RESUMO
IMPORTANCE: Sepsis is the consequence of a systemic bacterial infection that exacerbates the immune cell's activation via bacterial products, resulting in the augmented release of inflammatory mediators. A critical factor in the pathogenesis of sepsis is the primary component of the outer membrane of Gram-negative bacteria known as lipopolysaccharide (LPS), which is sensed by TLR4. For this reason, scientists have aimed to develop antagonists able to block TLR4 and, thereby the cytokine storm. We report here that a mixture of mu-class isoforms from the F. hepatica GST protein family administered intraperitoneally 1 h prior to a lethal LPS injection can modulate the dynamics and abundance of large peritoneal macrophages in the peritoneal cavity of septic mice while significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock. These results suggest that native F. hepatica glutathione S-transferase is a promising candidate for drug development against endotoxemia and other inflammatory diseases.
Assuntos
Fasciola hepatica , Sepse , Animais , Camundongos , Macrófagos Peritoneais/metabolismo , Lipopolissacarídeos/metabolismo , Fasciola hepatica/metabolismo , Escherichia coli/metabolismo , Síndrome da Liberação de Citocina/metabolismo , Receptor 4 Toll-Like/metabolismo , MacrófagosRESUMO
BACKGROUND: Clonorchiasis, caused by the infection of Clonorchis sinensis (C. sinensis), is a kind of neglected tropical disease, but it is highly related to cholangiocarcinoma. It has been well known that NO from chronic inflammation responses are thought to be a major component of the damage and ultimate carcinogenesis ESPs such as nitric oxide synthase interacting protein (NOSIP) are thought to enhance the damage. The objective of this study was to identify the protein candidates interact with recombinant CsNOSIP (rCsNOSIP) and explore their role involved in CCA development or progression. METHODS: We applied HuProt microarray containing 21,000 probe sets for a systematic identification of rCsNOSIP-binding proteins and grouped binding hits by gene function. Pull-down assays were used to confirm the interaction of rCsNOSIP with alveolar soft part sarcoma (ASPSCR-1) and sirtuins 5 (Sirt-5). ASPSCR-1/Sirt-5 over-expression and siRNA knockdown experiments were employed for obtain of ASPSCR-1/Sirt-5 high or low expression (ASP-oe/Sirt5-oe or ASP-si/Sirt5-si) cholangiocarcinoma cell line (CCLP-1) cells. Nitric oxide (NO) and reactive oxygen species assay (ROS) as well as cell proliferation and wound-healing assays were performed to observe the effect of rCsNOSIP on ASP-oe/Sirt5-oe or ASP-si/Sirt5-si CCLP-1 cells. RESULTS: Seventy candidate proteins protein "hits" were detected as rCsNOSIP-binding proteins by HuProt microarray and bioinformatics analysis. Pull down assay showed that ASPSCR-1 and Sirt-5 could interact with rCsNOSIP. In addition, endotoxin-free-rCsNOSIP could increase the production of NO and ROS and promote the migration of CCLP-1 cells, while its effect on enhancing cell proliferation was not significant. Furthermore, ROS/NO production, proliferation, or migration were increased in ASP-si or Sirt5-si CCLP-1 cells but decreased in Asp-oe or Sirt5-oe CCLP-1 cells when stimulated with rCsNOSIP. CONCLUSIONS: Our findings suggest that CsNOSIP as a component of CsESPs might promote the development and invasion of CCA and Sirt5/ ASPSCR1 as host molecules might play a novel protective role against adverse stimulus during C. sinensis infection. This work supports the idea that CsESPs induce the occurrence and progression of CCA through ROS/RNS-induced oxidative and nitrative DNA damage.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Clonorquíase , Clonorchis sinensis , Fasciola hepatica , Sarcoma Alveolar de Partes Moles , Animais , Humanos , Fasciola hepatica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sarcoma Alveolar de Partes Moles/metabolismo , Clonorchis sinensis/genética , Estresse Oxidativo , Proteínas de Transporte/metabolismo , Proliferação de Células , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologiaRESUMO
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. HCC with liver fluke infection could harbor unique biological behaviors. This study was aimed at investigating radiomics features of HCC with liver fluke infection and establishing a model to predict the expression of cytokeratin 7 (CK7) and cytokeratin 19 (CK19) as well as prognosis at the same time. A total of 134 HCC patients were included. Gadoxetic acid-enhanced magnetic resonance imaging (MRI) images of all patients were acquired. Radiomics features of the tumor were extracted and then data dimensionality was reduced. The radiomics model was established to predict liver fluke infection and the radiomics score (Radscore) was calculated. There were 11 features in the four-phase combined model. The efficiency of the combined model increased significantly compared to each single-phase MRI model. Radscore was an independent predictor of liver fluke infection. It was also significantly different between different expression of CK7/ CK19. Meanwhile, liver fluke infection was associated with CK7/CK19 expression. A cut-off value was set up and all patients were divided into high risk and low risk groups of CK7/CK19 positive expression. Radscore was also an independent predictor of these two biomarkers. Overall survival (OS) and recurrence free survival (RFS) of negative liver fluke infection group were significantly better than the positive group. OS and RFS of negative CK7 and CK19 expression were also better, though not significantly. Positive liver fluke infection and CK19 expression prediction groups harbored significantly worse OS and RFS, survival of positive CK7 expression prediction was unsatisfying as well. A radiomics model was established to predict liver fluke infection among HCC patients. This model could also predict CK7 and CK19 expression. OS and RFS could be foreseen by this model at the same time.
Assuntos
Carcinoma Hepatocelular , Fasciola hepatica , Neoplasias Hepáticas , Humanos , Animais , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Queratina-19/metabolismo , Queratina-7/metabolismo , Fasciola hepatica/metabolismo , Imageamento por Ressonância Magnética/métodos , Estudos RetrospectivosRESUMO
Extracellular vesicles (EVs) released by the helminths Dicrocoelium dendriticum and Fasciola hepatica are important modulators of the host immune response, contributing to the establishment of the infection. Monocytes and, in particular, macrophages are major regulators of the inflammatory response and are likely responsible for the phagocytosis of most of the parasite EVs. In this study, we isolated EVs from F. hepatica (FhEVs) and D. dendriticum (DdEVs) by size exclusion chromatography (SEC) and characterized them by nanoparticle tracking analysis, transmission electron microscopy and LC-MS/MS, and analyzed the cohort of proteins. The treatment of monocytes/macrophages with FhEVs, DdEVs or EV-depleted fractions from SEC, demonstrated species-specific effects of the EVs. In particular, FhEVs reduce the migratory capacity of monocytes and the analysis of the cytokine profile showed that they induce a mixed M1/M2 response, exerting anti-inflammatory properties in Lipopolysaccharide-activated macrophages. In contrast, DdEVs do not affect monocyte migration and seem to have pro-inflammatory properties. These results correlate with the differences in the life cycle of both parasites, suggesting different host immune responses. Only F. hepatica migrates to the bile duct through the liver parenchyma, driving the host immune response to heal deep erosions. Furthermore, the proteomic analysis of the macrophages upon FhEV treatment identified several proteins that might be involved in FhEV-macrophage interactions.
Assuntos
Dicrocoelium , Vesículas Extracelulares , Fasciola hepatica , Animais , Humanos , Fasciola hepatica/metabolismo , Vesículas Extracelulares/metabolismo , Proteômica , Cromatografia Líquida , Espectrometria de Massas em Tandem , Macrófagos/metabolismoRESUMO
BACKGROUND: Clonorchis sinensis requires bile acid transporters as this fluke inhabits bile juice-filled biliary ducts, which provide an extreme environment. Clonorchis sinensis sodium-bile acid co-transporter (CsSBAT) is indispensable for the fluke's survival in the final host, as it circulates taurocholate and prevents bile toxicity in the fluke; hence, it is recognized as a useful drug target. METHODOLOGY AND PRINCIPAL FINDINGS: In the present study, using structure-based virtual screening approach, we presented inhibitor candidates targeting a bile acid-binding pocket of CsSBAT. CsSBAT models were built using tertiary structure modeling based on a bile acid transporter template (PDB ID: 3zuy and 4n7x) and were applied into AutoDock Vina for competitive docking simulation. First, potential compounds were identified from PubChem (holding more than 100,000 compounds) by applying three criteria: i) interacting more favorably with CsSBAT than with a human homolog, ii) intimate interaction to the inward- and outward-facing conformational states, iii) binding with CsSBAT preferably to natural bile acids. Second, two compounds were identified following the Lipinski's rule of five. Third, other two compounds of molecular weight higher than 500 Da (Mr > 500 Da) were presumed to efficiently block the transporter via a feasible rational screening strategy. Of these candidates, compound 9806452 exhibited the least hepatotoxicity that may enhance drug-likeness properties. CONCLUSIONS: It is proposed that compound 9806452 act as a potential inhibitor toward CsSBAT and further studies are warranted for drug development process against clonorchiasis.
Assuntos
Clonorquíase , Clonorchis sinensis , Fasciola hepatica , Simportadores , Animais , Humanos , Clonorchis sinensis/metabolismo , Fasciola hepatica/metabolismo , Simulação de Dinâmica Molecular , Sódio , Carcinógenos , Proteínas de Helminto/metabolismo , Clonorquíase/tratamento farmacológico , Clonorquíase/diagnóstico , Ácidos e Sais Biliares/farmacologiaRESUMO
Fasciola hepatica is a trematode parasite that infects animals and humans causing fasciolosis, a worldwide-distributed disease responsible for important economic losses and health problems. This disease is of growing public health concern since parasite isolates resistant to the current treatment (triclabendazole) have increasingly been described. F. hepatica infects its vertebrate host after ingestion of the encysted parasite (metacercariae), which are found in the water or attached to plants. Upon ingestion, newly excysted juveniles of F. hepatica (FhNEJ) emerge in the intestinal lumen and cross the intestinal barrier, reach the peritoneum and migrate to the biliary ducts, where adult worms fully develop. Despite the efforts made to develop new therapeutic and preventive tools, to date, protection against F. hepatica obtained in different animal models is far from optimal. Early events of host-FhNEJ interactions are of paramount importance for the infection progress in fasciolosis, especially those occurring at the host-parasite interface. Nevertheless, studies of FhNEJ responses to the changing host environment encountered during migration across host tissues are still scarce. Here, we set-up an ex vivo model coupled with quantitative SWATH-MS proteomics to study early host-parasite interaction events in fasciolosis. After comparing tegument and somatic fractions from control parasites and FhNEJ that managed to cross a mouse intestinal section ex vivo, a set of parasite proteins whose expression was statistically different were found. These included upregulation of cathepsins L3 and L4, proteolytic inhibitor Fh serpin 2, and a number of molecules linked with nutrient uptake and metabolism, including histone H4, H2A and H2B, low density lipoprotein receptor, tetraspanin, fatty acid binding protein a and glutathione-S-transferase. Downregulated proteins in FhNEJ after gut passage were more numerous than the upregulated ones, and included the heath shock proteins HSP90 and alpha crystallin, amongst others. This study brings new insights into early host-parasite interactions in fasciolosis and sheds light on the proteomic changes in FhNEJ triggered upon excystment and intestinal wall crossing, which could serve to define new targets for the prevention and treatment of this widespread parasitic disease.
Assuntos
Fasciola hepatica , Fasciolíase , alfa-Cristalinas , Animais , Catepsinas , Fasciola hepatica/metabolismo , Fasciolíase/parasitologia , Proteínas de Ligação a Ácido Graxo , Glutationa/metabolismo , Proteínas de Helminto/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Proteômica , Receptores de LDL/metabolismo , Transferases/metabolismo , Triclabendazol , alfa-Cristalinas/metabolismoRESUMO
Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Fasciola hepatica , Nitrosaminas , Opistorquíase , Opisthorchis , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/parasitologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/parasitologia , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/genética , Colangiocarcinoma/parasitologia , Cricetinae , Fasciola hepatica/genética , Fasciola hepatica/metabolismo , Fibrose , Granulinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Opistorquíase/complicações , Opistorquíase/parasitologia , Opistorquíase/patologia , Opisthorchis/genética , Opisthorchis/metabolismo , Infecção Persistente , RNA Guia de CinetoplastídeosRESUMO
The identification of extracellular vesicles (EVs) in Fasciola hepatica has provided a new way to understand parasite-host communication. Most of the studies on EVs have focused on the adult stage of F. hepatica, but recently, the presence of EVs from different developmental stages has been reported. To better understand the potential role of EVs in the biology of the parasite and in the infection process, the protein cargo of EVs from embryonated eggs and newly-excysted juvenile (NEJs) flukes cultured up to 28 days, has been analyzed. EVs were isolated by size exclusion chromatography and evaluated by nanoparticle tracking analysis and transmission electron microscopy. LC-MS/MS proteomic analysis of EVs revealed the presence of 23 different proteins from embryonated egg-derived EVs and 29 different proteins from NEJ-derived EVs. Most of the identified proteins had been previously described in EVs from F. hepatica adults, including cytoskeletal proteins, glycolytic enzymes, stress-related proteins and tetraspanins. Nevertheless, EVs from hatching eggs and NEJs exhibited qualitative differences in composition, when compared to EVs form adults, including the absence of cathepsin cysteine peptidases. The differential content of the EVs released by the different developmental stages of the parasite reflect the intense activity of NEJs at this early stage, with several proteins involved in membrane traffic and cell physiology. This new set of identified proteins could help to understand key metabolic, biochemical and molecular mechanisms mediated by EVs that take place upon egg hatching and after parasite excystment.
Assuntos
Vesículas Extracelulares , Fasciola hepatica , Animais , Cromatografia Líquida , Vesículas Extracelulares/metabolismo , Fasciola hepatica/química , Fasciola hepatica/metabolismo , Proteínas de Helminto/metabolismo , Proteômica , Espectrometria de Massas em TandemRESUMO
Chronic infection with liver flukes (such as Clonorchis sinensis) can induce severe biliary injuries, which can cause cholangitis, biliary fibrosis, and even cholangiocarcinoma. The release of extracellular vesicles by C. sinensis (CsEVs) is of importance in the long-distance communication between the hosts and worms. However, the biological effects of EVs from liver fluke on biliary injuries and the underlying molecular mechanisms remain poorly characterized. In the present study, we found that CsEVs induced M1-like activation. In addition, the mice that were administrated with CsEVs showed severe biliary injuries associated with remarkable activation of M1-like macrophages. We further characterized the signatures of miRNAs packaged in CsEVs and identified a miRNA Csi-let-7a-5p, which was highly enriched. Further study showed that Csi-let-7a-5p facilitated the activation of M1-like macrophages by targeting Socs1 and Clec7a; however, CsEVs with silencing Csi-let-7a-5p showed a decrease in proinflammatory responses and biliary injuries, which involved in the Socs1- and Clec7a-regulated NF-κB signaling pathway. Our study demonstrates that Csi-let-7a-5p delivered by CsEVs plays a critical role in the activation of M1-like macrophages and contributes to the biliary injuries by targeting the Socs1- and Clec7a-mediated NF-κB signaling pathway, which indicates a mechanism contributing to biliary injuries caused by fluke infection. However, molecules other than Csi-let-7a-5p from CsEVs that may also promote M1-like polarization and exacerbate biliary injuries are not excluded.
Assuntos
Vesículas Extracelulares/metabolismo , Fasciola hepatica/metabolismo , Macrófagos/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Infecção Persistente/parasitologia , Transdução de Sinais/fisiologiaRESUMO
Fasciola hepatica is a global parasite of humans and their livestock. Regulation of parasite-secreted cathepsin L-like cysteine proteases associated with virulence is important to fine-tune parasite-host interaction. We uncovered a family of seven Kunitz-type (FhKT) inhibitors dispersed into five phylogenetic groups. The most highly expressed FhKT genes (group FhKT1) are secreted by the newly excysted juveniles (NEJs), the stage responsible for host infection. The FhKT1 inhibitors do not inhibit serine proteases but are potent inhibitors of parasite cathepsins L and host lysosomal cathepsin L, S and K cysteine proteases (inhibition constants < 10 nM). Their unusual inhibitory properties are due to (a) Leu15 in the reactive site loop P1 position that sits at the water-exposed interface of the S1 and S1' subsites of the cathepsin protease, and (b) Arg19 which forms cation-π interactions with Trp291 of the S1' subsite and electrostatic interactions with Asp125 of the S2' subsite. FhKT1.3 is exceptional, however, as it also inhibits the serine protease trypsin due to replacement of the P1 Leu15 in the reactive loop with Arg15. The atypical Kunitz-type inhibitor family likely regulate parasite cathepsin L proteases and/or impairs host immune cell activation by blocking lysosomal cathepsin proteases involved in antigen processing and presentation.
Assuntos
Inibidores de Cisteína Proteinase/metabolismo , Fasciola hepatica/metabolismo , Proteínas de Helminto/metabolismo , Helmintos/metabolismo , Parasitos/metabolismo , Inibidores de Serina Proteinase/metabolismo , Animais , Catepsinas/metabolismo , Bovinos , Cisteína/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Humanos , Lisossomos/metabolismo , Filogenia , Serina Proteases/metabolismo , Tripsina/metabolismoRESUMO
Parasite-released extracellular vesicles (EVs) deliver signals to the host immune system that are critical to maintaining the long-term relationship between parasite and host. In the present study, total EVs (FhEVs) released in vitro by adults of the helminth parasite Fasciola hepatica were isolated using a recently described gravity flow method that protects their structural integrity. The FhEVs molecular cargo was defined using proteomic analysis and their surface topology characterised by glycan microarrays. The proteomic analysis identified 618 proteins, 121 of which contained putative N-linked glycosylation sites while 132 proteins contained putative O-linked glycosylation sites. Glycan arrays revealed surface-exposed glycans with a high affinity for mannose-binding lectins indicating the predominance of oligo mannose-rich glycoproteins, as well as other glycans with a high affinity for complex-type N-glycans. When added to bone-marrow derived dendritic cells isolated FhEV induced a novel phenotype that was categorised by the secretion of low levels of TNF, enhanced expression of cell surface markers (CD80, CD86, CD40, OX40L, and SIGNR1) and elevation of intracellular markers (SOCS1 and SOCS3). When FhEV-stimulated BMDCs were introduced into OT-II mice by adoptive transfer, IL-2 secretion from skin draining lymph nodes and spleen cells was inhibited in response to both specific and non-specific antigen stimulation. Immunisation of mice with a suspension of FhEV did not elicit significant immune responses; however, in the presence of alum, FhEVs induced a mixed Th1/Th2 immune response with high antigen specific antibody titres. Thus, we have demonstrated that FhEVs induce a unique phentotype in DC capable of suppressing IL-2 secretion from T-cells. Our studies add to the growing immuno-proteomic database that will be an important source for the discovery of future parasite vaccines and immunotherapeutic biologicals.
Assuntos
Células Dendríticas/metabolismo , Vesículas Extracelulares/metabolismo , Fasciola hepatica/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Fenótipo , Animais , Antígenos de Helmintos/análise , Biomarcadores , Medula Óssea , Citocinas/metabolismo , Modelos Animais de Doenças , Fasciola hepatica/isolamento & purificação , Fasciolíase/imunologia , Fasciolíase/parasitologia , Glicoproteínas , Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polissacarídeos/metabolismo , Proteômica , Linfócitos T/imunologiaRESUMO
The synthetic peroxides OZ78 and MT04 recently emerged as fasciocidal drug candidates. However, the effect of iron on fasciocidal activity and hepatocellular toxicity of these compounds is unknown. We investigated the in vitro fasciocidal activity and hepatocellular toxicity of OZ78 and MT04 in absence and presence of Fe(II)chloride and hemin, and conducted a toxicological study in mice. Studies were performed in comparison with the antimalarial artesunate (AS), a semisynthetic peroxide. Fasciocidal effects of OZ78 and MT04 were confirmed and enhanced by Fe2+ or hemin. In HepG2 cells, AS reduced cellular ATP and impaired membrane integrity concentration-dependently. In comparison, OZ78 or MT04 were not toxic at 100 µM and reduced the cellular ATP by 13% and 19%, respectively, but were not membrane-toxic at 500 µM. The addition of Fe2+ or hemin increased the toxicity of OZ78 and MT04 significantly. AS inhibited complex I, II, and IV of the mitochondrial electron transport chain, and MT04 impaired complex I and II, whereas OZ78 was not toxic. All three compounds increased cellular reactive oxygen species (ROS) concentration-dependently, with a further increase by Fe2+ or hemin. Mice treated orally with up to 800 mg OZ78, or MT04 showed no relevant hepatotoxicity. In conclusion, we confirmed fasciocidal activity of OZ78 and MT04, which was increased by Fe2+ or hemin. OZ78 and MT04 were toxic to HepG2 cells, which was explained by mitochondrial damage associated with ROS generation in the presence of iron. No relevant hepatotoxicity was observed in mice in vivo, possibly due to limited exposure and/or high antioxidative hepatic capacity.
Assuntos
Adamantano/análogos & derivados , Fasciola hepatica/efeitos dos fármacos , Fasciola hepatica/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Ferro/metabolismo , Compostos de Espiro/farmacologia , Adamantano/síntese química , Adamantano/química , Adamantano/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Cromatografia Líquida , Células Hep G2 , Humanos , Ferro/farmacologia , Microssomos Hepáticos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Compostos de Espiro/síntese química , Compostos de Espiro/química , Espectrometria de Massas em TandemRESUMO
The characteristics of parasitic infections are often tied to host behavior. Although most studies have investigated definitive hosts, intermediate hosts can also play a role in shaping the distribution and accumulation of parasites. This is particularly relevant in larval stages, where intermediate host's behavior could potentially interfere in the molecules secreted by the parasite into the next host during infection. To investigate this hypothesis, we used a proteomic approach to analyze excretion/secretion products (ESP) from Fasciola hepatica newly excysted juveniles (NEJ) derived from two intermediate host species, Lymnaea viatrix and Pseudosuccinea columella. The two analyzed proteomes showed differences in identity, abundance, and functional classification of the proteins. This observation could be due to differences in the biological cycle of the parasite in the host, environmental aspects, and/or host-dependent factors. Categories such as protein modification machinery, protease inhibitors, signal transduction, and cysteine-rich proteins showed different abundance between samples. More specifically, differences in abundance of individual proteins such as peptidyl-prolyl cis-trans isomerase, thioredoxin, cathepsin B, cathepsin L, and Kunitz-type inhibitors were identified. Based on the differences identified between NEJ ESP samples, we can conclude that the intermediate host is a factor influencing the proteomic profile of ESP in F. hepatica.
Assuntos
Fasciola hepatica/metabolismo , Proteínas de Helminto/metabolismo , Lymnaea/parasitologia , Proteômica , Caramujos/parasitologia , Animais , Anidrases Carbônicas/classificação , Anidrases Carbônicas/metabolismo , Proteínas de Helminto/classificação , Larva/metabolismo , Peptídeo Hidrolases/classificação , Peptídeo Hidrolases/metabolismo , Peroxirredoxinas/classificação , Peroxirredoxinas/metabolismo , Inibidores de Proteases/classificação , Inibidores de Proteases/metabolismo , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/metabolismoRESUMO
Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.
Assuntos
Endocitose , Vesículas Extracelulares/metabolismo , Fasciola hepatica/metabolismo , Proteínas de Helminto/metabolismo , Proteínas de Membrana/metabolismo , Animais , Células Cultivadas , Imuno-Histoquímica , Macrófagos/metabolismo , Proteômica , RatosRESUMO
The secreted growth factor granulin (GRN) is upregulated during diverse epithelial cancers. GRN stimulates cell growth and development while inhibiting apoptosis. Orthologues of vertebrate granulins evolved in other animals including the liver fluke Opisthorchis viverrini. Curiously, liver fluke granulin, termed Ov-GRN-1 promotes cholangiocarcinogenesis during chronic opisthorchiasis but, by contrast, limited information is available concerning mammalian GRN during liver fluke infection-induced cholangiocarcinoma (CCA). Here we investigated the expression of mammalian granulin in the O. viverrini-associated a hamster model of opisthorchiasis and liver fluke infection-induced CCA. Male Syrian golden hamsters were assigned to one of four treatment groups, each group included 30 hamsters: 1) normal (control), 2) infected with O. viverrini (OV); 3) exposed to N-dimethylnitrosamine in drinking water (DMN); and 4) infected with O. viverrini and exposed to DMN (OVDMN). Immunohistochemistry using an anti-granulin specific probe for mammalian granulin was undertaken to monitor expression and location in hepatobiliary tissues of the hamsters. In parallel, cognate studies of transcription of mRNA and protein. Histopathological examination revealed development of proliferative lesions from the onset and eruption of CCA onwards, an outcome that was most prominent in the OVDMN hamsters. Proliferating cell nuclear antigen (PCNA) index rose continuously from initiation of infection and increased with lesion progression in OV, DMN and markedly in OVDMN hamsters. Expression of GRN in biliary was elevated in biliary epithelial cells in CCA lesions in hamsters in the DMN and OVDMN groups. Expression of GRN as assayed by western blot and RT-PCR reflected the same trend as seen with PCNA. Together the histopathogical and molecular assay based findings revealed marked expression of granulin during cholangiocarcinoma in these hamsters, and highlighted the prospect that granulin represents a potential prognostic marker for cholangiocarcinoma.
Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Carcinogênese/metabolismo , Colangiocarcinoma/metabolismo , Granulinas/metabolismo , Opistorquíase/metabolismo , Opisthorchis/patogenicidade , Animais , Neoplasias dos Ductos Biliares/patologia , Carcinogênese/patologia , Proliferação de Células/fisiologia , Colangiocarcinoma/patologia , Cricetinae , Células Epiteliais/patologia , Células Epiteliais/virologia , Fasciola hepatica/metabolismo , Fasciola hepatica/patogenicidade , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Opistorquíase/parasitologia , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
Granulins are a family of growth factors involved in cell proliferation. The liver-fluke granulin, Ov-GRN-1, isolated from a carcinogenic liver fluke Opisthorchis viverrini, can significantly accelerate wound repair in vivo and in vitro. However, it is difficult to express Ov-GRN-1 in recombinant form at high yield, impeding its utility as a drug lead. Previously we reported that a truncated analogue ( Ov-GRN12-35_3s) promotes healing of cutaneous wounds in mice. NMR analysis of this analogue indicates the presence of multiple conformations, most likely as a result of proline cis/ trans isomerization. To further investigate whether the proline residues are involved in adopting the multiple confirmations, we have synthesized analogues involving mutation of the proline residues. We have shown that the proline residues have a significant influence on the structure, activity, and folding of Ov-GRN12-35_3s. These results provide insight into improving the oxidative folding yield and bioactivity of Ov-GRN12-35_3s and might facilitate the development of a novel wound healing agent.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fasciola hepatica/química , Granulinas/farmacologia , Proteínas de Helminto/farmacologia , Fragmentos de Peptídeos/farmacologia , Dermatopatias/prevenção & controle , Cicatrização/efeitos dos fármacos , Animais , Fasciola hepatica/metabolismo , Fasciolíase/parasitologia , Feminino , Granulinas/química , Proteínas de Helminto/química , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/química , Conformação ProteicaRESUMO
Glycans present in biological glycoconjugates have several structural and functional roles. Elucidation of glycan structure and biological function is critical to understand their role in physiological and pathogenic process, enabling the development of diagnostic methods and disease treatment. Immobilized glycosidases are powerful tools for glycan analysis, as they are able to remove specific carbohydrates without altering the protein structure. Here we describe the individual immobilization of Aspergillus oryzae ß-galactosidase and Canavalia ensiformis α-mannosidase onto agarose and silica magnetic nanoparticles activated with cyanate ester groups. High immobilization yields (70-90%) were achieved, keeping above 60% of its original activity. Immobilized glycosidases were effective in the selective deglycosylation of model glycoproteins and a Fasciola hepatica lysate, evidenced by a decrease in specific lectin recognition of 40-50% after enzymatic deglycosylation. Immobilized glycosidases were reused for several deglycosylation cycles without loss of effectiveness. Their use was extended to the elucidation of the glycan role of native glycoconjugates. A decrease in the recognition of lactoferrin treated with α-mannosidase by a C-type lectin receptor, DC-SIGN was found. Also the specific deglycosylation of a F. hepatica lysate demonstrated the relevance of mannosylated glycans in the induction of Th2/Treg immune responses during the infection. Our results show successful immobilization of specific glycosidases in nano-supports and validate their utility to identify glycans biological functions.
Assuntos
Enzimas Imobilizadas/química , Glicoconjugados/análise , Glicômica , Nanopartículas de Magnetita , alfa-Manosidase/química , beta-Galactosidase/química , Animais , Aspergillus oryzae/enzimologia , Medula Óssea/metabolismo , Canavalia/enzimologia , Bovinos , Moléculas de Adesão Celular/metabolismo , Células Dendríticas/metabolismo , Enzimas Imobilizadas/metabolismo , Fasciola hepatica/metabolismo , Glicoconjugados/isolamento & purificação , Glicoproteínas/metabolismo , Glicosilação , Lactoferrina/metabolismo , Lectinas/metabolismo , Lectinas Tipo C/metabolismo , Camundongos Endogâmicos BALB C , Polissacarídeos/metabolismo , Receptores de Superfície Celular/metabolismo , alfa-Manosidase/metabolismo , beta-Galactosidase/metabolismoRESUMO
Fascioliasis is a parasitic disease of grazing livestock and a threat to global food security by significantly reducing the production value of sheep, goats and cattle. Moreover, the zoonotic parasite is also a re-emerging food borne threat to human populations. Driven by climate change, the prevalence of fascioliasis is set to increase. Efforts to control the main causative agent, Fasciola hepatica, are hampered by short lived chemotherapy approaches that are becoming increasingly obsolete due to therapeutic failure and resistance. A protective vaccine is urgently needed. A recombinant form of Sigma class glutathione transferase (Hematopoietic Prostaglandin D synthase) from F. hepatica (FhGSTS1) with confirmed prostaglandin synthase activity shows immune-modulation activity via suppression of Th17 responses in host dendritic cells. In vaccine trials recombinant FhGSTS1 reduces liver pathology but not worm burden. Native FhGSTS1 is yet to be tested for immune-modulation activities or for vaccine potential, primarily due to the technical difficulty in purifying FhGST-S1 away from the other more abundant GST members in F. hepatica cytosol. This paper reports a pipeline for the purification of native FhGSTS1 using a two-step process consisting of glutathione-agarose affinity and cationic exchange chromatography. The methodology allows for the isolation of purified and active FhGSTS1 or Sigma GSTs from other sources for analytical biochemical and immunological studies.
Assuntos
Fasciola hepatica/enzimologia , Fasciolíase/veterinária , Glutationa Transferase/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Doenças dos Ovinos/parasitologia , Animais , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Fasciola hepatica/química , Fasciola hepatica/genética , Fasciola hepatica/metabolismo , Fasciolíase/parasitologia , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Ponto Isoelétrico , OvinosRESUMO
Fasciola hepatica are trematodes that reside in the bile ducts of mammals. Infection causes US$3 billion in losses annually in animal production and is considered a zoonosis of growing importance. An under-represented area in F. hepatica research has been the examination of the different immunomodulatory abilities of various parasite isolates on the host immune system. In this paper, this issue was explored, with the bovine macrophage cell line "BOMA". The cells were matured by LPS treatment and stimulated with excretory/secretory antigens (ES) from two Fasciola hepatica isolates: a laboratory isolate "Weybridge" (Fh-WeyES) and a wild isolate (Fh-WildES). As expected, stimulation with antigen mixtures with highly similar compositions resulted in mild transcriptomic differences. However, there were significant differences in cytokine levels. Compared to Fh-WeyES, exposure to Fh-WildES upregulated 27 and downregulated 30 genes. Fh-ES from both isolates diminished the release of TNF-α, whereas only Fh-WildES decreased IL-10 secretion. Neither Fh-WeyES nor Fh-WildES had an impact on IL-12 release. Our results indicate that various isolates can have different immunomodulatory abilities and impacts on the bovine immune system.
Assuntos
Antígenos de Helmintos/metabolismo , Doenças dos Bovinos/genética , Doenças dos Bovinos/parasitologia , Fasciola hepatica/metabolismo , Fasciolíase/veterinária , Interleucina-10/metabolismo , Macrófagos/parasitologia , Animais , Antígenos de Helmintos/genética , Bovinos , Doenças dos Bovinos/metabolismo , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Fasciola hepatica/genética , Fasciola hepatica/isolamento & purificação , Fasciolíase/genética , Fasciolíase/metabolismo , Fasciolíase/parasitologia , Interações Hospedeiro-Parasita , Interleucina-10/genética , Interleucina-12/genética , Interleucina-12/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Recently, we reported that a native Fasciola hepatica fatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markers in vivo and in vitro. Because the purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up, this study accomplished the task of optimizing the expression and purification of a recombinant form of FABP (Fh15). Additionally, we ascertained whether this molecule could exhibit a similar suppressive effect on TLR-stimulation and inflammatory cytokine expression from macrophages than those previously demonstrated for the native molecule. Results demonstrated that Fh15 suppresses the expression of IL-1ß and TNFα in murine macrophages and THP1 Blue CD14 cells. Additionally, Fh15 suppress the LPS-induced TLR4 stimulation. This effect was not impaired by a thermal denaturing process or blocked by the presence of anti-Fh12 antibodies. Fh15 also suppressed the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti-inflammatory drug in preclinical studies in the near future.