Role of fasciola hepatica small RNAs in the interaction with the mammalian host

Material complementario: https://www.frontiersin.org/articles/10.3389/fcimb.2021.812141/full#supplementary-materialMicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression being involved in many different biological processes and play a key role in developmental timing. Additionally, recent studies have shown that miRNAs released from parasites are capable of regulating the expression of host genes. In the present work, we studied the expression patterns of ncRNAs of various intra-mammalian life-cycle stages of the liver fluke, Fasciola hepatica, as well as those packaged into extracellular vesicles and shed by the adult fluke. The miRNA expression profile of the intra-mammalian stages shows important variations, despite a set of predominant miRNAs that are highly expressed across all stages. No substantial variations in miRNA expression between dormant and activated metacercariae were detected, suggesting that they might not be central players in regulating fluke gene expression during this crucial step in the invasion of the definitive host. We generated a curated pipeline for the prediction of putative target genes that reports only sites conserved between three different prediction approaches. This pipeline was tested against an iso-seq curated database of the 3’ UTR regions of F. hepatica genes to detect miRNA regulation networks within liver fluke. Several functions related to the host immune response or modulation were enriched among the targets of the most highly expressed parasite miRNAs, stressing that they might be key players during the establishment and maintenance of infection. Additionally, we detected fragments derived from the processing of tRNAs, in all developmental stages analyzed, and documented the presence of novel long tRNA fragments enriched in vesicles. We confirmed the presence of at least 5 putative vault RNAs (vtRNAs), that are expressed across different stages and enriched in vesicles. The presence of tRNA fragments and vtRNAs in vesicles raise the possibility that they could be involved in the host-parasite interaction.CSIC: I+D 2019_52

Proteoma de la saliva de Ornithodoros moubata: comparativa entre machos y hembras

Comunicaciones a congreso

Construcción de microarrays de proteínas a partir de genotecas de expresión para el estudio de la interfase molecular garrapata-hospedador

Comunicaciones a congreso

The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular "machinery" required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack

Fasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism

Publication history: Accepted - 12 May, 2022; Published - 15 may 2022.The liver fluke, Fasciola hepatica, is an obligate blood-feeder, and the gastrodermal cells of the parasite form the interface with the host’s blood. Despite their importance in the host–parasite interaction, in-depth proteomic analysis of the gastrodermal cells is lacking. Here, we used laser microdissection of F. hepatica tissue sections to generate unique and biologically exclusive tissue fractions of the gastrodermal cells and tegument for analysis by mass spectrometry. A total of 226 gastrodermal cell proteins were identified, with proteases that degrade haemoglobin being the most abundant. Other detected proteins included those such as proton pumps and anticoagulants which maintain a microenvironment that facilitates digestion. By comparing the gastrodermal cell proteome and the 102 proteins identified in the laser microdissected tegument with previously published tegument proteomic datasets, we showed that one-quarter of proteins (removed by freeze– thaw extraction) or one-third of proteins (removed by detergent extraction) previously identified as tegumental were instead derived from the gastrodermal cells. Comparative analysis of the laser microdissected gastrodermal cells, tegument, and F. hepatica secretome revealed that the gastrodermal cells are the principal source of secreted proteins, as well as showed that both the gastrodermal cells and the tegument are likely to release subpopulations of extracellular vesicles (EVs). Microscopical examination of the gut caeca from flukes fixed immediately after their removal from the host bile ducts showed that selected gastrodermal cells underwent a progressive thinning of the apical plasma membrane which ruptured to release secretory vesicles en masse into the gut lumen. Our findings suggest that gut-derived EVs are released via a novel atypical secretory route and highlight the importance of the gastrodermal cells in nutrient acquisition and possible immunomodulation by the parasite.This work was supported by a grant to M.W.R. (BB/L019612/1) from the Biotechnology and Biological Sciences Research Council (BBSRC). A.P.S.B. was supported by a postgraduate studentship from the Department for the Economy (DfE) Northern Ireland

Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells.

<div><p>Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult <i>F</i>. <i>hepatica</i>, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the <i>F</i>. <i>hepatica</i> EVs were resistant to <i>exo</i>- and <i>endo</i>-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by <i>F</i>. <i>hepatica</i> and sheds light on the molecular interaction between parasite EVs and host cells.</p></div

Caracterización molecular y funcional de proteínas de Schistosoma bovis implicadas en la regulación de los mecanismos hemostáticos del hospedador

[ES] La esquistosomosis, constituye un importante problema sanitario y veterinario en numerosas zonas tropicales y subtropicales del mundo. Schistosoma bovis es un parásito sanguíneo de rumiantes, considerado uno de los principales causantes de la esquistosomosis animal. Esta parasitosis, que afecta a 150 millones de animales en Asia y África y también está presente en nuestro país, provoca pérdidas significativas en las explotaciones ganaderas al reducir la tasa de crecimiento de los animales infectados y aumentar la susceptibilidad de los mismos a otras enfermedades. Además de su importancia veterinaria, S. bovis constituye un buen modelo para el estudio de la esquistosomosis humana, dada la proximidad filogenética que existe entre las especies del género. Para sobrevivir en el lecho vascular del hospedador, S. bovis debe neutralizar no solo la respuesta inmune sino también los mecanismos hemostáticos de dicho hospedador, para lo cual debe contar con las oportunas moléculas anti-hemostáticas. Pese a su importancia, las moléculas anti-hemostáticas de los esquistosomas apenas han sido estudiadas. Los pocos datos conocidos acerca de la interacción entre S. bovis y el sistema hemostático de sus hospedadores son los obtenidos por nuestro grupo de investigación en estudios previos. En dichos estudios se comprobó que S. bovis manipula el sistema fibrinolítico del hospedador mediante la fijación y activación de plasminógeno y se identificaron diez proteínas fijadoras de plasminógeno, además de otras proteínas que podrían también estar relacionadas con la regulación de los mecanismos hemostáticos del hospedador, entre ellas la anexina y la Sb22.6. El objetivo de la presente tesis doctoral fue la caracterización molecular y funcional de cinco proteínas de S. bovis, por su interés como potenciales reguladores de los mecanismos hemostáticos del hospedador. Las cinco proteínas seleccionadas son: la enolasa, la gliceraldehído-3-fosfato deshidrogenasa y la fructosa-1,6-bisfosfato aldolasa por su previsible función profibrinolítica como ligandos de plasminógeno, y la anexina y el antígeno Sb22.6 por sus potenciales funciones anticoagulantes. La caracterización molecular y funcional de estas moléculas no solo ha permitido profundizar en el conocimiento de la biología del parásito y de sus relaciones con el hospedador, sino que, dado que la neutralización de tales moléculas antihemostáticas podría traducirse en una interrupción de la supervivencia del parásito, abre nuevas perspectivas para el desarrollo de vacunas o fármacos frente a S. bovis[EN] Schistosomiasis is an important health and veterinary problem in many tropical and subtropical areas of the world. Schistosoma bovis is a ruminant blood parasite, considered one of the main causes of schistosomiasis animal. This parasitic disease that affects 150 million animals in Asia and Africa and is also present in our country, causing significant losses in livestock farms to reduce the growth rate of infected animals and increase susceptibility to other diseases themselves. Besides its importance veterinary, S. bovis is a good model for studying human schistosomiasis, given the phylogenetic proximity between the species of the genus. To survive in the vascular bed of the host, S. bovis should neutralize not only the immune response but also hemostatic mechanisms of the host, for which you must have the appropriate anti-hemostatic molecules. Despite its importance, anti-hemostatic molecules of schistosomes have hardly been studied. The few details known about the interaction between S. bovis and the hemostatic system of their hosts are those obtained by our research group in previous studies. These studies found that S. bovis manipulates the host fibrinolytic system by fixing and plasminogen activation and identified ten plasminogen binding proteins, and other proteins that could also be related to the regulation of host hemostatic mechanisms, including annexin and SB22. June. The aim of this thesis was the molecular and functional characterization of five proteins of S. bovis, for their interest as potential regulators of host hemostatic mechanisms. The five selected proteins include enolase, glyceraldehyde-3-phosphate dehydrogenase and fructose-1 ,6-bisphosphate aldolase for its expected function as ligands fibrinolytic plasminogen and annexin and antigen for their potential Sb22.6 anticoagulant functions . The molecular and functional characterization of these molecules has not only enabled a deeper understanding of the biology of the parasite and its relationship with the host, but, since neutralization of such molecules antihemostáticas could result in a disruption of the parasite's survival opens new perspectives for the development of vaccines or drugs against S. bovi