Abstract
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.
Highlights
Just as antimicrobial resistance poses a serious threat to the treatment of bacterial infections, anthelmintic resistance is a major challenge for the control of parasitic nematodes
We show that the level of a single miRNA is significantly up-regulated in parental and backcrossed resistant strains compared to the susceptible MHco3(ISE) and identify possible target mRNAs of this miRNA, the expression levels of which correlate inversely with miRNA levels
The array was probed with a 1:1 mix of RNA from male and female H. contortus worms recovered from three individual sheep per strain, yielding triplicate biological replicates of IVM sensitive strain MHco3(ISE) and IVM resistant strains MHco4(WRS) and MHco10(CAVR) and the relevant backcrosses, MHco3/4.BC4 and MHco3/10.BC4
Summary
Just as antimicrobial resistance poses a serious threat to the treatment of bacterial infections, anthelmintic resistance is a major challenge for the control of parasitic nematodes. In some parts of the world, including the United Kingdom, parasites resistant to all three drug classes have been identified (Sargison et al, 2007), posing a threat to the viability of livestock farming and compromising global food security. Of these broad-spectrum compounds, the macrocyclic lactones are the most recent and the most widely used. Whether or not similar mechanisms underlie IVM resistance in parasitic nematodes is the focus of much debate (Gilleard and Beech, 2007)
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