microRNAs of parasitic helminths – identification, characterization and potential as drug targets

microRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation. They were first identified in the free-living nematode Caenorhabditis elegans, where the miRNAs lin-4 and let-7 were shown to be essential for regulating correct developmental progression. The sequence of let-7 was subsequently found to be conserved in higher organisms and changes in expression of let-7, as well as other miRNAs, are associated with certain cancers, indicating important regulatory roles. Some miRNAs have been shown to have essential functions, but the roles of many are currently unknown. With the increasing availability of genome sequence data, miRNAs have now been identified from a number of parasitic helminths, by deep sequencing of small RNA libraries and bioinformatic approaches. While some miRNAs are widely conserved in a range of organisms, others are helminth-specific and many are novel to each species. Here we review the potential roles of miRNAs in regulating helminth development, in interacting with the host environment and in development of drug resistance. Use of fluorescently-labeled small RNAs demonstrates uptake by parasites, at least in vitro. Therefore delivery of miRNA inhibitors or mimics has potential to alter miRNA activity, providing a useful tool for probing the roles of miRNAs and suggesting novel routes to therapeutics for parasite control

Alien Registration- Winter, Alan John D. (Bangor, Penobscot County)

https://digitalmaine.com/alien_docs/13968/thumbnail.jp

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

Characterisation of nematode prolyl 4-hydroxylase collagen modifying enzymes

The function of prolyl 4-hydroxylases (P4H) in the formation of the nematode cuticle was studied. The cuticle is one of the two major forms of extracellular matrix (ECM) in the nematode and performs vital roles in these animals including acting as an exoskeleton to maintain body morphology. Nematodes develop from an embryo through four larval moults to the adult stage. Each larval stage is characterised by the synthesis of a new cuticle and shedding of the existing structure. The nematode cuticle is a complex multi-layered structure formed principally from collagens that are synthesised by the underlying hypodermal tissue. Collagens are characterised by repeats of the amino acid sequence Gly-X-Y, where Gly is glycine and X and Y can be any residue but are most commonly proline and 4-hydroxyproline respectively. Three collagen monomers combine to form a triple helix, with the presence of 4- hydroxyproline residues stabilising the structure. The enzyme P4H modifies Y position proline residues in newly synthesised collagen molecules within the endoplasmic reticulum (ER) of the cell to produce 4-hydroxyproline. P4H role in cuticular ECM formation was examined primarily using the free-living species Caenorhabditis elegans due to its ease of handling and culture in the laboratory, the range of genetic and transgenic techniques available, and the complete genome sequence. Characterised collagen P4H from other species are most often multi-enzyme complexes formed from catalytically active beta subunits with the alpha subunit being the enzyme protein disulphide isomerase (PDI). The role of PDI in these complexes is not connected to its enzymatic activity but instead functions to keep the beta subunits in a catalytically active form within the ER of the cell. The described vertebrate P4Hs are alpha2beta2 tetramer complexes. Two different alpha subunits have been identified which form into separate enzyme complexes with a common PDI beta subunit. In this study the genes Ce-phy-1, Ce-phy-2 and Ce-pdi-2 were examined for their role in cuticular ECM formation in C. elegans. These genes were shown to be expressed throughout development in cuticle collagen synthesising hypodermal tissue in a manner reflecting the expression of their substrate, placing the encoded enzymes in the appropriate tissue for collagen modification, at times of maximal collagen synthesis. Disruption of Ce-phy-1 gene function produced nematodes with a mutant body shape known as dumpy (Dpy). This demonstrated that this gene is required for correct body morphology and led to the identification of the strain dpy-18 as a Ce-phy-1 mutant. This represented the first identification of a P4H mutant in any organism. Double disruption of Ce-phy-1 and Ce-phy-2 or Ce-pdi-2 singly resulted in an embryonic lethal phenotype due to the loss of P4H activity resulting in a cuticle unable to maintain nematode body shape. Disruption of these genes was demonstrated to have an affect on the localisation of specific cuticle collagens. The forms of P4H complex present were examined which revealed the presence of a unique mixed a subunit tetramer, in addition to the already described dimer form of the enzyme. Examination of three divergent putative Ce phy genes did not reveal any role for these in modification of the major ECMs in this nematode and showed that only Ce-PHY-1, Ce-PHY-2 and Ce-PDI-2 perform the essential modification of cuticle collagens. P4H was also studied in the human infective filarial nematode Brugia malayi which is one of the causative agents of lymphatic filariasis in humans, a disease that affects over 120 million people. A phy gene homologue, named Bm phy-1, was cloned and characterised from this organism. In contrast to the both human a subunit-encoding genes, which were shown to rescue the C. elegans dpy-18 P4H mutant, expression of Bm-phy-1 was not sufficient to repair the mutant form of these nematodes. Expression of this gene was demonstrated in all life cycle stages examined, with the gene promoter directing expression of a reporter gene to the hypodermal cells of C. elegans

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

Prolyl 4-hydroxlase activity is essential for development and cuticle formation in the human infective parasitic nematode Brugia malayi

Collagen prolyl 4-hydroxylases (C-P4H) are required for formation of extracellular matrices in higher eukaryotes. These enzymes convert proline residues within the repeat regions of collagen polypeptides to 4-hydroxyproline, a modification essential for the stability of the triple helix. C-P4Hs are most often oligomeric complexes, with enzymatic activity contributed by the α subunits, and the β subunits formed by protein disulfide isomerase (PDI). Here we characterise this enzyme class in the important human parasitic nematode Brugia malayi. All potential C-P4H subunits were identified by detailed bioinformatic analysis of sequence databases, function was investigated both by RNAi in the parasite and heterologous expression in Caenorhabditis elegans, while biochemical activity and complex formation were examined via co-expression in insect cells. Simultaneous RNAi of two B. malayi C-P4H α subunit-like genes resulted in a striking, highly penetrant body morphology phenotype in parasite larvae. This was replicated by single RNAi of a B. malayi C-P4H β subunit-like PDI. Surprisingly however, the B. malayi proteins were not capable of rescuing a C. elegans α subunit mutant, whereas the human enzymes could. In contrast, the B. malayi PDI did functionally complement the lethal phenotype of a C. elegans β subunit mutant. Comparison of recombinant and parasite derived material indicates that enzymatic activity may be dependent on a non-reducible, inter-subunit cross-link, present only in the parasite. We therefore demonstrate that C-P4H activity is essential for development of B. malayi and uncover a novel parasite-specific feature of these collagen biosynthetic enzymes that may be exploited in future parasite control

Loss of secretory pathway FK506-binding proteins results in cold-sensitive lethality in caenorhabditis elegans

The FK506-binding proteins (FKBs) represent ubiquitous enzymes that catalyse the rate-limiting peptidyl prolyl cis-trans isomerization step in protein folding. The nematode <i>Caenorhabditis elegans</i> has eight FKBs, three of which (FKB-3, -4 and -5) have dual peptidyl prolyl <i>cis-trans</i> isomerase (PPIase) domains, signal peptides and ER-retention signals. PPIase activity has been detected for recombinant FKB-3. Both FKB-3 and -5 are expressed in the exoskeleton-synthesising hypodermis with transcript peaks that correspond to the molting and collagen synthesis cycles. FKB-4 is expressed at a low level throughout development. No phenotypes were observed in deletion mutants in each of the secretory pathway FKBs. Combined triple and <i>fkb-4/-5</i> double deletion mutants were found to arrest at 12°C, but developed normally at 15-25°C. This cold-sensitive larval lethal effect was not maternally-derived, occurred during embryogenesis and could be rescued following the transgenic introduction of a wild type copy of either <i>fkb-4 or fkb-5</i>. The temperature-sensitive defects also affected molting, cuticle collagen expression, hypodermal seam cell morphology and the structural integrity of the cuticular extracellular matrix. This study establishes that the secretory pathway FK506-binding PPIase enzymes are essential for normal nematode development, collagen biogenesis and the formation of an intact exoskeleton under adverse physiological conditions

DNA methylation-associated colonic mucosal immune and defense responses in treatment-naïve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P < 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD

Protein disulfide isomerase activity is essential for viability and extracellular matrix formation in the nematode Caenorhabditis elegans.

Protein disulfide isomerase (PDI) is a multifunctional protein required for many aspects of protein folding and transit through the endoplasmic reticulum. A conserved family of three PDIs have been functionally analysed using genetic mutants of the model organism Caenorhabditis elegans. PDI-1 and PDI-3 are individually nonessential, whereas PDI-2 is required for normal post-embryonic development. In combination, all three genes are synergistically essential for embryonic development in this nematode. Mutations in pdi-2 result in severe body morphology defects, uncoordinated movement, adult sterility, abnormal molting and aberrant collagen deposition. Many of these phenotypes are consistent with a role in collagen biogenesis and extracellular matrix formation. PDI-2 is required for the normal function of prolyl 4-hydroxylase, a key collagen-modifying enzyme. Site-directed mutagenesis indicates that the independent catalytic activity of PDI-2 may also perform an essential developmental function. PDI-2 therefore performs two critical roles during morphogenesis. The role of PDI-2 in collagen biogenesis can be partially restored following complementation of the mutant with human PDI

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