Antigens from the Helminth Fasciola hepatica Exert Antiviral Effects against SARS-CoV-2 In Vitro.

SARS-CoV-2, the causal agent of COVID-19, is a new coronavirus that has rapidly spread worldwide and significantly impacted human health by causing a severe acute respiratory syndrome boosted by a pulmonary hyperinflammatory response. Previous data from our lab showed that the newly excysted juveniles of the helminth parasite Fasciola hepatica (FhNEJ) modulate molecular routes within host cells related to vesicle-mediated transport and components of the innate immune response, which could potentially be relevant during viral infections. Therefore, the aim of the present study was to determine whether FhNEJ-derived molecules influence SARS-CoV-2 infection efficiency in Vero cells. Pre-treatment of Vero cells with a tegument-enriched antigenic extract of FhNEJ (FhNEJ-TEG) significantly reduced infection by both vesicular stomatitis virus particles pseudotyped with the SARS-CoV-2 Spike protein (VSV-S2) and live SARS-CoV-2. Pre-treatment of the virus itself with FhNEJ-TEG prior to infection also resulted in reduced infection efficiency similar to that obtained by remdesivir pre-treatment. Remarkably, treatment of Vero cells with FhNEJ-TEG after VSV-S2 entry also resulted in reduced infection efficiency, suggesting that FhNEJ-TEG may also affect post-entry steps of the VSV replication cycle. Altogether, our results could potentially encourage the production of FhNEJ-derived molecules in a safe, synthetic format for their application as therapeutic agents against SARS-CoV-2 and other related respiratory viruses.

Study of the cross-talk between Fasciola hepatica juveniles and the intestinal epithelial cells of the host by transcriptomics in an in vitro model.

Fasciolosis is a globally widespread trematodiasis with a major economic and veterinary impact. Therefore, this disease is responsible for millions of dollars in losses to the livestock industry, and also constitutes an emerging human health problem in endemic areas. The ubiquitous nature of Fasciola hepatica, the main causative agent, is one of the key factors for the success of fasciolosis. Accordingly, this parasite is able to subsist in a wide variety of ecosystems and hosts, thanks to the development of a plethora of strategies for adaption and immune evasion. Fasciolosis comprises a growing concern due to its high prevalence rates, together with the emergence of strains of the parasite resistant to the treatment of choice (triclabendazole). These facts highlight the importance of developing novel control measures which allow for an effective protection against the disease before F. hepatica settles in a niche inaccessible to the immune system. However, knowledge about the initial phases of the infection, including the migration mechanisms of the parasite and the early innate host response, is still scarce. Recently, our group developed an in vitro host-parasite interaction model that allowed the early events to be unveiled after the first contact between the both actors. This occurs shortly upon ingestion of F. hepatica metacercariae and the emergence of the newly excysted juveniles (FhNEJ) in the host duodenum. Here, we present a transcriptomic analysis of such model using an approach based on RNA sequencing (RNA-Seq), which reveals changes in gene expression related to proteolysis and uptake of metabolites in FhNEJ. Additionally, contact with the parasite triggered changes in host intestinal cells related to pseudogenes expression and host defence mechanisms, including immune response, among others. In sum, these results provide a better understanding of the early stages of fasciolosis at molecular level, and a pool of targets that could be used in future therapeutic strategies against the disease.

Molecular Characterization of the Interplay between Fasciola hepatica Juveniles and Laminin as a Mechanism to Adhere to and Break through the Host Intestinal Wall.

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.

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

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.

Proteomics coupled with in vitro model to study the early crosstalk occurring between newly excysted juveniles of Fasciola hepatica and host intestinal cells.

Fasciolosis caused by the trematode Fasciola hepatica is a zoonotic neglected disease affecting animals and humans worldwide. Infection occurs upon ingestion of aquatic plants or water contaminated with metacercariae. These release the newly excysted juveniles (FhNEJ) in the host duodenum, where they establish contact with the epithelium and cross the intestinal barrier to reach the peritoneum within 2-3 h after infection. Juveniles crawl up the peritoneum towards the liver, and migrate through the hepatic tissue before reaching their definitive location inside the major biliary ducts, where they mature into adult worms. Fasciolosis is treated with triclabendazole, although resistant isolates of the parasite are increasingly being reported. This, together with the limited efficacy of the assayed vaccines against this infection, poses fasciolosis as a veterinary and human health problem of growing concern. In this context, the study of early host-parasite interactions is of paramount importance for the definition of new targets for the treatment and prevention of fasciolosis. Here, we develop a new in vitro model that replicates the first interaction between FhNEJ and mouse primary small intestinal epithelial cells (MPSIEC). FhNEJ and MPSIEC were co-incubated for 3 h and protein extracts (tegument and soma of FhNEJ and membrane and cytosol of MPSIEC) were subjected to quantitative SWATH-MS proteomics and compared to respective controls (MPSIEC and FhNEJ left alone for 3h in culture medium) to evaluate protein expression changes in both the parasite and the host. Results show that the interaction between FhNEJ and MPSIEC triggers a rapid protein expression change of FhNEJ in response to the host epithelial barrier, including cathepsins L3 and L4 and several immunoregulatory proteins. Regarding MPSIEC, stimulation with FhNEJ results in alterations in the protein profile related to immunomodulation and cell-cell interactions, together with a drastic reduction in the expression of proteins linked with ribosome function. The molecules identified in this model of early host-parasite interactions could help define new tools against fasciolosis.

Study of the migration of Fasciola hepatica juveniles across the intestinal barrier of the host by quantitative proteomics in an ex vivo model.

Fasciola hepatica is a trematode parasite that infects animals and humans causing fasciolosis, a worldwide-distributed disease responsible for important economic losses and health problems. This disease is of growing public health concern since parasite isolates resistant to the current treatment (triclabendazole) have increasingly been described. F. hepatica infects its vertebrate host after ingestion of the encysted parasite (metacercariae), which are found in the water or attached to plants. Upon ingestion, newly excysted juveniles of F. hepatica (FhNEJ) emerge in the intestinal lumen and cross the intestinal barrier, reach the peritoneum and migrate to the biliary ducts, where adult worms fully develop. Despite the efforts made to develop new therapeutic and preventive tools, to date, protection against F. hepatica obtained in different animal models is far from optimal. Early events of host-FhNEJ interactions are of paramount importance for the infection progress in fasciolosis, especially those occurring at the host-parasite interface. Nevertheless, studies of FhNEJ responses to the changing host environment encountered during migration across host tissues are still scarce. Here, we set-up an ex vivo model coupled with quantitative SWATH-MS proteomics to study early host-parasite interaction events in fasciolosis. After comparing tegument and somatic fractions from control parasites and FhNEJ that managed to cross a mouse intestinal section ex vivo, a set of parasite proteins whose expression was statistically different were found. These included upregulation of cathepsins L3 and L4, proteolytic inhibitor Fh serpin 2, and a number of molecules linked with nutrient uptake and metabolism, including histone H4, H2A and H2B, low density lipoprotein receptor, tetraspanin, fatty acid binding protein a and glutathione-S-transferase. Downregulated proteins in FhNEJ after gut passage were more numerous than the upregulated ones, and included the heath shock proteins HSP90 and alpha crystallin, amongst others. This study brings new insights into early host-parasite interactions in fasciolosis and sheds light on the proteomic changes in FhNEJ triggered upon excystment and intestinal wall crossing, which could serve to define new targets for the prevention and treatment of this widespread parasitic disease.

Characterization of Microbial Diversity in Decayed Wood from a Spanish Forest: An Environmental Source of Industrially Relevant Microorganisms.

Rotting wood is inhabited by a large diversity of bacteria, fungi, and insects with complex environmental relationships. The aim of this work was to study the composition of the microbiota (bacteria and fungi) in decaying wood from a northwest Spanish forest as a source of industrially relevant microorganisms. The analyzed forest is situated in a well-defined biogeographic area combining Mediterranean and temperate macrobioclimates. Bacterial diversity, determined by metagenome analyses, was higher than fungal heterogeneity. However, a total of 194 different cultivable bacterial isolates (mainly Bacillaceae, Streptomycetaceae, Paenibacillaceae, and Microbacteriaceae) were obtained, in contrast to 343 fungal strains (mainly Aspergillaceae, Hypocreaceae, and Coniochaetaceae). Isolates traditionally known as secondary metabolite producers, such as Actinobacteria and members of the Penicillium genus, were screened for their antimicrobial activity by the detection of antibiotic biosynthetic clusters and competitive bioassays against fungi involved in wood decay. In addition, the ability of Penicillium isolates to degrade cellulose and release ferulic acid from wood was also examined. These results present decaying wood as an ecologically rich niche and a promising source of biotechnologically interesting microorganisms.

Recognition Pattern of the Fasciola hepatica Excretome/Secretome during the Course of an Experimental Infection in Sheep by 2D Immunoproteomics.

Excretory/secretory products released by helminth parasites have been widely studied for their diagnostic utility, immunomodulatory properties, as well as for their use as vaccines. Due to their location at the host/parasite interface, the characterization of parasite secretions is important to unravel the molecular interactions governing the relationships between helminth parasites and their hosts. In this study, the excretory/secretory products from adult worms of the trematode Fasciola hepatica (FhES) were employed in a combination of two-dimensional electrophoresis, immunoblot and mass spectrometry, to analyze the immune response elicited in sheep during the course of an experimental infection. Ten different immunogenic proteins from FhES recognized by serum samples from infected sheep at 4, 8, and/or 12 weeks post-infection were identified. Among these, different isoforms of cathepsin L and B, peroxiredoxin, calmodulin, or glutathione S-transferase were recognized from the beginning to the end of the experimental infection, suggesting their potential role as immunomodulatory antigens. Furthermore, four FhES proteins (C2H2-type domain-containing protein, ferritin, superoxide dismutase, and globin-3) were identified for the first time as non-immunogenic proteins. These results may help to further understand host/parasite relationships in fasciolosis, and to identify potential diagnostic molecules and drug target candidates of F. hepatica.

Insights into Fasciola hepatica Juveniles: Crossing the Fasciolosis Rubicon.

Unraveling the molecular interactions governing the first contact between parasite and host tissues is of paramount importance to the development of effective control strategies against parasites. In fasciolosis, a foodborne trematodiasis caused mainly by Fasciola hepatica, these early interactions occur between the juvenile worm and the host intestinal wall a few hours after ingestion of metacercariae, the infectious stage of the parasite. However, research on these early events is still scarce and the majority of studies have focused on the adult worm. Here, we review current knowledge on the biology and biochemistry of F. hepatica juveniles and their molecular relationships with the host tissues and identify the research needs and gaps to be covered in the future.

Fascioliasis and fasciolopsiasis: Current knowledge and future trends.

Food-borne zoonotic trematodiases are classified as neglected diseases by the World Health Organization. Among them, fascioliasis is caused worldwide by Fasciola hepatica and F. gigantica, and represent a huge problem in livestock production and human health in endemic areas. Fasciolopsis buski, restricted to specific regions of Asia, causes fasciolopsiasis. The incidence of these trematodiases is underestimated due to under-reporting and to the lack of sensitive and widely accepted tool for their diagnosis. This, together with a rising trend in reporting of drug resistance and the need for an effective vaccine against these parasites, pose a challenge in the effective control of these diseases. Here, the latest reports on fascioliasis outbreaks between 2000 and 2020 and the most recent advances in their epidemiology, diagnosis, treatment and control are revised. Finally, future needs in the field of fascioliasis and fasciolopsiasis are presented, which could be addressed based on current knowledge and by means of new emerging technologies.

Evaluation of the sensitivity and specificity of GST-tagged recombinant antigens 2B2t, Ag5t and DIPOL in ELISA for the diagnosis and follow up of patients with cystic echinococcosis.

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus sensu lato. Diagnosis and monitoring of CE rely primarily on imaging while serology is used as a confirmatory test. However, imaging is not always conclusive and currently available serological assays have suboptimal sensitivity and specificity, lack standardization, and are not useful for patients´ follow-up. Seroassays for CE are usually based on hydatid fluid (HF), a complex, variable antigenic mixture, and cross-reactivity exists especially with alveolar echinococcosis. Recombinant proteins based on immunogenic antigens most abundant in HF, such as AgB1, AgB2 and Ag5, have been used to overcome these limitations. None of them so far showed potential to replace HF; however, their performance have been largely tested on a limited number of samples, and comparison of different antigens using the same cohort has been rarely performed. The combination of several immunogenic epitopes in a single recombinant protein could enhance test sensitivity. For the diagnosis and follow-up of patients with CE, we compared the performance of the crude HF, previously described recombinant 2B2t antigen, and GST-tagged version of 2B2t, and novel designed recombinants (GST-Ag5t and the GST-DIPOL chimera containing AgB1, AgBB2 and Ag5 epitopes) by IgG-ELISA format. Samples belong to a retrospective cohort of 253 well-characterized patients with CE, previously described for the evaluation of the 2B2t antigen, 92 patients with alveolar echinococcosis, and 82 healthy donors. The reference standard for CE diagnosis was the presence of a CE lesion as diagnosed by ultrasonography. The highest sensitivity was obtained with HF [86.7%, 95% confidence interval (CI): 81.2-91.0], followed by GST-2B2t (70.0%, 95% CI: 63.1-76.2), 2B2t (65.5%, 95% CI: 58.5-72.0), GST-Ag5t (64.5%, 95% CI: 57.5-71.1) and GST-DIPOL (63.1%, 95% CI: 56.0-69.7). The GST-2B2t had the best specificity (95.8%, 95% CI: 88.3-99.1) and the lowest cross-reactivity (38.7%, 95% CI: 27.6-50.6). Good response to treatment also correlated to negative test results in the GST-2B2t ELISA. While none of the tested recombinant antigen appears suitable to replace HF for the diagnosis of CE, GST-2B2t should be further explored as a confirmation test, based on its high specificity and low cross-reactivity, and for the follow-up after treatment in those patients with positive serology for this antigen.

Set up of an in vitro model to study early host-parasite interactions between newly excysted juveniles of Fasciola hepatica and host intestinal cells using a quantitative proteomics approach.

Fasciola hepatica is the causative agent of fasciolosis, a parasitic zoonosis of global distribution causing significant economic losses in animal production and a human public health problem in low-income countries. Hosts are infected by ingestion of aquatic plants carrying metacercariae. Once ingested, the juvenile parasites excyst in the small intestine and, after crossing it, they follow a complex migratory route that lead the parasites to their definitive location in the bile ducts. Despite being a critical event in the progression of the infection, the available data on the cross-talk relationships between the parasite and the host at an early stage of the infection are scarce. The objective of the present work is to characterize the proteomic changes occurring in both the parasite and the host, through the development of a novel in vitro model, to shed light on the molecular pathways of communication between the newly excysted juveniles (NEJ) from F. hepatica and the host's intestinal epithelium. For this, in vitro excystation of F. hepatica metacercariae was carried out and NEJ were obtained. Additionally, optimal conditions of growth and expansion of mouse primary small intestinal epithelial cells (MPSIEC) in culture were fine-tuned. Tegumentary and somatic parasite antigens (NEJ-Teg and NEJ-Som), as well as host cell protein lysate (MPSIEC-Lys) were obtained before and after 24 h co-culture of NEJ with MPSIEC. We used an isobaric tags for relative and absolute quantitation (iTRAQ)-based strategy to detect 191 and 62 up-regulated, and 112 and 57 down-regulated proteins in the NEJ-Teg and NEJ-Som extracts, respectively. Similarly, 87 up-regulated and 73 down-regulated proteins in the MPSIEC-Lys extract were identified. Taking into account the biological processes in which these proteins were involved, interesting mechanisms related to parasite development, invasion and evasion, as well as manipulation of the host intestinal epithelial cell adhesion, immunity and apoptosis pathways, among others, could be inferred, taking place at the host-parasite interface. The further understanding of these processes could constitute promising therapeutic targets in the future against fasciolosis.