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The miRNome of Fasciola hepatica juveniles endorse the existence of a reduced set of highly divergent miRNAs in parasitic flatworms.

ABSTRACT: The liver fluke Fasciola hepatica is a foodborne zoonotic parasite affecting livestock worldwide with increasing relevance in human health. The first developmental stage that the host meets after ingestion of the parasite is the newly excysted juvenile (NEJ), that actively transverse the gut wall and migrates to their final location in the liver. The regulation of the early developmental events in NEJs is still poorly understood and a relevant target for control strategies. Here we investigated the putative involvement of small regulatory RNAs in the invasion process. The small RNA population of the NEJ fall into two classes, one represented by miRNAs and a secondary group of larger (32- 33 nucleotides) tRNA derived sequences. We identified more than 30 different miRNAs most of them belonging to ancient miRNAs conserved in protostomes and metazoans, notably with an miR-125b variant as highly predominant. Remarkably, several protostomian and metazoan conserved families were not detected in consonance with previous reports of drastic miRNome reduction in parasitic flatworms. Additionally, a set of 11 novel miRNAs was identified, probably associated with specific gene regulation expression needs in F. hepatica. While sequence conservation in mature miRNA is high across the metazoan tree, we observed that flatworm miRNAs are more divergent suggesting that mutation rates in parasitic flatworms could be high. Finally, the distinctive presence of tRNA derived sequences, mostly 5' tRNA halves of selected tRNAs in the small RNA population of NEJs could indicate that this parasite uses both miRNA and tRNA fragments for the regulation of gene expression.

ORGANISM(S): Fasciola hepatica

PROVIDER: GSE66490 | GEO | 2015/10/02

SECONDARY ACCESSION(S): PRJNA277065

REPOSITORIES: GEO

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Project description:Effective management and control of parasitic infections on farms depends on their early detection. Traditional serological diagnostic methods for Fasciola hepatica infection in livestock are specific and sensitive, but currently the earliest detection of the parasite only occurs at approximately three weeks post-infection. At this timepoint, parasites have already entered the liver and caused the tissue damage and immunopathology that results in reduced body weight and loss in productivity. Here, we investigated whether the differential abundance of micro(mi)miRNAs in sera of F. hepatica-infected sheep has potential as a tool for the early diagnosis of infection. Using miRNA sequencing analysis, we discovered specific profiles of sheep miRNAs at both the pre-hepatic and hepatic infection phases in comparison to non-infected sheep. In addition, six F. hepatica-derived miRNAs were specifically identified in sera from infected sheep. Thus, a panel of differentially expressed miRNAs comprising four sheep (miR-3231-3p; miR133-5p; 3957-5p; 1197-3p) and two parasite miRNAs (miR-124-3p; miR-Novel-11-5p) were selected as potential biomarkers. The expression of these candidates in sera samples from longitudinal sheep infection studies collected between 7 days and 23 weeks was quantified using RT-qPCR and compared to samples from age-matched non-infected sheep. We identified oar-miR-133-5p and oar-miR-3957-5p as promising biomarkers of fasciolosis, detecting infection as early as 7 days. The differential expression of the other selected miRNAs was not sufficient to diagnose infection; however, our analysis found that the most abundant forms of fhe-miR-124-3p in sera were sequence variants (IsomiRs) of the canonical miRNA, highlighting the critical importance of primer design for accurate diagnostic RT-qPCR. Accordingly, this investigative study suggests that certain miRNAs are biomarkers of F. hepatica infection and validates miRNA-based diagnostics for the detection of fasciolosis in sheep.

Project description:Background: Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with Fasciola hepatica metacercariae using a microarray-based methodology. Methodology: A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21). Results: We found that Fasciola hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death. Conclusion: The present study provides us insights at the molecular level about the underlying mechanisms used by Fasciola hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma. However, more studies should be performed to confirm this hypothesis. We used three experimental groups each containing 3 mice. Group 1 remains untreated and served as control. Group 2 was infected with Fasciola hepatica metacercariae on day 0 and humanely necropsied at 7 days post-infection. Group 3 was infected with Fasciola hepatica metacercariae on day 0 and humanely necropsied at 21 days post- infection. At the time of necropsy, liver of each mice were removed and the RNA was isolated. We compared the gene expression profile in the liver of mice infected with Fasciola hepatica.

Project description:Parasitic worms (helminths) ensure their survival to establish chronic infection within mammalian hosts by strategically regulating their host s immune responses. Deciphering the mechanisms behind this immune modulation will inform the development of novel strategies for infection control. It is now understood that the co-ordinated control of gene expression by non-coding RNAs (ncRNA) underpins the activation and regulation of immune cells. However, it is also clear that pathogens (including helminths) secrete their own ncRNAs to manipulate host gene expression and influence immune outcomes to infection. To discriminate the contribution of host and helminth derived ncRNAs to the activation and/or regulation of innate immune responses during infection, we examined ncRNA expression in the peritoneal macrophages from mice infected with Fasciola hepatica. This revealed an enrichment of several parasitic-derived miRNAs (fhe-miR-277b-3p, fhe-miR-71a-5p, fhe-miR-125b-5p, fhe-miR-125a-5p) within macrophages at 6hr and 18hr after infection which is coincident with the parasite s migration through the peritoneal cavity. Target prediction analysis indicated that these Fasciola miRNAs regulated host genes associated with the activation of host pro-inflammatory macrophages (Hif1 , Nod1, Nlrp6 and P2rx7). Concomitantly, there was a distinct shift in host ncRNA expression in response to infection, which was most significant at 5 days post-infection. Although, analysis suggested that these host ncRNAs target a different cohort of genes (Adar1, Igf1 and Plec) to the parasite miRNAs, the functional outcome was predicted to be the same, with a reduced pro-inflammatory response and the promotion of a reparative/tolerant phenotype. Combined, these observations uncover the interplay between host and parasitic ncRNAs and reveals a complementary regulation of the immune response that allows the parasite to evade immune detection and promotes tissue repair for the host. This level of characterisation of the ncRNA landscape during helminth infection is highly novel and provides a new understanding of the molecular impact that these parasites have on host immune cells.

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The miRNome of Fasciola hepatica juveniles endorse the existence of a reduced set of highly divergent miRNAs in parasitic flatworms.

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