alleviation of tissue injury. The EgCF conferred therapeutic efficacy was associated with upregulated anti-inflammatory cytokines (IL-10 and TGF-ß) and reduced pro-inflammatory cytokines (TNF-α and INF-γ). Treatment with EgCF induced Arg-1-expressed M2, and inhibited iNOS-expressed M1 macrophages. The expression of TLR-2 and MyD88 in EgCF-treated mice was reduced. CONCLUSIONS: The results demonstrated that EgCF confers a therapeutic effect on sepsis by inhibiting the production of pro-inflammatory cytokines and inducing regulatory cytokines. The anti-inflammatory effect of EgCF is carried out possibly through inducing macrophage polarization from pro-inflammatory M1 to regulatory M2 phenotype to reduce excessive inflammation of sepsis and subsequent multi-organ damage. The role of EgCF in regulating macrophage polarization may be achieved by inhibiting the TLR2/MyD88 signaling pathway.

Echinococcus granulosus cyst fluid inhibits KDM6B-mediated demethylation of trimethylated histone H3 lysine 27 and interleukin-1ß production in macrophages.

BACKGROUND: Echinococcus granulosus can manipulate its host's immune response to ensure its own survival. However, the effect of histone modifications on the regulation of the NOD-like receptor protein 3 (NLRP3) inflammasome and downstream interleukin-1ß (IL-1ß) production in response to the parasite is not fully understood. METHODS: We evaluated IL-1ß secretion through enzyme-linked immunosorbent assay and assessed reactive oxygen species levels using the dichlorodihydrofluorescein diacetate probe. Western blotting and quantitative real-time polymerase chain reaction were performed to examine the expression of NLRP3 and IL-1ß in mouse peritoneal macrophages and Tohoku Hospital Pediatrics-1 cells, a human macrophage cell line. The presence of trimethylated histone H3 lysine 27 (H3K27me3) modification on NLRP3 and IL-1ß promoters was studied by chromatin immunoprecipitation. RESULTS: Treatment with E. granulosus cyst fluid (EgCF) considerably reduced IL-1ß secretion in mouse and human macrophages, although reactive oxygen species production increased. EgCF also suppressed the expression of NLRP3 and IL-1ß. Mechanistically, EgCF prompted the enrichment of repressive H3K27me3 modification on the promoters of both NLRP3 and IL-1ß in macrophages. Notably, the presence of EgCF led to a significant reduction in the expression of the H3K27me3 demethylase KDM6B. CONCLUSIONS: Our study revealed that EgCF inhibits KDM6B expression and H3K27me3 demethylation, resulting in the transcriptional inhibition of NLRP3 and IL-1ß. These results provide new insights into the immune evasion mechanisms of E. granulosus.

Tirapazamine combined with photodynamic therapy improves the efficacy of ABZSO nanoparticles on Echinococcosis granulosus via further enhancing "breaking-then-curing".

BACKGROUND: Photodynamic therapy (PDT) has a promising application prospect in Echinococcus granulosus (Egs), however, the hypoxic environment of Egs and the hypoxia associated with PDT will greatly limit its effects. As a hypoxic-activated pre-chemotherapeutic drug, tirapazamine (TPZ) can be only activated and produce cytotoxicity under hypoxia environment. Albendazole sulfoxide (ABZSO) is the first choice for the treatment of Egs. This study aimed to explore the effects of ABZSO nanoparticles (ABZSO NPs), TPZ combined with PDT on the activity of Egs in vitro and in vivo. METHODS: The Egs were divided into control, ABZSO NPs, ABZSO NPs + PDT, and ABZSO NPs + TPZ + PDT groups, and the viability of Egs was determined using methylene blue staining. Then, the ROS, LDH and ATP levels were measured using their corresponding assay kit, and H2AX and TopoI protein expression was detected by western blot. The morphology of Egs with different treatments was observed using hematoxylin eosin (HE) staining and scanning electron microscopy (SEM). After that, the in vivo efficacy of ABZSO NPs, TPZ and PDT on Egs was determined in a Egs infected mouse model. RESULTS: In vitro experiments showed that the combined treatment of TPZ, ABZSO NPs and PDT significantly inhibited Egs viability; and significantly increased ROS levels and LDH contents, while decreased ATP contents in Egs; as well as up-regulated H2AX and down-regulated TopoI protein expression. HE staining and SEM results showed that breaking-then-curing treatment seriously damaged the Egs wall. Additionally, in vivo experiments found that the combination of ABZSO NPs, PDT and TPZ had more serious calcification and damage of the wall structure of cysts. CONCLUSIONS: ABZSO NPs combined with TPZ and PDT has a better inhibitory effect on the growth of Egs in vitro and in vivo based on the strategy of "breaking-then-curing".

Design of ion channel blocking, toxin-like Kunitz inhibitor peptides from the tapeworm, Echinococcus granulosus, with potential anti-cancer activity.

Over-expression of K+ channels has been reported in human cancers and is associated with the poor prognosis of several malignancies. EAG1, a particular potassium ion channel, is widely expressed in the brain but poorly expressed in other normal tissues. Kunitz proteins are dominant in metazoan including the dog tapeworm, Echinococcus granulosus. Using computational analyses on one A-type potassium channel, EAG1, and in vitro cellular methods, including major cancer cell biomarkers expression, immunocytochemistry and whole-cell patch clamp, we demonstrated the anti-tumor activity of three synthetic small peptides derived from E. granulosus Kunitz4 protease inhibitors. Experiments showed induced significant apoptosis and inhibition of proliferation in both cancer cell lines via disruption in cell-cycle transition from the G0/G1 to S phase. Western blotting showed that the levels of cell cycle-related proteins including P27 and P53 were altered upon kunitz4-a and kunitz4-c treatment. Patch clamp analysis demonstrated a significant increase in spontaneous firing frequency in Purkinje neurons, and exposure to kunitz4-c was associated with an increase in the number of rebound action potentials after hyperpolarized current. This noteworthy component in nature could act as an ion channel blocker and is a potential candidate for cancer chemotherapy based on potassium channel blockage.

[Echinococcus granulosus cyst fluid(EgCF) inhibits the migration and phagocytic function of mouse macrophages induced by LPS via inducing cytoskeletal rearrangement].

Objective To investigate the effect of Echinococcus granulosus cyst fluid(EgCF) on the cytoskeletal rearrangement and phagocytosis and the migration of macrophages induced by lipopolysaccharide(LPS). Methods Peritoneal macrophages of C57BL/6 mice were isolated and cultured in vitro, and divided into control group and LPS group and LPS combined with EgCF group. After 48 hours of treatment, filamentous actin (F-actin) changes were observed with rhodamine-labelled phalloidin staining and fluorescence microscopy; TranswellTM chamber was used to test cell migration ability and flow cytometry to test cell phagocytosis. After 1 hour of treatment, PI3K and AKT, phosphorylated AKT (p-AKT), Rac1, guanosine triphospho-Rac1 (GTP-Rac1), WASP and Arp2 protein expressions were detected with Western blot analysis. Results Compared with the control group, after LPS stimulation, macrophages were deformed significantly; pseudopodia increased; actin cytoskeleton increased and was more distributed in pseudopodia; the ability of migration and phagocytosis were significantly improved, and the expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 proteins significantly increased. EgCF treatment caused cell shrinkage and disappearance of pseudopodia protrusions of LPS-activated cells, and led to the reduced phagocytic and migratory of cells; the protein expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 decreased significantly compared with the LPS group. Conclusion LPS induces the migration and enhances phagocytosis of macrophages while EgCF inhibits these effects, which is related to actin cytoskeleton rearrangement.

MicroRNA-Transcription factor regulatory networks in the early strobilar development of Echinococcus granulosus protoscoleces.

BACKGROUND: Echinococcus granulosus sensu lato has a complex developmental biology with a variety of factors relating to both intermediate and final hosts. To achieve maximum parasite adaptability, the development of the cestode is dependent on essential changes in transcript regulation. Transcription factors (TFs) and miRNAs are known as master regulators that affect the expression of downstream genes through a wide range of metabolic and signaling pathways. In this study, we aimed to develop a regulatory miRNA-Transcription factor (miRNA-TF) network across early developmental stages of E. granulosus protoscoleces by performing in silico analysis, and to experimentally validate TFs expression in protoscoleces obtained from in vitro culture, and from in vivo experiments. RESULTS: We obtained list of 394 unique E. granulosus TFs and matched them with 818 differentially expressed genes which identified 41 predicted TFs with differential expression. These TFs were used to predict the potential targets of 31 differentially expressed miRNAs. As a result, eight miRNAs and eight TFs were found, and the predicted network was constructed using Cytoscape. At least four miRNAs (egr-miR-124a, egr-miR-124b-3p, egr-miR-745-3p, and egr-miR-87-3p) and their corresponding differentially expressed TFs (Zinc finger protein 45, Early growth response protein 3, Ecdysone induced protein 78c and ETS transcription factor elf 2) were highlighted in this investigation. The expression of predicted differentially expressed TFs obtained from in vitro and in vivo experiments, were experimentally validated by quantitative polymerase chain reaction. This confirmed findings of RNA-seq data. CONCLUSION: miRNA-TF networks presented in this study control some of the most important metabolic and signaling pathways in the development and life cycle of E. granulosus, providing a potential approach for disrupting the early hours of dog infection and preventing the development of the helminth in the final host.

Modeling, molecular dynamics and docking studies of a full-length Echinococcus granulosus 2DBD nuclear receptor.

Nuclear receptors are ligand-activated transcription factors capable of regulating the expression of complex gene networks. The family includes seven subfamilies of protein with a wide phylogenetic distribution. A novel subfamily with two DNA-binding domains (2DBDs) has been first reported in Schistosoma mansoni (Platyhelminth, Trematoda). Employing an ab initio protocol and homology modeling methods, the full-length 3D structure of the Eg2DBDα.1 nuclear receptor from Echinococcus granulosus (Platyhelminth, Cestoda) was generated. The model analysis reveals the presence of the conserved three-layered alpha-helical sandwich structure in the ligand binding domain, and a particularly long and flexible hinge region. Molecular dynamics simulations were performed previous to dock a conformational library of fatty acids and retinoic acids. Our results indicate that oleic and linoleic acids are suitable ligands to this receptor. The ligand-protein complex is stabilized mainly by hydrogen bonds and hydrophobic interactions. The fact that 2DBD nuclear receptors have not been identified in vertebrates confers particular interest to these nuclear receptors, not only concerning their structure and function but as targets of new anthelmintic drugs.Communicated by Ramaswamy H. Sarma.

Echinococcus granulosus cyst fluid(EgCF) inhibits the migration and phagocytic function of mouse macrophages induced by LPS via inducing cytoskeletal rearrangement / 细胞与分子免疫学杂志

Objective To investigate the effect of Echinococcus granulosus cyst fluid(EgCF) on the cytoskeletal rearrangement and phagocytosis and the migration of macrophages induced by lipopolysaccharide(LPS). Methods Peritoneal macrophages of C57BL/6 mice were isolated and cultured in vitro, and divided into control group and LPS group and LPS combined with EgCF group. After 48 hours of treatment, filamentous actin (F-actin) changes were observed with rhodamine-labelled phalloidin staining and fluorescence microscopy; TranswellTM chamber was used to test cell migration ability and flow cytometry to test cell phagocytosis. After 1 hour of treatment, PI3K and AKT, phosphorylated AKT (p-AKT), Rac1, guanosine triphospho-Rac1 (GTP-Rac1), WASP and Arp2 protein expressions were detected with Western blot analysis. Results Compared with the control group, after LPS stimulation, macrophages were deformed significantly; pseudopodia increased; actin cytoskeleton increased and was more distributed in pseudopodia; the ability of migration and phagocytosis were significantly improved, and the expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 proteins significantly increased. EgCF treatment caused cell shrinkage and disappearance of pseudopodia protrusions of LPS-activated cells, and led to the reduced phagocytic and migratory of cells; the protein expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 decreased significantly compared with the LPS group. Conclusion LPS induces the migration and enhances phagocytosis of macrophages while EgCF inhibits these effects, which is related to actin cytoskeleton rearrangement.

Echinococcus granulosus Protoscoleces-Derived Exosome-like Vesicles and Egr-miR-277a-3p Promote Dendritic Cell Maturation and Differentiation.

Cystic echinococcosis, a major parasitic disease caused by Echinococcus granulosus, seriously threatens human health. The excretory-secretory (ES) products of E. granulosus can induce immune tolerance in dendritic cells (DCs) to downregulate the host's immune response; however, the effect of exosomes in the ES products on the DCs has remained unclear. This study showed that E. granulosus protoscoleces-derived exosome-like vesicles (PSC-ELVs) could be internalized by bone marrow-derived dendritic cells (BMDCs), allowing for the delivery of the parasite microRNAs to the BMDCs. Moreover, PSC-ELVs induced BMDCs to produce the proinflammatory cytokinesinterleukin (IL)-6, IL-12, IL-ß, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ). PSC-ELVs also upregulated the BMDCs surface marker major histocompatibility complex class II (MHC II), as well as costimulatory molecules CD40, CD80, and CD86. PSC-ELV-derived egr-miR-277a-3p upregulated the IL-6, IL-12, and TNF-α mRNA levels in BMDCs. Moreover, egr-miR-277a-3p directly targeted Nfkb1 (encoding nuclear factor kappa B 1) to significantly suppress the mRNA and protein levels of NF-κB1 in BMDCs, while the expression of NF-κB p65 significantly increased, suggesting that egr-miR-277a-3p induces the production of proinflammatory cytokines by the modification of the NF-kB p65/p50 ratio in BMDCs. These results demonstrated that PSC-ELVs and egr-miR-277a-3p might enhance DCs maturation and differentiation in a cross-species manner, which in turn may modulate the host immune responses and offer a new approach to echinococcosis prevention and treatment.

Bioinformatics analysis and experimental verification of Notch signalling pathway-related miRNA-mRNA subnetwork in extracellular vesicles during Echinococcus granulosus encystation.

BACKGROUND: Encystation of the protoscoleces (PSCs) of Echinococcus granulosus is the main cause of secondary hydatid dissemination in the intermediate host. Extracellular vesicles (EVs) can transfer miRNAs into parasite cells to regulate mRNA expression. However, loading of developmental pathway-related miRNAs, such as those related to the Notch signalling pathway in EVs is unclear. Thus, we screened the miRNA-mRNA subnetwork involved in the Notch pathway during E. granulosus encystation in vitro and assessed changes in expression in the parasite and EVs. METHODS: mRNAs and miRNAs differentially expressed (DE) between PSCs and microcysts (MCs) were screened using high-throughput sequencing. DE mRNAs obtained from transcriptome analysis were intersected with mRNAs predicted to be targets of the conserved DE miRNAs of a small RNA library. DE miRNA functions were analysed using public databases, and a miRNA-mRNA subnetwork related to the Notch pathway was established. Notch pathway-related mRNA and miRNA expression of worms and EVs at different times was verified. RESULTS: In total, 1445 DE mRNAs between MCs and PSCs were screened after the intersection between 1586 DE mRNAs from the transcriptome and 9439 target mRNAs predicted using 39 DE miRNAs from the small RNA library. The DE mRNAs were clustered into 94 metabolic pathways, including the Notch pathway. Five DE miRNAs, including the most significantly expressed new DE miRNA, egr-new-mir0694-3p, corresponding to four target mRNAs (EgrG_000892700, EgrG_001029400, EgrG_001081400 and EgrG_000465800) were all enriched in the Notch pathway. The expression of the above mRNAs and miRNAs was consistent with the results of high-throughput sequencing, and the expression of each miRNA in EVs was verified. Annotated as ADAM17/TACE in the Notch pathway, EgrG_000892700 was down-regulated during PSC encystation. egr-miR-4989-3p and egr-miR-277a-3p expression in EVs after encystation was nearly five times that in EVs before encystation, which might regulate the expression of EgrG_000892700. CONCLUSIONS: Five miRNAs corresponding to four target mRNAs may be involved in regulating the Notch pathway during the PSC encystation. EVs may regulate the expression of EgrG_000892700 in PSCs because of continuous targeting of egr-miR-4989-3p and egr-miR-277a-3p and participate in the regulation the Notch pathway. The study might expand new ideas for blocking the secondary infection of E. granulosus PSCs via EVs miRNAs.

Proteomic Analysis of Plasma-Derived Extracellular Vesicles From Mice With Echinococcus granulosus at Different Infection Stages and Their Immunomodulatory Functions.

The globally distributed cystic echinococcosis (CE) is caused by the larval stage of Echinococcus granulosus (E. granulosus), a cosmopolitan and zoonotic disease with potentially life-threatening complications in humans. The emerging roles for extracellular vesicles (EVs) in parasitic infection include transferring proteins and modifying host cell gene expression to modulate host immune responses. Few studies focused on the host-derived EVs and its protein profiles. We focused on the EVs from mouse infected with E. granulosus at different stages. ExoQuick kit was used for isolating EVs from mouse plasma and ExoEasy Maxi kit was used for isolating protoscolex culture supernatant (PCS) and hydatid cyst fluid (HCF). Firstly, EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and immunoblot. Secondly, the proteins of plasma EVs were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting LC-MS/MS data were processed using Maxquant search engine (v 1.5.2.8). Tandem mass spectra were researched against the mice and E. granulosus proteins database in the NCBI. The differentially expressed proteins are performed by proteomic label-free quantitative analysis and bioinformatics. Thirdly, in vitro experiment, the results of co-culture of plasma EVs and spleen mononuclear cells showed that 7W-EVs can increase the relative abundance of regulatory T (Treg) cells and IL-10. We further verified that EVs can be internalized by CD4+ and CD8+ T cells, B cells, and myeloid-derived suppressor cells (MDSC). These results implied host-derived EVs are multidirectional immune modulators. The findings can contribute to a better understanding of the role of host-derived EVs which are the optimal vehicle to transfer important cargo into host immune system. In addition, we have found several important proteins associated with E. granulosus and identified in infected mouse plasma at different stages. Furthermore, our study further highlighted the proteomics and immunological function of EVs from mouse infected with E. granulosus protoscoleces at different infection stages. We have laid a solid foundation for the role of EVs in cystic echinococcosis in the future research and supplemented a unique dataset for this E. granulosus.

Unraveling post-translational modifications in Echinococcus granulosus sensu lato.

Echinococcus granulosus sensu lato (s.l.) is the helminth parasite responsible for cystic echinococcosis, a neglected tropical disease currently affecting millions of people worldwide. Incomplete knowledge on the parasite biochemistry contributes, at least partially, to the limited development of useful biotechnological advances for the infection control. In this sense, little information is available regarding post-translational modifications (PTMs) occurring in E. granulosus s.l. proteins, which ultimately may affect the performance of biotechnological products to be developed. Therefore, we report here a proteomic analysis of the parasite PTMs identified through FindMod software applied to a set of tegumental proteins previously characterized by mass spectrometry (MALDI-TOF/TOF) analysis of protein spots from a 2D electrophoresis gel. Manual searches for already annotated proteins exhibiting such PTMs were also performed within proteome databases of E. granulosus s.l. and other platyhelminthes. In addition, key enzymes involved in PTMs modifications were searched for within E. granulosus s.l. proteome. Finally, the presence of selected PTMs was further confirmed by a high-resolution proteomic approach (nanoLC-MS/MS). A set of 22 different PTMs most likely to be present in the parasite was suggested, 9 of them with high confidence as they were identified in the same m/z fragment by both proteomic techniques (acetylation, deamidation, deamidation followed by methylation, mono- and di-hydroxylation, mono- and di-methylation, S-nitrosylation and phosphorylation). Interestingly, 5 PTMs were herein identified for the first time in E. granulosus s.l. proteins. Our results expand the scarcely studied topic of PTMs in platyhelminthes.

Parasite-Derived Excretory-Secretory Products Alleviate Gut Microbiota Dysbiosis and Improve Cognitive Impairment Induced by a High-Fat Diet.

High-fat (HF) diet-induced neuroinflammation and cognitive decline in humans and animals have been associated with microbiota dysbiosis via the gut-brain axis. Our previous studies revealed that excretory-secretory products (ESPs) derived from the larval Echinococcus granulosus (E. granulosus) function as immunomodulators to reduce the inflammatory response, while the parasitic infection alleviates metabolic disorders in the host. However, whether ESPs can improve cognitive impairment under obese conditions remain unknown. This study aimed to investigate the effects of E. granulosus-derived ESPs on cognitive function and the microbiota-gut-brain axis in obese mice. We demonstrated that ESPs supplementation prevented HF diet-induced cognitive impairment, which was assessed behaviorally by nest building, object location, novel object recognition, temporal order memory, and Y-maze memory tests. In the hippocampus (HIP) and prefrontal cortex (PFC), ESPs suppressed neuroinflammation and HF diet-induced activation of the microglia and astrocytes. Moreover, ESPs supplementation improved the synaptic ultrastructural impairments and increased both pre- and postsynaptic protein levels in the HIP and PFC compared to the HF diet-treated group. In the colon, ESPs reversed the HF diet-induced gut barrier dysfunction, increased the thickness of colonic mucus, upregulated the expression of zonula occludens-1 (ZO-1), attenuated the translocation of bacterial endotoxins, and decreased the colon inflammation. Notably, ESPs supplementation alleviated the HF diet-induced microbiota dysbiosis. After clarifying the role of antibiotics in obese mice, we found that broad-spectrum antibiotic intervention abrogated the effects of ESPs on improving the gut microbiota dysbiosis and cognitive decline. Overall, the present study revealed for the first time that the parasite-derived ESPs alleviate gut microbiota dysbiosis and improve cognitive impairment induced by a high-fat diet. This finding suggests that parasite-derived molecules may be used to explore novel drug candidates against obesity-associated neurodegenerative diseases.

Genome-wide transcriptome analysis of the early developmental stages of Echinococcus granulosus protoscoleces reveals extensive alternative splicing events in the spliceosome pathway.

BACKGROUND: The complex life cycle of Echinococcus granulosus involves numerous environmental conditions within different intermediate and definitive hosts. This requires adaptation at different levels of transcript regulation. Alternative splicing (AS) and the related cellular functions as one of the major fields of post-genomics has been poorly studied in tapeworms. In the present study, we investigated AS events and their potential biological effects in E. granulosus. METHODS: Whole transcriptome sequencing data of four groups of protoscoleces were prepared for RNA-seq library construction. Fresh protoscoleces were either used as non-induced controls (NT group) or incubated for 15 min with pepsin (PEP group) and cultivated in a biphasic medium for 12 and 24 h (12 and 24 h groups). The frequency and different types of AS events were identified using rMATS software. Functional annotations and gene ontology of differential AS (DAS) genes were performed using Blast2GO software. AS events were experimentally validated by PCR on the protoscolex cDNAs using specific primers for each gene. RESULTS: At least one AS event was found in 38.1% of the genes (3904 out of 10,245) in the protoscoleces during early strobilar development. The genes were associated primarily with cellular and metabolic processes and binding and catalytic activity. KEGG pathway analysis of DAS events revealed a number of genes belonging to different components of the spliceosome complex. These genes tended to belong to common SR proteins, U1-related factors, U2-related factors, complex A-specific factors and other splicing-related proteins. CONCLUSIONS: The high number of AS events in the transcriptome regulatory mechanisms indicates the essential rapid molecular processes required by the parasite for adaptation in different environments.

Expression and distribution of glutathione transferases in protoscoleces of Echinococcus granulosus sensu lato.

Glutathione transferases (GSTs) belong to a diverse superfamily of multifunctional proteins involved in metabolic detoxification. In helminth parasite, GSTs are particularly relevant since they are also involved in host immunomodulation. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least three phylogenetically distant cytosolic GSTs: EgGST1 and EgGST2 previously grouped within Mu and Sigma classes, respectively; and EgGST3 related to both Omega and Sigma classes. To better characterize E. granulosus s.l. GSTs, herein their expression and distribution were assessed in the pre-adult protoscolex (PSC) parasite stage. Potential transcriptional regulatory mechanisms of the corresponding EgGST genes were also explored. Firstly, the transcription of the three EgGSTs was significantly induced during the early stages of the murine model of infection, suggesting a potential role during parasite establishment. EgGST1 was detected in the parenchyma of PSCs and its expression increased after H2O2 exposure, supporting its role in detoxification. EgGST2 was mainly detected on the PSCs tegument, strategically localized for potential immunoregulation functions due to its Sigma-class characteristics. In addition, its expression increased after anthelmintic treatment, suggesting a role in chemotherapy resistance. Finally, the Omega-related EgGST3 was localized throughout the entire PSC body, including suckers and tegument, and since its expression also increased after H2O2 treatment, a potential role in oxidative stress response could also be ascribed. On the other hand, known cis-acting regulatory motifs were detected in EgGST genes, suggesting similar transcription processes to other eukaryotes. The results herein reported provide additional data regarding the roles of EgGSTs in E. granulosus s.l. biology, contributing to a better understanding of its host-parasite interaction.

In vitro effects of tropisetron and granisetron against Echinococcus granulosus (s.s.) protoscoleces by involvement of calcineurin and calmodulin.

BACKGROUND: Cystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato  (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs. METHODS: Protoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively. RESULTS: At 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 µM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 µM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations. CONCLUSIONS: Tropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE.

The Effects of Nitric Oxide and Inhibitor, and Combination of Albendazole and Praziquantel On Liver in Mice Injected with Echinococcus granulosus Larvae.

In this study, the role of nitric oxide (NO) in the pathogenesis of hydatidosis and the interaction with effects of anthelmintic drugs, albendazole and praziquantel, were examined in larval infection caused by protoscolices obtained from hydatid cysts of sheep liver in Albino Balb/c mice. Animals were divided into ten groups including controls and infected groups. Larval infection was established with intraperitoneal injection of protoscolices. Eight months after infection with protoscolices, the infected animals were divided into 6 groups. The infected animals were given a selective inhibitor of inducible nitric oxide synthase (iNOS) L-N6-(1-Iminoethyl) lysine-hydrochloride (L-NIL), NO donor sodium nitroprusside (SNP), albendazole and praziquantel as anthelmintic drugs for 7 days. In addition, control groups were composed of intact group, control, anthelmintic drugs + L-NIL, and anthelmintic drugs + SNP. The liver and blood samples were taken for cytological, histological, immunohistochemical and biochemical analyses 7 days after treatments at the end of experiment. The animals injected with protoscolices showed histopathological changes including inflammation areas, infiltration and accumulation of leukocytes, dilation of sinusoids, and damage in endothelial cells and hepatocytes at light microscopy. Electron microscopy were revealed severe damage in sinusoidal endothelial cells, leukocytes especially eosinophils in sinusoid lumens and disorganization in endoplasmic reticulum and nuclear membrane. Endothelial nitric oxide synthase (eNOS) and iNOS reactions were increased in the tissue. Anthelmintic drugs decreased inflammation areas and damages; however, it did not change NOS reactions in the animals given protoscolices. L-NIL and SNP diminished both iNOS and eNOS reactions. Unlike the group administered the inhibitor, SNP treated group exhibited less inflammation areas. Combination of these substances and drugs resulted in decreased inflammation areas. eNOS and iNOS reactions decreased in the drugs and SNP administered group, while only iNOS reaction was decreased in L-NIL given infection group. In addition, the infected groups which received SNP displayed expanded sinusoids and hepatocytes with vacuoles, intriguingly. While levels of serum nitrite/nitrate elevated only in the infection group given drugs and SNP, it decreased in the L-NIL administered group. Tissue level of malondialdehyde increased in infection groups with drugs and SNP. In conclusion, the results indicated that NO plays an important role in the pathogenesis of hydatidosis.

Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model.

BACKGROUND: Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. METHODS: The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. RESULTS: In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 µg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. CONCLUSIONS: Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.