Molecular characterization of the interplay between Fasciola hepatica juveniles and laminin as a mechanism to adhere to and break through the host intestinal wall

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

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