Developmental Regulation and Functional Prediction of microRNAs in an Expanded Fasciola hepatica miRNome

Abstract

The liver fluke, Fasciola hepatica, is a global burden on the wellbeing and productivity of farmed ruminants, and a zoonotic threat to human health. Despite the clear need for accelerated discovery of new drug and vaccine treatments for this pathogen, we still have a relatively limited understanding of liver fluke biology and host interactions. Noncoding RNAs, including micro (mi)RNAs, are key to transcriptional regulation in all eukaryotes, such that an understanding of miRNA biology can shed light on organismal function at a systems level. Four previous publications have reported up to 89 mature miRNA sequences from F. hepatica, but our data show that this does not represent a full account of this species miRNome. We have expanded on previous studies by sequencing, for the first time, miRNAs from multiple life stages (adult, newly excysted juvenile (NEJ), metacercariae and adult-derived extracellular vesicles (EVs)). These experiments detected an additional 61 high-confidence miRNAs, most of which have not been described in any other species, expanding the F. hepatica miRNome to 150 mature sequences. We used quantitative (q)PCR assays to provide the first developmental profile of miRNA expression across metacercariae, NEJ, adult and adult-derived Evs. The majority of miRNAs were expressed most highly in metacercariae, with at least six distinct expression clusters apparent across life stages. Intracellular miRNAs were functionally analyzed to identify target mRNAs with inversely correlated expression in F. hepatica tissue transcriptomes, highlighting regulatory interactions with key virulence transcripts including cathepsin proteases, and neuromuscular genes that control parasite growth, development and motility. We also linked 28 adult-derived EV miRNAs with downregulation of 397 host genes in F. hepatica-infected transcriptomes from ruminant lymph node, peripheral blood mononuclear cell (PBMC) and liver tissue transcriptomes. These included genes involved in signal transduction, immune and metabolic pathways, adding to the evidence for miRNA-based immunosuppression during fasciolosis. These data expand our understanding of the F. hepatica miRNome, provide the first data on developmental miRNA regulation in this species, and provide a set of testable hypotheses for functional genomics interrogations of liver fluke miRNA biology.

Transcriptomic analysis supports a role for the nervous system in regulating growth and development of Fasciola hepatica juveniles

“…Small RNA FASTQ files were aligned to a Bowtie index of F. hepatica genome PRJEB25283 using miRDeep2 (v 2.0.1.2) [37]. miRNAs were defined using the following criteria; present in at least 2 out of 3 replicates for either in vitro or in vivo transcriptomes, a minimum of 10 reads mapped to the mature sequence, at least 1 read mapped to the star sequence, a significant randfold value and a minimum miRDeep2 score of 5. miRNA naming was consistent with that presented in Herron et al [38]. Differential expression of identified miRNAs was carried out using DESeq2 (version 1.28.1), with an adjusted p-value of 0.001 for significance.…”

“…To determine the role of miRNAs in the regulation of transcripts associated with growth and development, miRNA sequencing was carried out on RNA extracted from in vitro and in vivo maintained juvenile liver fluke. This study identified 103 miRNAs in liver fluke, including 14 novel PLOS NEGLECTED TROPICAL DISEASES miRNAs (Sheet A in S9 Table) and 89 miRNAs reported in previous studies on F. hepatica [38,[64][65][66][67]. Recently, Herron et al [38] highlighted considerable redundancy across published miRNAs and moved to refine the current cohort of F. hepatica miRNAs by removing duplicate sequences of high similarity and retaining the longer sequence as individual miRNAs.…”

“…This study identified 103 miRNAs in liver fluke, including 14 novel PLOS NEGLECTED TROPICAL DISEASES miRNAs (Sheet A in S9 Table) and 89 miRNAs reported in previous studies on F. hepatica [38,[64][65][66][67]. Recently, Herron et al [38] highlighted considerable redundancy across published miRNAs and moved to refine the current cohort of F. hepatica miRNAs by removing duplicate sequences of high similarity and retaining the longer sequence as individual miRNAs. Applying this approach to our dataset refines our final miRNA dataset to 89 miRNAs (75 previously published [64][65][66][67]; 14 novel).…”

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

“…The reason behind such an atypical secretory mechanism remains unclear although it would enable delivery of gut-derived EVs en masse into the host bile/bloodstream during the regular regurgitation of fluke ESPs that occurs during feeding [ 22 ]. Given the variety of protein and RNA cargo associated with F. hepatica EVs [ 50 , 51 , 52 ], it is conceivable that EVs released in this way may exert immunomodulatory or other effects on host cells at locations quite distant from their bile duct niche. Whether the ruptured gastrodermal cells are degraded entirely and replaced with new cells is unclear.…”

A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance

“…Interrogation of in vivo RNA-seq datasets from isolates of known phenotype would inform on whether any candidate genes show differential expression on TCBZ exposure. Similarly, as small non-coding microRNA (miRNA) are known to regulate gene expression it would be prudent to look for predicted miRNA binding sites in candidate genes, particularly given that they have been linked to drug resistance in nematodes [76] and a large dataset of miRNAs has been reported in F. hepatica [77].…”