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BackgroundThe trematodeFasciola hepaticais the most widespread causative agent of fasciolosis, a parasitic disease that mainly affects humans and ruminants worldwide. DuringF. hepaticainfection, newly excysted juveniles (FhNEJ) emerge in the duodenum of the mammalian host and migrate towards the definitive location of the parasite, the intra-hepatic biliary ducts. Understanding howF. hepaticatraverses the intestinal wall and migrates towards the liver is pivotal for the development of more successful strategies against fasciolosis. The central enzyme of the mammalian fibrinolytic system is plasmin, a serine protease whose functions are exploited by a number of parasite species owing to its broad spectrum of substrates, including components of tissue extracellular matrices. The aim of the present work is to understand whether FhNEJ co-opt the functions of their host fibrinolytic system as a mechanism to facilitate trans-intestinal migration.Methodology/Principal FindingsAn FhNEJ tegument protein extract (FhNEJ-Teg) was obtainedin vitro, and its capability to bind the zymogen plasminogen (PLG) and enhance its conversion to the active protease, plasmin, were analyzed by a combination of enzyme-linked immunosorbent, chromogenic and immunofluorescence assays. Additionally, PLG-binding proteins in FhNEJ-Teg were identified by 2D electrophoresis coupled to mass-spectrometry analysis, and the interactions were validated using FhNEJ recombinant proteins.Conclusions/SignificanceOur results show that FhNEJ-Teg contains proteins that bind PLG and stimulate its activation to plasmin, which could facilitate the traversal of the intestinal wall by FhNEJ and contribute to the successful establishment of the parasite within its mammalian host. Altogether, our findings contribute to a better understanding of host-parasite relationships during early fasciolosis and may be exploited from a pharmacological and/or immunological perspective for the development of treatment and control strategies against this global disease.Author SummaryFasciolosis is a disease caused by parasites of the genusFasciola, of whichF. hepaticastands out as it has successfully spread all over the world and infects humans and animals throughout the entire global geography. Definitive hosts become infected by ingestion of aquatic plants or water contaminated with metacercariae, which excyst in the duodenum and release the so-called newly excysted juvenile flukes (FhNEJ). FhNEJ traverse the intestinal wall and evolve into immature parasites that migrate through the peritoneum and liver parenchyma until they reach their definitive location inside the major biliary ducts, where adult worms develop and egg shedding starts. In order to cross the intestinal wall, FhNEJ are endowed with a repertoire of proteases that degrade components of the intestinal extracellular matrix, and we hypothesized that they may also co-opt the proteolytic functions of plasmin, the central enzyme of the mammalian fibrinolytic system, to migrate more efficiently across host tissues. In this study, we demonstrate that FhNEJ express proteins on their tegument surface that interact with plasminogen, the zymogen of plasmin, and stimulate its conversion into its active form, which could potentially be used for trans-intestinal migration and contribute to the successful establishment of the parasite within its mammalian host.

19 páginas, 6 figuras, 1 tabla, Fasciola hepatica is the main causative agent of fasciolosis, a zoonotic parasitic disease of growing public health concern. F. hepatica metacercariae are ingested by the host and excyst in the intestine, thereby releasing the newly excysted juveniles (FhNEJ), which traverse the gut wall and migrate towards the biliary ducts. Since blocking F. hepatica development is challenging after crossing of the intestinal wall, targeting this first step of migration might result in increased therapeutic success. The intestinal extracellular matrix (ECM) is constituted by a network of structural proteins, including laminin (LM) and fibronectin (FN), that provide mechanical support while acting as physical barrier against intestinal pathogens. Here, we employed ELISA and immunofluorescent assays to test for the presence of LM- and FN-binding proteins on a tegument-enriched antigenic fraction of FhNEJ, and further determined their identity by two-dimensional electrophoresis coupled to mass spectrometry. Additionally, we performed enzymatic assays that revealed for the first time the capability of the juvenile-specific cathepsin L3 to degrade LM, and that LM degradation by FhNEJ proteins is further potentiated in the presence of host plasminogen. Finally, a proteomic analysis showed that the interaction with LM triggers protein changes in FhNEJ that may be relevant for parasite growth and adaptation inside the mammalian host. Altogether, our study provides valuable insights into the molecular interplay between FhNEJ and the intestinal ECM, which may lead to the identification of targetable candidates for the development of more effective control strategies against fasciolosis., This work was supported by the JIN project ‘ULYSSES’ (RTI2018-093463-J-100) funded by the Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER, UE). We also acknowledge the financial support of the project “CLU-2019-05–IRNASA-CSIC Unit of Excellence”, funded by the Junta de Castilla y León and co-funded by the European Union (FEDER “Europe drives our growth”), and the Programme for strengthening research structures “Stairway to excellence” internationalisation aid, co-funded by the European Regional Development Fund. M.S.L. acknowledges the financial support of the Spanish Ministry of Science and Innovation (Project PID2019-108782RB-C22). J.S. and D.B.R. acknowledge the support of the Junta de Castilla y León for their Predoctoral contracts. M.L.G. acknowledges the support of the Spanish Ministry of Science and Innovation for her FPU Predoctoral contract. J.G.M. is supported by the ‘Ramón y Cajal’ program (RYC2020-030575-I) of the Ministerio de Ciencia e Innovación. The proteomics laboratory where LC-MS/MS analysis was performed is a member of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I + D+i 2013–2016, funded by the Instituto de Salud Carlos III (ISCIII) and the European Regional Development Fund (ERDF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

[EN] The opioid system is well conserved among species and plays a critical role in pain and addiction systems. The use of zebrafish as an experimental model to study development and genetics is extraordinary and has been proven to be relevant for the study of different diseases. The main drawback to its use for the analysis of different pathologies is the lack of protein tools. Antibodies that work in other models are not suitable for zebrafish due to the low degree of homology that exists among the opioid receptor protein sequences in different species. Here we report the successful generation and characterization of antibodies against the mu, delta 1 and delta 2 opioid receptors in zebrafish. The antibodies obtained, which are specific for each receptor due to the use of the C-terminus as antigens, work for Western blotting and immunohistochemistry. In addition, the antibodies against mu and delta 1 opioid receptors, but not those against delta 2, are able to immunoprecipitate the corresponding receptor from zebrafish lysates. The development of opioid receptor antibodies is an asset to the further study of the endogenous opioid system in zebrafish.