Estudio proteómico y transcriptómico comparado de la interfase parásito-hospedador en la fasciolosis

Tesis por compendio de publicaciones.[ES] La fasciolosis es una zoonosis parasitaria causada por vermes trematodos del género Fasciola, principalmente por las especies F. hepatica y F. gigantica. Está considerada como la enfermedad de transmisión alimentaria con la distribución geográfica más amplia conocida, con casos documentados en los 5 continentes. El carácter ubicuo de la enfermedad es, en parte, debido a la capacidad de los vermes para establecerse sobre una amplia variedad de hospedadores, tanto intermediarios como definitivos. La fasciolosis causa anualmente pérdidas millonarias a la industria ganadera en forma de muertes de cabezas de ganado o reducción en la productividad de derivados animales (carne, leche, lana, etc.). Además, los costes indirectos como consecuencia del tratamiento farmacológico o la disminución de la fertilidad como resultado de la infección incrementan aún más la cifra. Por otra parte, aunque los hospedadores más habituales del parásito se identifican con mamíferos rumiantes, el ser humano también puede actuar como hospedador definitivo, por lo que la fasciolosis puede suponer un problema de salud pública, con varias regiones endémicas en todo el mundo. El control de la enfermedad resulta complicado por la naturaleza inespecífica de los signos y síntomas clínicos, la variabilidad de los mismos y la escasez de alternativas terapéuticas. Además, el tratamiento farmacológico presenta problemas como la persistencia de los fármacos en los tejidos del hospedador, lo que dificulta su aplicación en animales destinados al consumo, la contaminación ambiental, y la aparición de resistencias frente a los principales antihelmínticos conocidos, lo que puede desembocar en la ineficacia del tratamiento. La fasciolosis ha sido designada por la Organización Mundial de la Salud (OMS) como enfermedad abordable mediante la perspectiva One Health. Según este enfoque, el tratamiento eficaz de la enfermedad a nivel humano, veterinario o ambiental redundaría positivamente en los demás niveles, siendo requisito imprescindible aumentar el conocimiento sobre las interacciones que se establecen a nivel molecular entre parásito y hospedador, especialmente en las fases tempranas de la infección. En este sentido, la propuesta de nuevos modelos de laboratorio que permitan replicar el contacto y la relación entre ambos organismos durante estas fases, y su estudio mediante tecnologías -ómicas, indispensables para identificar las moléculas que rigen dichas interacciones, supondrían herramientas de gran utilidad para desentrañar estos importantes mecanismos. En la presente Tesis Doctoral, se llevó a cabo la puesta a punto de dos modelos de interacción parásito-hospedador que buscan replicar las condiciones de la infección temprana en la fasciolosis, la cual se produce tras la ingesta de las formas de resistencia del parásito, denominadas metacercarias, que liberan en el intestino delgado del hospedador los juveniles recién excistados (NEJ): - El primer modelo reproduce el primer contacto entre los NEJ y el intestino de su hospedador definitivo, representado por un cultivo primario de células epiteliales intestinales de ratón (MPSIEC), mediante un abordaje in vitro. Para ello, los NEJ previamente excistados se pusieron en contacto con un cultivo en placa de MPSIEC, y tras un tiempo de contacto comprendido entre las 3 y las 24 horas se separaron para analizar los cambios a nivel molecular en ambos organismos. - El segundo modelo recurre a una aproximación ex vivo con el fin de recrear el paso de los NEJ a través de la pared intestinal de su hospedador. En dicho modelo, se obtuvieron fragmentos de intestino delgado procedentes de ratones de laboratorio, en cuyo interior se introdujeron los NEJ previamente excistados. Pasadas dos horas y media, los NEJ que lograron atravesar el intestino se recolectaron y analizaron. La caracterización molecular de los cambios en parásito y hospedador tras su interacción se realizó mediante técnicas proteómicas y transcriptómicas. En concreto, el perfil de expresión diferencial a nivel de proteína en ambos organismos se evaluó mediante las metodologías iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) y SWATH-MS (Sequential Window Acquisition of All Theoretical Mass Spectra), las cuales hacen uso de la cromatografía líquida de alta resolución (HPLC) acoplada a espectrometría de masas (MS) para la determinación del perfil proteómico de las muestras estimuladas y no estimuladas. Por su parte, los estudios transcriptómicos se realizaron mediante secuenciación de ARN (RNA-Seq) y su posterior mapeo sobre los correspondientes genomas de referencia. La utilización de estas técnicas permitió identificar un alto número de proteínas y transcritos cuyos niveles de expresión en muestras estimuladas se modificaban significativamente respecto a las no estimuladas. En los NEJ, estos se correspondieron con procesos biológicos importantes para la supervivencia del parásito, tales como la proteolisis y su regulación, la respuesta al estrés oxidativo, el metabolismo energético y el crecimiento o los mecanismos de evasión inmune, mientras que, en las MPSIEC, las proteínas y genes diferencialmente expresados se relacionaron principalmente con procesos de transporte intra y extracelular, la modulación de la respuesta inmune o el control de la expresión génica, además de incluir un elevado número de pseudogenes. El análisis e interpretación de los resultados de este trabajo proporcionan una mejor comprensión de la interacción parásito-hospedador a nivel temprano en la fasciolosis, y señalan una serie de antígenos importantes para la misma que podrían permitir la identificación de nuevas dianas terapéuticas y la formulación de nuevas vacunas, con el fin de lograr un control eficaz de la enfermedad en el futuro

Insights into Fasciola hepatica Juveniles: Crossing the Fasciolosis Rubicon.

13 páginas, 1 tabla, 2 cajas, 2 figurasUnraveling 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.J.G.M. is supported by the 'Juan de la Cierva-Incorporación' program (IJC2018-036660-I) of the Ministerio de Ciencia,Innovación y Universidades (MCIU) and by the JIN project 'ULYSSES' (RTI2018-093463-J-100) funded by MCIU, AgenciaEstatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER, UE). D.B.R. is supported by a predoctoralfellowship from Junta de Castilla y León and the European Social Fund. Authors acknowledgefinancial support from MCIU(Projects AGL2015-67023-C2-2-R and PID2019-108782RB-C22),Peer reviewe

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

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

Set up of an in vitro platform for the study of the therapeutic potential of helminth molecules against viruses

Resumen del trabajo presentado a las III Jornadas Científicas PTI+ Salud Global, celebradas en el Centro de Ciencias Humanas y Sociales (CCHS), CSIC (Madrid) del 20 al 22 de noviembre de 2023.[Background] Helminth parasites like the trematode Fasciola hepatica, a veterinary parasite with an important zoonotic potential, have adapted to their mammalian hosts during long co-evolution processes by establishing host-parasite relationships that modulate different aspects of the host physiology. In line with this, previous data from our lab showed that F. hepatica newly excysted juveniles (FhNEJ) modulate cellular routes within host cells related to vesiclemediated transport and components of the innate immune response, which could potentially be relevant during the course of viral infections. Therefore, the aim of the present study was to set up an in vitro platform based on the use of viral particles pseudotyped with the envelope protein of highly pathogenic human viruses to screen for the capability of F. hepatica molecules to alter their infective potential.[Methods] We produced viral particles derived from the vesicular-stomatitis virus (VSV) pseudotyped with Spike, the envelope protein of the recently emerged severe acute respiratory syndrome 2 (SARS-CoV-2), to test for the applicability of this in vitro platform, and validated the results using genuine SARS-CoV-2 infections in Vero cells. Results: Our compound screen revealed that a tegument-enriched antigenic fraction of FhNEJ contains proteins with antiviral potential against both Spike-pseudotyped VSV viral particles and live SARS-CoV-2.[Conclusions] First, our results revealed that FhNEJ express molecules that are capable of mediating virus entry and/or replication of SARS-CoV-2 virus and potentially other enveloped viruses with zoonotic potential. Second, our results evidenced that the in vitro platform that we established for virus pseudotyping is valid for the interrogation of antiviral molecules from F. hepatica. Altogether, this platform could lead to the identification of antiviral molecules in helminth parasites and encourage their production in a safe, synthetic format for their application as therapeutics against viruses of growing public health concern.Peer reviewe

Molecular characterization of the interplay between Fasciola hepatica juveniles and laminin as a mechanism to adhere to and break through the host intestinal wall

Comunicación oral presentada en el 29th International Conference of the World Association for the Advancement of Veterinary Parasitology celebrado en Chennai (India), del 20 al 24 de agosto de 2023.Peer reviewe

Molecular Characterization of the Interplay between <i>Fasciola hepatica</i> 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

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

24 páginas, 9 figurasFasciolosis 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.Financial support of the Spanish Ministry of Science Innovation (Projects AGL2015-67023-C2-2-R and PID2019-108782RB-C22), JIN project (RTI2018-093463-J-100) funded by Ministerio de Ciencia, Innovación y Universidades (MCIU), and the Project“CLU-2019-05 – IRNASA/CSIC Unit of Excellence”, funded by the Junta de Castilla y León and cofinanced by the European Union (ERDF “Europe drives our growth”).Peer reviewe

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

9 páginas, 5 figurasFasciolosis 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.M.S.L. acknowledges the financial support of the Spanish Ministry of Science and Innovation (grant numbers AGL2015-67023-C2-2-R and PID2019-108782RB-C22) and the Junta de Castilla y Leon ´ “IRNASA/ CSIC Unit of Excellence” co-financed by the European Union (ERDF “Europe drives our growth”) (grant number CLU-2019-05). D.B.R. and J. S. acknowledge the support of the Junta de Castilla y León for their Predoctoral contracts. M.L.G. acknowledges the support of the Spanish Ministry of Science and Innovation for her FPU Predoctoral contract. J. G.M. is supported by the ‘Ramón y Cajal’ program of the Spanish Ministerio de Ciencia e Innovación (grant number RYC2020-030575-I). M.T. V. acknowledges the support of the European Commission NextGenerationEU Fund (grant number EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global)Peer reviewe

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.</jats:p