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

Abstract

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 impede this function by vaccination or drug design.

Fig 1. Recombinant expression and purification of F. hepatica enolase (rFhENO).

(A) 4–20% SDS-PAGE gel of time points for recombinant expression of rFhENO in BL21 ClearColi system. T0, T1, T2, T3, and T4 refer to 0, 1, 2, 3, and 4 hours post induction with IPTG, respectively. (B) Corresponding immunoblot of time points of recombinant expression probed with an anti-polyhistidine antibody at 1:10,000 dilution. (C) 4–20% SDS-PAGE gel of NiNTA-Agarose affinity chromatography purification steps of rFhENO; (1) cell pellet of T4, (2) supernatant after cell lysis, (3) run-through of cell lysate, (4) wash of the purification column, and (5) rFhENO eluted from the column with 250 mM imidazole. (D) Corresponding immunoblot of affinity chromatography purification steps probed with an anti-polyhistidine antibody at 1:10,000 dilution. MW = molecular weight in kilodaltons.

Fig 2. Size-exclusion chromatography (SEC) and enzyme activity assay of rFhENO.

A SEC readout of separated dimeric and monomeric peaks of rFhENO are calculated to be ~94 kDa and ~47 kDa, respectively. The black arrows indicate the molecular weight size markers, alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa), resolved on the same SEC. Peak 9.81 (dimer) eluted at 9.81 mL between the molecular markers 150 and 66 kDa, indicating a dimeric form. Peak 12.16 (monomer) eluted at 12.16 mL between the molecular weight markers 66 and 29 kDa, indicative of the monomeric form. The fractions that eluted within these two peaks were pooled and assayed for enzyme activity at 0.1 μM. The rFhENO activity is graphically represented in orange and expressed as nmoles H₂O₂ generated per 1 μM of enzyme. Total purified rFhENO not separated by SEC was also assayed at 0.1 μM and its activity is expressed in the figure (inset on the left), to serve as a comparison of enzyme activity of the eluted peaks.

Fig 3. Detection of F. hepatica enolase in adult fluke extracts by immunoblot.

Samples of the different adult liver fluke extracts (2 μg/lane) were probed with (A) Rabbit anti-rFhENO polyclonal antibody at a 1:1,000 dilution. (B) Pre–immune rabbit sera at a dilution of 1:1,000. MW: molecular weight in kilodaltons, (1) adult somatic extract (FhSoma), (2) adult tegumental extract (FhTeg), (3) total adult excretory/secretory products (FhES), and (4) concentrated size-separated fraction excretory secretory products (fFhES).

Fig 4. Immunolocalisation of native FhENO in F. hepatica newly excysted juveniles (FhNEJ).

Confocal microscopy of FhNEJ probed with pre-immune anti-rFhCL3 sera (FhCL3 PI), anti-rFhCL3 polyclonal antibody (FhCL3), pre-immune anti-rFhENO sera (FhENO PI), and anti-rFhENO polyclonal antibody (FhENO), from left to right. Positive antibody binding is show in green, whilst the musculature of the parasites was counter-stained with TRITC, which appears as red labelling. OUT and IN indicate the external and internal surfaces of the parasites, respectively. The main FhNEJ’s features are highlighted: G: Gut; OS: Oral sucker; VS: Ventral sucker. Scale bar: 50 μM.

Fig 5. Immunolocalisation of native FhENO in the tegument of adult F. hepatica.

F. hepatica adult JB-4 resin-embedded sections in the area of the tegument (A–C) and gut (D–F). Sections were probed with (A and D) pre-immune anti-rFhCL1pp sera, (B and E) anti-rFhCL1pp polyclonal antibody, (C and F) anti-rFhENO polyclonal antibody. Scale bar: 50 μM.

Fig 6. Immunogenicity of FhENO in sheep experimentally infected with F. hepatica.

(A) Sera collected at different time points from sheep experimentally infected with F. hepatica was analysed for the presence of antibodies against rFhENO, and, for comparison, to another glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase (rFhGAPDH2). (i) 0 weeks post infection (WPI), (ii) 7 WPI, (iii) 11 WPI, and (iv) 15 WPI. The recombinant antigens, rFhGAPDH2 (lane 1) and rFhENO (lane 2). (B) Serum from experimentally infected sheep (5) was analysed by ELISA for IgG response to rFhENO and cathepsin L1 (rFhCL1) at 0 WPI, 4 WPI, 13 WPI, and 20WPI. MW: molecular weight in kilodaltons.

Fig 7. Protein-protein interaction assays to examine ability of rFhENO to bind fibronectin (FN) and laminin (LM).

Binding of rFhENO to FN (A) and LM (B) was analysed over a range of FN and LM concentrations using a microtiter plate method. Wells coated with 1% BSA serve as negative controls, and wells coated with FN or LM serve as positive controls for antibody binding. Each data point represents the mean ± SD of three technical replicates. Asterisks indicate significant differences between rFhENO and 1% BSA (**p≤0.01, ***p≤0.001; Student’s t-test).

Fig 8. Plasminogen (PLG) binding to rFhENO and stimulation of plasmin generation from bound PLG.

PLG binding to rFhENO was analysed using a microtiter plate method (A) in the presence or absence of the lysine analogue ε-ACA (B). rFhENO was incubated with PLG in the presence of t-PA (C) or u-PA (D) and a plasmin-specific chromogenic substrate and plasmin generation was measured by monitoring substrate cleavage over time. Wells containing 1% BSA instead of rFhENO serve as negative controls. Data points represent the mean ± SD of three technical replicates. Asterisks indicate significant differences between rFhENO and 1% BSA (*p≤0.05, **p≤0.01, ***p≤0.001; Student’s t-test [A], one-way ANOVA [B-D]).