Profiling microRNAs through development of the parasitic nematode Haemonchus identifies nematode-specific miRNAs that suppress larval development.

Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.

Increased Expression of a MicroRNA Correlates with Anthelmintic Resistance in Parasitic Nematodes.

Resistance to anthelmintic drugs is a major problem in the global fight against parasitic nematodes infecting humans and animals. While previous studies have identified mutations in drug target genes in resistant parasites, changes in the expression levels of both targets and transporters have also been reported. The mechanisms underlying these changes in gene expression are unresolved. Here, we take a novel approach to this problem by investigating the role of small regulatory RNAs in drug resistant strains of the important parasite Haemonchus contortus. microRNAs (miRNAs) are small (22 nt) non-coding RNAs that regulate gene expression by binding predominantly to the 3' UTR of mRNAs. Changes in miRNA expression have been implicated in drug resistance in a variety of tumor cells. In this study, we focused on two geographically distinct ivermectin resistant strains of H. contortus and two lines generated by multiple rounds of backcrossing between susceptible and resistant parents, with ivermectin selection. All four resistant strains showed significantly increased expression of a single miRNA, hco-miR-9551, compared to the susceptible strain. This same miRNA is also upregulated in a multi-drug-resistant strain of the related nematode Teladorsagia circumcincta. hco-miR-9551 is enriched in female worms, is likely to be located on the X chromosome and is restricted to clade V parasitic nematodes. Genes containing predicted binding sites for hco-miR-9551 were identified computationally and refined based on differential expression in a transcriptomic dataset prepared from the same drug resistant and susceptible strains. This analysis identified three putative target mRNAs, one of which, a CHAC domain containing protein, is located in a region of the H. contortus genome introgressed from the resistant parent. hco-miR-9551 was shown to interact with the 3' UTR of this gene by dual luciferase assay. This study is the first to suggest a role for miRNAs and the genes they regulate in drug resistant parasitic nematodes. miR-9551 also has potential as a biomarker of resistance in different nematode species.

Conservation of a microRNA cluster in parasitic nematodes and profiling of miRNAs in excretory-secretory products and microvesicles of Haemonchus contortus.

microRNAs are small non-coding RNAs that are important regulators of gene expression in a range of animals, including nematodes. We have analysed a cluster of four miRNAs from the pathogenic nematode species Haemonchus contortus that are closely linked in the genome. We find that the cluster is conserved only in clade V parasitic nematodes and in some ascarids, but not in other clade III species nor in clade V free-living nematodes. Members of the cluster are present in parasite excretory-secretory products and can be detected in the abomasum and draining lymph nodes of infected sheep, indicating their release in vitro and in vivo. As observed for other parasitic nematodes, H. contortus adult worms release extracellular vesicles (EV). Small RNA libraries were prepared from vesicle-enriched and vesicle-depleted supernatants from both adult worms and L4 stage larvae. Comparison of the miRNA species in the different fractions indicated that specific miRNAs are packaged within vesicles, while others are more abundant in vesicle-depleted supernatant. Hierarchical clustering analysis indicated that the gut is the likely source of vesicle-associated miRNAs in the L4 stage, but not in the adult worm. These findings add to the growing body of work demonstrating that miRNAs released from parasitic helminths may play an important role in host-parasite interactions.

Application of small RNA technology for improved control of parasitic helminths.

Over the last decade microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important regulators of post-transcriptional gene expression. miRNAs are short, non-coding RNAs that regulate a variety of processes including cancer, organ development and immune function. This class of small RNAs bind with partial complementarity to their target mRNA sequences, most often in the 3'UTR, to negatively regulate gene expression. In parasitic helminths, miRNAs are being increasingly studied for their potential roles in development and host-parasite interactions. The availability of genome data, combined with small RNA sequencing, has paved the way to profile miRNAs expressed at particular developmental stages for many parasitic helminths. While some miRNAs are conserved across species, others appear to be unique to specific parasites, suggesting important roles in adaptation and survival in the host environment. Some miRNAs are released from parasites, in exosomes or in protein complexes, and the potential effects of these on host immune function are being increasingly studied. In addition, release of miRNAs from schistosome and filarial parasites into host plasma can be exploited for the development of specific and sensitive diagnostic biomarkers of infection. Interfering with miRNA function, as well as silencing key components of the pathways they regulate, will progress our understanding of parasite development and provide a novel approach to therapeutic control. RNA interference (RNAi) by siRNAs has proven to be inconsistent in parasitic nematodes. However, the recent successes reported for schistosome and liver fluke RNAi, encourage further efforts to enhance delivery of RNA and improve in vitro culture systems and assays to monitor phenotypic effects in nematodes. These improvements are important for the establishment of reliable functional genomic platforms for novel drug and vaccine development. In this review we focus on the important roles of miRNAs and siRNAs in post-transcriptional gene regulation in veterinary parasitic helminths and the potential value of these in parasite diagnosis and control.