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

Abstract

Background The 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 their definitive location, 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 Findings A tegument-enriched antigenic extract of FhNEJ (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 bidimensional electrophoresis coupled to mass spectrometry analysis, and the interactions were validated using FhNEJ recombinant proteins. Conclusions/Significance Our 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.

Moonlighting on theFasciola hepaticategument: enolase, a glycolytic enzyme, interacts with the extracellular matrix and fibrinolytic system of the host

“…In fact, enolase has been recently reported as the most commonly identified PLG receptor in parasitic helminths in a scoping review conducted by our group [19]. Our results also confirm those obtained twenty years ago by Bernal et al [64], who identified an enolase in the excretory/secretory products of F. hepatica as a potential 20 PLG receptor, and complement our own findings in which we characterize the potential of the tegument of F. hepatica juvenile [26] and adult worms [65] to interact with the fibrinolytic system of their host. Indeed, the utilization of this mechanism throughout the life cycle of F. hepatica could have different functions adapted to the changing environments faced by the parasite.…”

“…rFhENO was tested in a PLG activation assay to explore the enhancement of plasmin generation by this recombinant protein following the methodology described by Serrat et al [26]. Briefly, wells containing 2 µg of human PLG (Origene) were incubated with 3 µg of D-Val-Leu-Lys 4-nitroanilide dihydrochloride chromogenic substrate (S-2251) (Sigma-Aldrich) in the presence of 1 µg of rFhENO or 1% BSA as negative control in a test volume of 100 µL.…”

“…The ability of rFhENO to bind host FN, LM and/or PLG was assayed by enzyme-linked immunosorbent assays (ELISA) as described in Serrat et al [26,27]. Briefly, multi-well microplates (Costar) were coated with 0.5 µg/well of rFhENO or 1% BSA as negative control, blocked and then incubated with increasing amounts of human FN (Santa Cruz Biotechnology) (from 0 µg to 2 µg), LM (Santa Cruz Biotechnology) (from 0 µg to 2 µg) or PLG (Origene) (from 0 µg to 3 µg).…”

“…Briefly, multi-well microplates (Costar) were coated with 0.5 µg/well of rFhENO or 1% BSA as negative control, blocked and then incubated with increasing amounts of human FN (Santa Cruz Biotechnology) (from 0 µg to 2 µg), LM (Santa Cruz Biotechnology) (from 0 µg to 2 µg) or PLG (Origene) (from 0 µg to 3 µg). After incubation with the corresponding antibodies and chromogen [26,27], optical densities (OD) were measured at 492 nm in a Multiskan GO spectrophotometer (Thermo Fisher Scientific). A competition assay to study the involvement of lysine residues in PLG binding was performed by including 0-100 mM of the lysine analogue ε-aminocaproic acid (ε-ACA) during PLG incubation with rFhENO.…”

“…Recently, we have described how F. hepatica interacts with host ECM and the fibrinolytic system at the intestinal level as a potential strategy used by the parasite to initiate the migration process [26,27]. Nevertheless, further research is needed to define and characterize the parasitic molecules involved in these adhesin-like mechanisms.…”