Project description:O-glycosylation is probably one of the most varied sets of post-translational modifications across all organisms, but amongst the most refractory to analyze. In animals, O-xylosylation of serine residues represents the first stage in the synthesis of glycosaminoglycans, whose repeat regions are generally analyzed as fragments resulting from enzymatic or chemical degradation, whereas their core regions can be isolated by β-elimination or endo-β-xylosidase digestion. In the present study, we show that hydrazinolysis can be employed for release of glycosaminoglycan-type oligosaccharides from nematodes prior to fluorescent labeling with 2-aminopyridine. While various [HexNAcHexA]nGal2Xyl oligosaccharides were isolated from the model organism Caenorhabditis elegans, more unusual glycosaminoglycan-type glycans were found to be present in the porcine parasite Oesophagostomum dentatum. In this case, as judged by MS/MS before and after hydrofluoric acid or β-galactosidase digestion, core sequences with extra galactose and phosphorylcholine residues were detected as [(±PC)HexNAcHexA]n(±PC)Galβ3-(±Galβ4)Galβ4Xyl. Thus, hydrazinolysis and fluorescent labeling can be combined to analyze unique forms of O-xylosylation, including new examples of zwitterionic glycan modifications.
Project description:The porcine nodule worm Oesophagostomum dentatum is a strongylid class V nematode rather closely related to the model organism Caenorhabditis elegans. However, in contrast to the non-parasitic C. elegans, the parasitic O. dentatum is an obligate sexual organism, which makes both a gender and developmental glycomic comparison possible.Different enzymatic and chemical methods were used to release N-glycans from male and female O. dentatum as well as from L3 and L4 larvae. Glycans were analysed by MALDI-TOF MS after either 2D-HPLC (normal then reversed phase) or fused core RP-HPLC.Whereas the L3 N-glycome was simpler and more dominated by phosphorylcholine-modified structures, the male and female worms express a wide range of core fucosylated N-glycans with up to three fucose residues. Seemingly, simple methylated paucimannosidic structures can be considered 'male', while methylation of fucosylated glycans was more pronounced in females. On the other hand, while many of the fucosylated paucimannosidic glycans are identical with examples from other nematode species, but simpler than the tetrafucosylated glycans of C. elegans, there is a wide range of phosphorylcholine-modified glycans with extended HexNAc2-4PC2-4 motifs not observed in our previous studies on other nematodes.The interspecies tendency of class V nematodes to share most, but not all, N-glycans applies also to O. dentatum; furthermore, we establish, for the first time in a parasitic nematode, that glycomes vary upon development and sexual differentiation.Unusual methylated, core fucosylated and phosphorylcholine-containing N-glycans vary between stages and genders in a parasitic nematode.
Project description:BackgroundGene identification and sequence determination are critical requirements for many biological, genomic, and bioinformatic studies. With the advent of next generation sequencing (NGS) technologies, such determinations are predominantly accomplished in silico for organisms for which the genome is known or for which there exists substantial gene sequence information. Without detailed genomic/gene information, in silico sequence determination is not straightforward, and full coding sequence determination typically involves time- and labor-intensive PCR-based amplification and cloning methods.ResultsAn improved method was developed with which to determine full length gene coding sequences in silico using de novo assembly of RNA-Seq data. The scheme improves upon initial contigs through contig-to-gene identification by BLAST nearest-neighbor comparison, and through single-contig refinement by iterative-binning and -assembly of reads. Application of the iterative method produced the gene identification and full coding sequence for 9 of 12 genes and improved the sequence of 3 of the 12 genes targeted by benzimidazole, macrocyclic lactone, and nicotinic agonist classes of anthelminthic drugs in the swine nodular parasite Oesophagostomum dentatum. The approach improved upon the initial optimized assembly with Velvet that only identified full coding sequences for 2 genes.ConclusionsOur reiterative methodology represents a simplified pipeline with which to determine longer gene sequences in silico from next generation sequence data for any nematode for which detailed genetic/gene information is lacking. The method significantly improved upon an initial Velvet assembly of RNA-Seq data that yielded only 2 full length sequences. The identified coding sequences for the 11 target genes enables further future examinations including: (i) the use of recombinant target protein in functional assays seeking a better understanding of the mechanism of drug resistance, and (ii) seeking comparative genomic and transcriptomic assessments between parasite isolates that exhibit varied drug sensitivities.
Project description:We explored molecules involved in in vitro exsheathment of Oesophagostomum dentatum L3s using a proteomic-transcriptomic-bioinformatic approach. Analysis of L3s before, during and after exsheathment identified 11 proteins that were over-expressed exclusively during exsheathment. These proteins (including key enzymes, heat shock, structural and nematode-specific proteins) were inferred to be involved in development, metabolism, structure, motility and/or host-parasite interactions. Some of these molecules represented homologues linked to entry into and exit from the dauer stage in the free-living nematode Caenorhabditis elegans. The approach established here provides a basis for investigations of ecdysis in other strongylid nematodes.
Project description:Nematode anthelminthic resistance is widespread for the 3 major drug classes commonly used in agriculture: benzamidazoles, macrocyclic lactones, and nicotinic agonists e.g. levamisole. In parasitic nematodes the genetics of resistance is unknown other than to the benzimidazoles which primarily involve a single gene. In previous work with a levamisole resistant Oesophagostomum dentatum isolate, the nicotinic acetylcholine receptor (nAChR) exhibited decreased levamisole sensitivity. Here, using a transcriptomic approach on the same isolate, we investigate whether that decreased nAChR sensitivity is achieved via a 1-gene mechanism involving 1 of 27 nAChR pathway genes. 3 nAChR receptor subunit genes exhibited ?2-fold change in transcript abundance: acr-21 and acr-25 increased, and unc-63 decreased. 4 SNPs having a ?2-fold change in frequency were also identified. These data suggest that resistance is likely polygenic, involving modulated abundance of multiple subunits comprising the heteropentameric nAChR, and is not due to a simple 1-gene mechanism.
Project description:Zolvix® is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel is a positive allosteric modulator of DEG-3/DES-2 type nicotinic acetylcholine receptors (nAChRs) in several nematode species. The drug has been reported to produce hypercontraction of Caenorhabditis elegans and Haemonchus contortus somatic muscle. We investigated the effects of monepantel on nAChRs from Ascaris suum and Oesophagostomum dentatum heterologously expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp electrophysiology, we studied the effects of monepantel on a nicotine preferring homomeric nAChR subtype from A. suum comprising of ACR-16; a pyrantel/tribendimidine preferring heteromeric subtype from O. dentatum comprising UNC-29, UNC-38 and UNC-63 subunits; and a levamisole preferring subtype (O. dentatum) comprising UNC-29, UNC-38, UNC-63 and ACR-8 subunits. For each subtype tested, monepantel applied in isolation produced no measurable currents thereby ruling out an agonist action. When monepantel was continuously applied, it reduced the amplitude of acetylcholine induced currents in a concentration-dependent manner. In all three subtypes, monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 from A. suum was particularly sensitive to monepantel inhibition (IC50 values: 1.6?±?3.1?nM and 0.2?±?2.3??M). We also investigated the effects of monepantel on muscle flaps isolated from adult A. suum. The drug did not significantly increase baseline tension when applied on its own. As with acetylcholine induced currents in the heterologously expressed receptors, contractions induced by acetylcholine were antagonized by monepantel. Further investigation revealed that the inhibition was a mixture of competitive and non-competitive antagonism. Our findings suggest that monepantel is active on multiple nAChR subtypes.
Project description:The characteristics of the intergenic spacer rDNAs (IGS rDNAs) of Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical locations in Mainland China were determined, and the phylogenetic relationships of the two species were reconstructed using the IGS rDNA sequences. The organization of the IGS rDNA sequences was similar to their organization in other eukaryotes. The 28S-18S IGS rDNA sequences of both O. dentatum and O. quadrispinulatum were found to have variable lengths, that is, 759-762 bp and 937-1128 bp, respectively. All of the sequences contained direct repeats and inverted repeats. The length polymorphisms were related to the different numbers and organization of repetitive elements. Different types and numbers of repeats were found between the two pig nodule species, and two IGS structures were found within O. quadrispinulatum. Phylogenetic analysis showed that all O. dentatum isolates were clustered into one clade, but O. quadrispinulatum isolates from different origins were grouped into two distinct clusters. These results suggested independent species and the existence of genotypes or subspecies within pig nodule worms. Different types and numbers of repeats and IGS rDNA structures could serve as potential markers for differentiating these two species of pig nodule worms.