Fasciola hepatica Cathepsin L Zymogens: Immuno-Proteomic Evidence for Highly Immunogenic Zymogen-Specific Conformational Epitopes to Support Diagnostics Development.

Fasciola hepatica, the common liver fluke and causative agent of zoonotic fasciolosis, impacts on food security with global economic losses of over $3.2 BN per annum through deterioration of animal health, productivity losses, and livestock death and is also re-emerging as a foodborne human disease. Cathepsin proteases present a major vaccine and diagnostic target of the F. hepatica excretory/secretory (ES) proteome, but utilization in diagnostics of the highly antigenic zymogen stage of these proteins is surprisingly yet to be fully exploited. Following an immuno-proteomic investigation of recombinant and native procathepsins ((r)FhpCL1), including mass spectrometric analyses (DOI: 10.6019/PXD030293), and using counterpart polyclonal antibodies to a recombinant mutant procathepsin L (anti-rFhΔpCL1), we have confirmed recombinant and native cathepsin L zymogens contain conserved, highly antigenic epitopes that are conformationally dependent. Furthermore, using diagnostic platforms, including pilot serum and fecal antigen capture enzyme-linked immunosorbent assay (ELISA) tests, the diagnostic capacities of cathepsin L zymogens were assessed and validated, offering promising efficacy as markers of infection and for monitoring treatment efficacy.

Proteomic Analysis of Extracellular Vesicles From Fasciola hepatica Hatching Eggs and Juveniles in Culture.

The identification of extracellular vesicles (EVs) in Fasciola hepatica has provided a new way to understand parasite-host communication. Most of the studies on EVs have focused on the adult stage of F. hepatica, but recently, the presence of EVs from different developmental stages has been reported. To better understand the potential role of EVs in the biology of the parasite and in the infection process, the protein cargo of EVs from embryonated eggs and newly-excysted juvenile (NEJs) flukes cultured up to 28 days, has been analyzed. EVs were isolated by size exclusion chromatography and evaluated by nanoparticle tracking analysis and transmission electron microscopy. LC-MS/MS proteomic analysis of EVs revealed the presence of 23 different proteins from embryonated egg-derived EVs and 29 different proteins from NEJ-derived EVs. Most of the identified proteins had been previously described in EVs from F. hepatica adults, including cytoskeletal proteins, glycolytic enzymes, stress-related proteins and tetraspanins. Nevertheless, EVs from hatching eggs and NEJs exhibited qualitative differences in composition, when compared to EVs form adults, including the absence of cathepsin cysteine peptidases. The differential content of the EVs released by the different developmental stages of the parasite reflect the intense activity of NEJs at this early stage, with several proteins involved in membrane traffic and cell physiology. This new set of identified proteins could help to understand key metabolic, biochemical and molecular mechanisms mediated by EVs that take place upon egg hatching and after parasite excystment.

Construction of a multiepitope vaccine candidate against Fasciola hepatica: an in silico design using various immunogenic excretory/secretory antigens.

BACKGROUND: Fasciola hepatica is an important pathogen that causes liver fluke disease in definitive hosts such as livestock animals and humans. Various excretory/secretory products have been used in serological diagnosis and vaccination studies targeting fasciolosis. There are no commercial vaccines against fasciolosis yet. Bioinformatic analysis based on computational methods have lower cost and provide faster output compared to conventional vaccine antigen discovery techniques. The aim of this study was to predict B- and T-cell specific epitopes of four excretory/secretory antigens (Kunitz-type serine protease inhibitor, cathepsin L1, helminth defense molecule, and glutathione S-transferase) of Fasciola hepatica and to construct a multiepitope vaccine candidate against fasciolosis. METHODS AND RESULTS: Initially, nonallergic and the highest antigenic B- and T- cell epitopes were selected and then, physico-chemical parameters, secondary and tertiary structures of designed multiepitope vaccine candidate were predicted. Tertiary structure was refined and validated using online bioinformatic tools. Linear and discontinuous B-cell epitopes and disulfide bonds were determined. Finally, molecular docking analysis for MHC-I and MHC-II receptors was performed. CONCLUSION: This multi-epitope vaccine candidate antigen, with high immunological properties, can be considered as a promising vaccine candidate for animal experiments and wet lab studies.

Recombinant Fasciola hepatica Fatty Acid Binding Protein as a Novel Anti-Inflammatory Biotherapeutic Drug in an Acute Gram-Negative Nonhuman Primate Sepsis Model.

Due to their phylogenetic proximity to humans, nonhuman primates (NHPs) are considered an adequate choice for a basic and preclinical model of sepsis. Gram-negative bacteria are the primary causative of sepsis. During infection, bacteria continuously release the potent toxin lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to death. Our previous research has demonstrated in vitro and in vivo using a mouse model of septic shock that Fh15, a recombinant variant of the Fasciola hepatica fatty acid binding protein, acts as an antagonist of Toll-like receptor 4 (TLR4) suppressing the LPS-induced proinflammatory cytokine storm. The present communication is a proof-of concept study aimed to demonstrate that a low-dose of Fh15 suppresses the cytokine storm and other inflammatory markers during the early phase of sepsis induced in rhesus macaques by intravenous (i.v.) infusion with lethal doses of live Escherichia coli. Fh15 was administered as an isotonic infusion 30 min prior to the bacterial infusion. Among the novel findings reported in this communication, Fh15 (i) significantly prevented bacteremia, suppressed LPS levels in plasma, and the production of C-reactive protein and procalcitonin, which are key signatures of inflammation and bacterial infection, respectively; (ii) reduced the production of proinflammatory cytokines; and (iii) increased innate immune cell populations in blood, which suggests a role in promoting a prolonged steady state in rhesus macaques even in the presence of inflammatory stimuli. This report is the first to demonstrate that a F. hepatica-derived molecule possesses potential as an anti-inflammatory drug against sepsis in an NHP model. IMPORTANCE Sepsis caused by Gram-negative bacteria affects 1.7 million adults annually in the United States and is one of the most important causes of death at intensive care units. Although the effective use of antibiotics has resulted in improved prognosis of sepsis, the pathological and deathly effects have been attributed to the persistent inflammatory cascade. There is a present need to develop anti-inflammatory agents that can suppress or neutralize the inflammatory responses and prevent the lethal consequences of sepsis. We demonstrated here that a small molecule of 14.5 kDa can suppress the bacteremia, endotoxemia, and many other inflammatory markers in an acute Gram-negative sepsis rhesus macaque model. These results reinforce the notion that Fh15 constitutes an excellent candidate for drug development against sepsis.

Fasciola hepatica fatty acid binding protein (Fh12) induces apoptosis and tolerogenic properties in murine bone marrow derived dendritic cells.

In a previous study we demonstrated that Fasciola hepatica fatty acid binding protein (Fh12) significantly suppress macrophage function by inhibiting IL-6, IL-1ß, tumor necrosis factor (TNF)-α and IL-12 production in TLR4-stimulated murine macrophages, an effect mediated through the signaling of CD14 co-receptor without affecting the viability of these cells. Given that dendritic cells (DCs) are immune cells that play a central role in the initiation of primary immune responses and that are the only antigen-presenting cells capable of stimulating naïve T-cells, in the present study we investigated the effect of Fh12 on DCs. We found that Fh12 exerts a strong suppressive effect on activation and function of DCs. However, in contrast to the effect observed on macrophages, Fh12 induces early and late apoptosis of DCs being this phenomenon dose-dependent and CD14-coreceptor independent. At low concentration Fh12 modulates the LPS-induced DCs maturation status by suppressing the MHC-II, and co-stimulatory molecules CD40 and CD80 surface expression together with the pro-inflammatory cytokines IL-12p70 and IL-6 production whereas increase the IL-10 levels. Besides, Fh12 decreased the ability of LPS-activated DCs to induce IFN-γ production against allogeneic splenocytes, while increasing IL-4 production. We have described for the first time the ability of Fh12 to modify selectively the viability of DCs by apoptosis induction. The selective diminution in DCs survival could be a F. hepatica strategy in order to prevent a host immune response during the earliest phases of infection.

The fatty acid-binding protein (FABP) decreases the clinical signs and modulates immune responses in a mouse model of experimental autoimmune encephalomyelitis (EAE).

BACKGROUND: An increasing body of studies has shown that Fasciola hepatica can affect immune responses. This study explored whether the fatty acid-binding protein (FABP) of F. hepatica can modulate the immune system in a mouse model of experimental autoimmune encephalomyelitis (EAE). METHODS: EAE-induced C57BL/6 mice were treated with vehicle, F. hepatica total extract (TE) or FABP. The clinical signs, body weights, and the expression of IFN-γ, T-bet, IL-4, GATA3, IL-17, RORγ, TGF-ß, FOXP3, IL-10, TNF-α genes and proteins were determined in the isolated CD4+ splenocytes. Besides, the percentage of Treg cells and degree of demyelination were evaluated. RESULTS: We found that TE and FABP treatments decreased the clinical scores, lymphocyte infiltration rate, and demyelinated plaques in EAE mice. The expressions of IL-4 and GATA3 were increased, whereas IL-17 and TNF-α were down-regulated. FABP did not affect the expression of IFN-γ, RORγ, IL-10, and TGF-ß genes or proteins but reduced the expression of T-bet. TE administration did not affect the expression of IL-10 and the Tbet genes, and increased the expression levels of IFN-γ and FOXP3 in CD4+ lymphocytes. Both FABP and TE treatment did not affect the Treg cell percentage. CONCLUSION: This study indicates that F. hepatica FABP and TE can suppress the inflammatory responses in EAE-induced mice and shift the immune system toward Th2 responses. However, FABP exerts stronger anti-inflammatory effects and seems to be more effective than TE for EAE treatment.

Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8.

BACKGROUND: Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms. RESULTS: We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents. CONCLUSIONS: We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

Isolation of Secreted and Tegumental Surface Proteins from Fasciola hepatica.

Proteins secreted by, or displayed on the surface tegument of, trematodes have key functions in the host-parasite interaction. As such, they are often leading targets for diagnostic tests or vaccine candidates. Here we describe methods for the isolation and analysis of soluble secreted proteins (i.e., the secretome) released during in vitro culture of adult Fasciola hepatica. We also describe two methods for the enrichment of proteins displayed on the outer tegumental surface of F. hepatica. These approaches enable downstream identification of the isolated proteins by mass spectrometry-based proteomics.

Isolation and Analysis of Fasciola hepatica Extracellular Vesicles.

The finding of extracellular vesicles (EVs) as important players in parasite-parasite and host-parasite communications has led to an increasing number of reports in the literature. Different protocols have been developed for isolation and further characterization of EVs from parasitic helminths. In this chapter, we describe step by step procedures to isolate EVs secreted by Fasciola hepatica adults in culture, which could be also applied for other developmental stages of the parasite, as well as EVs present in plasma and urine. Along with classical isolation methods like differential ultracentrifugation, and more recent techniques like size exclusion chromatography (SEC), here we also refer to the storage of EVs for further functional assays. In addition, characterization of F. hepatica by electron microscopy techniques like immuno-gold staining, as well as labeling techniques useful for functional assays, like in vitro uptake of fluorescent EVs by cells in culture are also described.

Purification of Native Fasciola hepatica Fatty Acid-Binding Protein and Induction of Alternative Activation of Human Macrophages.

This chapter presents the different techniques to purify the native forms of Fasciola hepatica fatty acid-binding protein (Fh12) using size exclusion chromatography and isoelectric focusing (IEF). Also, it presents the procedure to study the immunological effect of the purified protein Fh12 using monocyte-derived macrophages (MDM) obtained from healthy human donors. For this purpose, I present the procedure to isolate and culture peripheral blood mononuclear cells (PBMCs) to generate alternatively activated macrophages (AAMΦ) by in vitro exposure to Fh12.

Design and expression of polytopic construct of cathepsin-L1, SAP-2 and FhTP16.5 proteins of Fasciola hepatica.

The enzyme-linked immunosorbent assay (ELISA) technique can play an important role in the early detection of fascioliasis. However, they have some diagnostic limitations, including cross-reaction with other helminths. It seems that the combination of recombinant parasite proteins as antigen can reduce these problems. Hence, the present study was aimed to design and confirm the antigenic recombinant multi-epitope (rMEP) construct of three protein epitopes (linear and conformational B-cell epitopes) of the parasite using immunoinformatic tools. For this purpose, the tertiary structures of Fasciola hepatica cathepsin-L1, saposin-like protein 2 and 16.5-kDa tegument-associated protein were predicted using the I-TASSER server. Validation of the modelled structures was performed by Ramachandran plots. The antigenic epitopes of the proteins were achieved by analysing the features of the IEDB server. The synthesized gene was cloned into the pET-22b (+) expression vector and transformed into the Escherichia coli BL21. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to verify and analyse the expression of the rMEP protein. Western blotting was utilized to confirm rMEP protein immunogenicity in two forms, one using an anti-His tag antibody and the other with human pooled sera samples (fascioliasis, non-fascioliasis and negative control sera). Our results demonstrated that the rMEP designed for the three proteins of F. hepatica was highly antigenic, and immune-detection techniques confirmed the antigen specificity. In conclusion, the presented antigenic multi-epitope may be very helpful to develop serodiagnostic kits such as indirect ELISA to evaluate the proper diagnosis of fascioliasis in humans and ruminants.

Structural Variants of a Liver Fluke Derived Granulin Peptide Potently Stimulate Wound Healing.

Granulins are a family of growth factors involved in cell proliferation. The liver-fluke granulin, Ov-GRN-1, isolated from a carcinogenic liver fluke Opisthorchis viverrini, can significantly accelerate wound repair in vivo and in vitro. However, it is difficult to express Ov-GRN-1 in recombinant form at high yield, impeding its utility as a drug lead. Previously we reported that a truncated analogue ( Ov-GRN12-35_3s) promotes healing of cutaneous wounds in mice. NMR analysis of this analogue indicates the presence of multiple conformations, most likely as a result of proline cis/ trans isomerization. To further investigate whether the proline residues are involved in adopting the multiple confirmations, we have synthesized analogues involving mutation of the proline residues. We have shown that the proline residues have a significant influence on the structure, activity, and folding of Ov-GRN12-35_3s. These results provide insight into improving the oxidative folding yield and bioactivity of Ov-GRN12-35_3s and might facilitate the development of a novel wound healing agent.

Intranasal delivery of a formulation containing stage-specific recombinant proteins of Fasciola hepatica cathepsin L5 and cathepsin B2 triggers an anti-fecundity effect and an adjuvant-mediated reduction in fluke burden in sheep.

Fasciola hepatica infection continues to be a major problem in the agriculture sector, particularly in sheep and cattle. Cathepsin L and B proteases are major components of the excretory/secretory material of the parasite, and their roles in several important aspects of parasite invasion and survival has led to their use as targets in rational vaccine design. Previous studies in rats demonstrated that the use of stage-specific antigens, cathepsin B2 and cathepsin L5, as part of a multivalent vaccine, was able to confer significant protection against challenge. In the present study, recombinant versions of cathepsin L5 and cathepsin B2 produced in yeast were used in combination to vaccinate sheep. Intramuscular and intranasal forms of administration were applied, and sheep were subsequently challenged with 150 F. hepatica metacercariae. Intramuscular vaccination was able to induce a strong systemic antibody response against both antigens, but failed to confer significant protection. Conversely, no elevated antibody response was detected against the vaccine antigens following nasal vaccination; however, a reduction in parasite egg viability (>92%) and a statistically significant (p = 0.006), predominantly adjuvant-mediated reduction in worm burdens was observed.

Purification of native Sigma class glutathione transferase from Fasciola hepatica.

Fascioliasis is a parasitic disease of grazing livestock and a threat to global food security by significantly reducing the production value of sheep, goats and cattle. Moreover, the zoonotic parasite is also a re-emerging food borne threat to human populations. Driven by climate change, the prevalence of fascioliasis is set to increase. Efforts to control the main causative agent, Fasciola hepatica, are hampered by short lived chemotherapy approaches that are becoming increasingly obsolete due to therapeutic failure and resistance. A protective vaccine is urgently needed. A recombinant form of Sigma class glutathione transferase (Hematopoietic Prostaglandin D synthase) from F. hepatica (FhGSTS1) with confirmed prostaglandin synthase activity shows immune-modulation activity via suppression of Th17 responses in host dendritic cells. In vaccine trials recombinant FhGSTS1 reduces liver pathology but not worm burden. Native FhGSTS1 is yet to be tested for immune-modulation activities or for vaccine potential, primarily due to the technical difficulty in purifying FhGST-S1 away from the other more abundant GST members in F. hepatica cytosol. This paper reports a pipeline for the purification of native FhGSTS1 using a two-step process consisting of glutathione-agarose affinity and cationic exchange chromatography. The methodology allows for the isolation of purified and active FhGSTS1 or Sigma GSTs from other sources for analytical biochemical and immunological studies.

High yield production of human invariant chain CD74 constructs fused to solubility-enhancing peptides and characterization of their MIF-binding capacities.

The HLA class II histocompatibility antigen gamma chain, also known as HLA-DR antigen-associated invariant chain or CD74, has been shown to be involved in many biological processes amongst which antigen loading and transport of MHC class II molecules from the endoplasmic reticulum to the Golgi complex. It is also part of a receptor complex for macrophage migration inhibitory factor (MIF), and participates in inflammatory signaling. The inhibition of MIF-CD74 complex formation is regarded as a potentially attractive therapeutic target in inflammation, cancer and immune diseases. In order to be able to produce large quantities of the extracellular moiety of human CD74, which has been reported to be unstable and protease-sensitive, different constructs were made as fusions with two solubility enhancers: the well-known maltose-binding domain and Fh8, a small protein secreted by the parasite Fasciola hepatica. The fusion proteins could be purified with high yields from Escherichia coli and were demonstrated to be active in binding to MIF. Moreover, our results strongly suggest that the MIF binding site is located in the sequence between the transmembrane and the membrane-distal trimerisation domain of CD74, and comprises at least amino acids 113-125 of CD74.

Purification and biochemical characterization of a 22-kDa stable cysteine- like protease from the excretory-secretory product of the liver fluke Fasciola hepatica by using conventional techniques.

This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40-60%) followed by a gel filtration through Sephadex G-100 and DEAE- cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18.8%. The MW of the purified protease was estimated by reducing SDS-PAGE to be 22kDa while the proteolytic activity detection was carried out by zymography on non-denaturing SDS-PAGE containing the casein as substrate. Using this substrate, the protease showed extreme proteolytic activity at pH 5.5 and temperature 35-40°C and was highly stable over a wide range of pH, from 5.0 to 10.0. In addition to its preference for the Z-Phe-Arg-AMC fluorogenic substrate resulting in maximum proteolytic activity (99.7%) at pH 7.0, the pure protease exhibited highest cleavage activity against hemoglobin and casein substrates at pH 5.5 (85.6% and 82.8%, respectively). The Km values obtained for this protease were 5.4, 13, 160 and approximately 1000µM using respectively the fluorogenic substrate Z-Phe-Arg-AMC, hemoglobin, casein and albumin. The protease activity was completely inhibited either by E-64 inhibitor (5mM) or iodoacetamide (10mM), indicating its cysteine nature. The usefulness of the purified protease as an antigen was studied by immunoblotting. Thus, sera from sheep experimentally infected with F. hepatica recognized the protease band at 2 weeks post-infection (WPI) and strongly at 7 WPI. The early detection of antibodies anti- F. hepatica suggests the application of this molecule as a specific epitope for the serodiagnosis of fascioliasis disease.

Recombinant Fasciola hepatica fatty acid binding protein suppresses toll-like receptor stimulation in response to multiple bacterial ligands.

Recently, we reported that a native Fasciola hepatica fatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markers in vivo and in vitro. Because the purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up, this study accomplished the task of optimizing the expression and purification of a recombinant form of FABP (Fh15). Additionally, we ascertained whether this molecule could exhibit a similar suppressive effect on TLR-stimulation and inflammatory cytokine expression from macrophages than those previously demonstrated for the native molecule. Results demonstrated that Fh15 suppresses the expression of IL-1ß and TNFα in murine macrophages and THP1 Blue CD14 cells. Additionally, Fh15 suppress the LPS-induced TLR4 stimulation. This effect was not impaired by a thermal denaturing process or blocked by the presence of anti-Fh12 antibodies. Fh15 also suppressed the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti-inflammatory drug in preclinical studies in the near future.

Fasciola hepatica glycoconjugates immuneregulate dendritic cells through the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin inducing T cell anergy.

Dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) expressed on a variety of DCs, is a C-type lectin receptor that recognizes glycans on a diverse range of pathogens, including parasites. The interaction of DC-SIGN with pathogens triggers specific signaling events that modulate DC-maturation and activity and regulate T-cell activation by DCs. In this work we evaluate whether F. hepatica glycans can immune modulate DCs via DC-SIGN. We demonstrate that DC-SIGN interacts with F. hepatica glycoconjugates through mannose and fucose residues. We also show that mannose is present in high-mannose structures, hybrid and trimannosyl N-glycans with terminal GlcNAc. Furthermore, we demonstrate that F. hepatica glycans induce DC-SIGN triggering leading to a strong production of TLR-induced IL-10 and IL-27p28. In addition, parasite glycans induced regulatory DCs via DC-SIGN that decrease allogeneic T cell proliferation, via the induction of anergic/regulatory T cells, highlighting the role of DC-SIGN in the regulation of innate and adaptive immune responses by F. hepatica. Our data confirm the immunomodulatory properties of DC-SIGN triggered by pathogen-derived glycans and contribute to the identification of immunomodulatory glyans of helminths that might eventually be useful for the design of vaccines against fasciolosis.

Delineating distinct heme-scavenging and -binding functions of domains in MF6p/helminth defense molecule (HDM) proteins from parasitic flatworms.

MF6p/FhHDM-1 is a small protein secreted by the parasitic flatworm (trematode) Fasciola hepatica that belongs to a broad family of heme-binding proteins (MF6p/helminth defense molecules (HDMs)). MF6p/HDMs are of interest for understanding heme homeostasis in trematodes and as potential targets for the development of new flukicides. Moreover, interest in these molecules has also increased because of their immunomodulatory properties. Here we have extended our previous findings on the mechanism of MF6p/HDM-heme interactions and mapped the protein regions required for heme binding and for other biological functions. Our data revealed that MF6p/FhHDM-1 forms high-molecular-weight complexes when associated with heme and that these complexes are reorganized by a stacking procedure to form fibril-like and granular nanostructures. Furthermore, we showed that MF6p/FhHDM-1 is a transitory heme-binding protein as protein·heme complexes can be disrupted by contact with an apoprotein (e.g. apomyoglobin) with higher affinity for heme. We also demonstrated that (i) the heme-binding region is located in the MF6p/FhHDM-1 C-terminal moiety, which also inhibits the peroxidase-like activity of heme, and (ii) MF6p/HDMs from other trematodes, such as Opisthorchis viverrini and Paragonimus westermani, also bind heme. Finally, we observed that the N-terminal, but not the C-terminal, moiety of MF6p/HDMs has a predicted structural analogy with cell-penetrating peptides and that both the entire protein and the peptide corresponding to the N-terminal moiety of MF6p/FhHDM-1 interact in vitro with cell membranes in hemin-preconditioned erythrocytes. Our findings suggest that MF6p/HDMs can transport heme in trematodes and thereby shield the parasite from the harmful effects of heme.

Proteomic analysis of Fasciola hepatica excretory and secretory products (FhESPs) involved in interacting with host PBMCs and cytokines by shotgun LC-MS/MS.

Fasciola hepatica is a helminth parasite with a worldwide distribution, which can cause chronic liver disease, fasciolosis, leading to economic losses in the livestock and public health in many countries. Control is mostly reliant on the use of drugs, and as a result, drug resistance has now emerged. The identification of F. hepatica genes involved in interaction between the parasite and host immune system is utmost important to elucidate the evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, we aimed to identify molecules in F. hepatica excretory and secretory products (FhESPs) interacting with the host peripheral blood mononuclear cells (PBMCs), Th1-like cytokines (IL2 and IFN-γ), and Th17-like cytokines (IL17) by Co-IP combined with tandem mass spectrometry. The results showed that 14, 16, and 9 proteins in FhESPs could bind with IL2, IL17, and IFN-γ, respectively, which indicated that adult F. hepatica may evade the host immune responses through directly interplaying with cytokines. In addition, nine proteins in FhESPs could adhere to PBMCs. Our findings provided potential targets as immuno-regulators, and will be helpful to elucidate the molecular basis of host-parasite interactions and search for new potential proteins as vaccine and drug target candidates.