Biology subjects

Gonzalez-Miguel, J.

Publications and source records attributed to Gonzalez-Miguel, J..

2 recordsLinked to original sources

bioRxiv2024

Moonlighting on the Fasciola hepatica tegument: enolase, a glycolytic enzyme, interacts with the extracellular matrix and fibrinolytic system of the host

Enolase is a 47 kDa enzyme that functions within the glycolysis and gluconeogenesis pathways involved in the reversible conversion of D-2-phosphoglycerate (2PGA) to phosphoenolpyruvate (PEP). However, in the context of host-pathogen interactions, enolase from different species of parasites, fungi and bacteria have been shown to contribute to adhesion processes by binding to proteins of the host extracellular matrix (ECM), such as fibronectin (FN) or laminin (LM). In addition, enolase is a plasminogen (PLG)-binding protein and induces its activation to plasmin, the main protease of the host fibrinolytic system. These secondary moonlighting functions of enolase are suggested to facilitate pathogen migration through host tissues. This study aims to uncover the moonlighting role of enolase from the parasite Fasciola hepatica, shedding light on its relevance to host-parasite interactions in fasciolosis, a global zoonotic disease of increasing concern. A purified recombinant form of F. hepatica enolase (rFhENO), functioning as an active homodimeric glycolytic enzyme of [~]94 kDa, was successfully obtained, fulfilling its canonical role. Immunoblotting studies on adult worm extracts showed that the enzyme is present in the tegument and the excretory/secretory products of the parasite, which supports its key role at the host-parasite interface. Confocal immunolocalisation studies of the protein in newly excysted juveniles and adult worms also localised its expression within the parasite tegument. Finally, we showed by ELISA that rFhENO can act as a parasitic adhesin by binding host LM, but not FN. rFhENO also binds PLG and enhances its conversion to plasmin in the presence of the tissue-type and urokinase-type PLG activators (t-PA and u-PA). This moonlighting adhesion-like function of the glycolytic protein enolase could contribute to the mechanisms by which F. hepatica efficiently invades and migrates within its host and encourages further research efforts that are designed to impediment this function by vaccination or drug design. AUTHOR SUMMARYFasciola hepatica is a parasitic worm causing fasciolosis, primarily affecting herbivorous mammals and posing a significant veterinary problem. Furthermore, it is a zoonosis, meaning it can be transmitted to humans. F. hepatica enters the definitive host through ingestion of contaminated aquatic plants, migrating through the intestine to settle in the liver bile ducts, where it matures into the adult stage. To migrate, it utilizes various invasion strategies, including the use of multifunctional proteins, known as moonlighting. In this study, we produced and molecularly characterized the parasitic enzyme enolase as a moonlighting protein to understand F. hepatica invasion mechanisms. We produced recombinant enolase with glycolytic activity, its canonical function in parasite energy production. Additionally, we localised this enzyme in the parasites tegument, in direct contact with the host, and demonstrated its ability to elicit an immune response early in ovine infection. Finally, we demonstrated the ability of enolase to interact with the extracellular matrix and the hosts fibrinolysis, a proteolytic system responsible for dissolving blood clots. These secondary functions of F. hepatica enolase, described here for the first time, along with its localisation and immunogenicity, suggest this protein as an interesting antigen for fasciolosis diagnosis and/or control.

molecular biology↗

bioRxiv2022

Fasciola hepatica juveniles interact with the host fibrinolytic system as a potential early-stage invasion mechanism

BackgroundThe trematode Fasciola hepatica is the most widespread causative agent of fasciolosis, a parasitic disease that mainly affects humans and ruminants worldwide. During F. hepatica infection, 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 how F. hepatica traverses 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 obtained in 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 genus Fasciola, of which F. hepatica stands 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.

molecular biology↗