The parasitic nematode Oesophagostomum dentatum synthesizes unusual glycosaminoglycan-like O-glycans.

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 ß-elimination or endo-ß-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 ß-galactosidase digestion, core sequences with extra galactose and phosphorylcholine residues were detected as [(±PC)HexNAcHexA]n(±PC)Galß3-(±Galß4)Galß4Xyl. Thus, hydrazinolysis and fluorescent labeling can be combined to analyze unique forms of O-xylosylation, including new examples of zwitterionic glycan modifications.

Gender and developmental specific N-glycomes of the porcine parasite Oesophagostomum dentatum.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. GENERAL SIGNIFICANCE: Unusual methylated, core fucosylated and phosphorylcholine-containing N-glycans vary between stages and genders in a parasitic nematode.

Cracking the nodule worm code advances knowledge of parasite biology and biotechnology to tackle major diseases of livestock.

Many infectious diseases caused by eukaryotic pathogens have a devastating, long-term impact on animal health and welfare. Hundreds of millions of animals are affected by parasitic nematodes of the order Strongylida. Unlocking the molecular biology of representatives of this order, and understanding nematode-host interactions, drug resistance and disease using advanced technologies could lead to entirely new ways of controlling the diseases that they cause. Oesophagostomum dentatum (nodule worm; superfamily Strongyloidea) is an economically important strongylid nematode parasite of swine worldwide. The present article reports recent advances made in biology and animal biotechnology through the draft genome and developmental transcriptome of O. dentatum, in order to support biological research of this and related parasitic nematodes as well as the search for new and improved interventions. This first genome of any member of the Strongyloidea is 443 Mb in size and predicted to encode 25,291 protein-coding genes. Here, we review the dynamics of transcription throughout the life cycle of O. dentatum, describe double-stranded RNA interference (RNAi) machinery and infer molecules involved in development and reproduction, and in inducing or modulating immune responses or disease. The secretome predicted for O. dentatum is particularly rich in peptidases linked to interactions with host tissues and/or feeding activity, and a diverse array of molecules likely involved in immune responses. This research progress provides an important resource for future comparative genomic and molecular biological investigations as well as for biotechnological research toward new anthelmintics, vaccines and diagnostic tests.

Trichuris suis and Oesophagostomum dentatum show different sensitivity and accumulation of fenbendazole, albendazole and levamisole in vitro.

BACKGROUND: The single-dose benzimidazoles used against Trichuris trichiura infections in humans are not satisfactory. Likewise, the benzimidazole, fenbendazole, has varied efficacy against Trichuris suis whereas Oesophagostomum dentatum is highly sensitive to the drug. The reasons for low treatment efficacy of Trichuris spp. infections are not known. METHODOLOGY: We studied the effect of fenbendazole, albendazole and levamisole on the motility of T. suis and O. dentatum and measured concentrations of the parent drug compounds and metabolites of the benzimidazoles within worms in vitro. The motility and concentrations of drug compounds within worms were compared between species and the maximum specific binding capacity (Bmax) of T. suis and O. dentatum towards the benzimidazoles was estimated. Comparisons of drug uptake in living and killed worms were made for both species. PRINCIPAL FINDINGS: The motility of T. suis was generally less decreased than the motility of O. dentatum when incubated in benzimidazoles, but was more decreased when incubated in levamisole. The Bmax were significantly lower for T. suis (106.6, and 612.7 pmol/mg dry worm tissue) than O. dentatum (395.2, 958.1 pmol/mg dry worm tissue) when incubated for 72 hours in fenbendazole and albendazole respectively. The total drug concentrations (pmol/mg dry worm tissue) were significantly lower within T. suis than O. dentatum whether killed or alive when incubated in all tested drugs (except in living worms exposed to fenbendazole). Relatively high proportions of the anthelmintic inactive metabolite fenbendazole sulphone was measured within T. suis (6-17.2%) as compared to O. dentatum (0.8-0.9%). CONCLUSION/SIGNIFICANCE: The general lower sensitivity of T. suis towards BZs in vitro seems to be related to a lower drug uptake. Furthermore, the relatively high occurrence of fenbendazole sulphone suggests a higher detoxifying capacity of T. suis as compared to O. dentatum.

Galactosylated fucose epitopes in nematodes: increased expression in a Caenorhabditis mutant associated with altered lectin sensitivity and occurrence in parasitic species.

The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by ß1,4-linked galactose ("GalFuc" epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and ß1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.

Prostaglandin D(2) synthesis in Oesophagostomum dentatum is mediated by cytosolic glutathione S-transferase.

Glutathione S-transferases (GSTs) of Oesophagostomum dentatum possess considerable similarity to synthetic prostaglandin D synthase (PGDS), and therefore their ability to convert prostaglandin (PG) H(2) to PGD(2)in vitro was investigated with a commercial Prostaglandin D Synthase Inhibitor Screening Assay Kit. Fractioned homogenates of O. dentatum third-stage larvae only displayed cytosolic but not microsomal GST. Both total larval homogenate and isolated GST could metabolise PGH(2) to PGD(2), which could be inhibited by the GST inhibitor sulfobromophthalein (SBP) in a dose-dependent manner, whereas reactions to the specific PGDS inhibitor HQL-79 were not dose-dependent. Inhibition of larval development by SBP in vitro was abolished by the addition of PGD(2) but not by PGH(2), supporting the assumption that GST acts as PGDS and is important for nematode development. Since motility and viability of O. dentatum larvae are reduced in vitro by various inhibitors of eicosanoid metabolism, enzymes of this pathway, including GST, constitute putative intervention targets.

A practical, bioinformatic workflow system for large data sets generated by next-generation sequencing.

Transcriptomics (at the level of single cells, tissues and/or whole organisms) underpins many fields of biomedical science, from understanding the basic cellular function in model organisms, to the elucidation of the biological events that govern the development and progression of human diseases, and the exploration of the mechanisms of survival, drug-resistance and virulence of pathogens. Next-generation sequencing (NGS) technologies are contributing to a massive expansion of transcriptomics in all fields and are reducing the cost, time and performance barriers presented by conventional approaches. However, bioinformatic tools for the analysis of the sequence data sets produced by these technologies can be daunting to researchers with limited or no expertise in bioinformatics. Here, we constructed a semi-automated, bioinformatic workflow system, and critically evaluated it for the analysis and annotation of large-scale sequence data sets generated by NGS. We demonstrated its utility for the exploration of differences in the transcriptomes among various stages and both sexes of an economically important parasitic worm (Oesophagostomum dentatum) as well as the prediction and prioritization of essential molecules (including GTPases, protein kinases and phosphatases) as novel drug target candidates. This workflow system provides a practical tool for the assembly, annotation and analysis of NGS data sets, also to researchers with a limited bioinformatic expertise. The custom-written Perl, Python and Unix shell computer scripts used can be readily modified or adapted to suit many different applications. This system is now utilized routinely for the analysis of data sets from pathogens of major socio-economic importance and can, in principle, be applied to transcriptomics data sets from any organism.

Characterization of major sperm protein genes and their expression in Oesophagostomum dentatum (Nematoda: Strongylida).

Major sperm protein (msp) genes were isolated from complementary (cDNA) and genomic DNA libraries prepared from the parasitic nematode, Oesophagostomum dentatum, characterized at the nucleotide and amino acid (aa) levels, and their expression was investigated. Three different msp cDNA and 2 genomic sequences were determined, each with an open reading frame (ORF) of 381 nucleotides. Nucleotide variation was detected at 30 positions in the ORF among all 5 sequences. Conceptual translation of the full-length msp sequences inferred 4 different MSPs each of 126 aa. These predicted MSPs differed at aa positions 15 (serine <----> threonine), 101 (alanine <----> glycine), 103 (glutamine <----> leucine) and 126 (proline <----> leucine). Southern blot analysis of O. dentatum genomic DNA, digested separately with various restriction endonucleases, displayed multiple (n = 7-13) bands for each enzyme, providing support for a multigene family. Also, at the genomic level, sequence tracts consistent with a 'substitute' TATA box sequence motif were identified within a region (-1 to -123 nt) preceding the 2 msp genes. In contrast to other species of nematode investigated to date, no GATA transcription factor binding motif was detected immediately upstream of the msp coding region. Real-time PCR analysis demonstrated that msp mRNA was expressed exclusively in the males of both fourth-stage larvae (L4s) and adults of O. dentatum (raised in pigs after intragastric inoculation). The magnitude of expression in male O. dentatum raised in pigs in the presence of female worms was the same as in males in the absence of females. Comparative analyses showed aa sequence conservation among MSPs from various nematodes, suggesting similar functional roles for these proteins.

Genomics of reproduction in parasitic nematodes-fundamental and biotechnological implications.

Understanding reproductive and developmental processes of socioeconomically important parasitic nematodes is of fundamental scientific interest and could have important implications for developing novel methods for parasite control via the disruption or interruption of such processes. Central to investigating reproductive molecular biology is the identification and characterisation of genes with sex-specific expression profiles. However, there is currently a paucity of information on such genes and their expression patterns in parasitic nematodes. This article describes recent progress on the characterisation of sex-specific genes from a parasitic nematode of veterinary importance, and discusses the fundamental scientific and applied implications of this work.

Isolation and characterisation of sex-specific transcripts from Oesophagostomum dentatum by RNA arbitrarily-primed PCR.

In light of the lack of molecular data on the sexual differentiation, maturation and interaction of parasitic nematodes of livestock, the present study investigated sex-specific gene expression in the nodule worm, Oesophagostomum dentatum (Strongylida). Using the technique of RNA arbitrarily-primed polymerase chain reaction (RAP-PCR), 31 expressed sequence tags (ESTs) differentially-displayed between the sexes were cloned. Northern blot analysis proved ten ESTs to be expressed exclusively in males (adults and fourth-stage larvae), while two were expressed solely in female stages. None of the ESTs were expressed in infective third-stage larvae. Sequence analysis and subsequent database searches revealed two male-specific ESTs to have significant similarity to Caenorhabditis elegans (predicted) proteins, a protein containing an EGF-like cysteine motif and a serine/threonine phosphatase. Another two male-specific ESTs had similarity to non-nematode sequences. The two female-specific ESTs had similarity to vitellogenin-5 and endonuclease III (predicted) from C. elegans. The remaining ESTs had no similarity to any nucleic acid or protein sequences contained in the databases. The isolation and characterisation of sex-specific ESTs from O. dentatum provides a unique opportunity for studying the reproductive biology of parasitic nematodes at the molecular level, with a view toward novel approaches for parasite control.

The effect of increasing levels of insoluble dietary fibre on the establishment and persistence of Oesophagostomum dentatum in pigs.

This investigation compared the effect of diets with increasing content of insoluble dietary fibre (DF) on the establishment and persistence of Oesophagostomum dentatum in growing pigs. Twenty-eight worm-free pigs, from a specific pathogen-free farm were randomly divided to four groups of seven animals. The animals were assigned to the following diets: diet A, barley flour plus protein mixture (70%:30%); diet B, barley flour, oat husk meal plus protein mixture (65%:7%:28%); diet C, barley flour, oat husk meal plus protein mixture (60%:14%:26%) and diet D, barley flour, oat husk meal plus protein mixture (55%:21%:24%). The diets were formulated to provide increasing content of DF but constant levels of digestible protein per feeding unit for pigs. All pigs were experimentally inoculated with 6,000 infective O. dentatum larvae and followed coprologically for 11 weeks post infection, whereafter they were slaughtered. The experimental diets influenced the mean transit time and the metabolism in the large intestine significantly. Diets C and D, with highest content of insoluble DF, provided favourable conditions for establishment of O. dentatum, but diets A and B led to a significant lower worm numbers and fecundity.

Glucose uptake in Oesophagostomum dentatum and the effect of oxfendazole.

The uptake of 14C-glucose by adult Oesophagostomum dentatum was characterised. The uptake was a non-linear function of external glucose concentration. The maximum velocity of uptake (Vmax) was 0.964 nmol/100 mg dry weight (dw)/5 min, and the transport constant (Kt) was 10.02 microM. When phlorizin, phloretin and 3-O-methylglucose were tested for their effects on the uptake of 14C-glucose, phloretin and 3-O-methylglucose produced significant inhibitions, indicating that the uptake was mediated and occurred by facilitated diffusion. Exposure of the worms to oxfendazole prior to incubation with 14C-glucose did not affect the uptake of glucose. In another experiment worms were incubated with unlabelled glucose and the external glucose concentration was measured enzymatically. During a 7 h incubation period, the quantity of glucose remaining in the incubation medium of oxfendazole exposed worms was significantly greater than in the control group. It was concluded that oxfendazole did not influence the process of 14C-glucose uptake, but might induce changes in the parasite leading to a reduced ability to deplete the incubation medium of glucose.

Investigations into the production and function of leukotrienes during histotropic development of Oesophagostomum dentatum.

The dynamics of production and excretion of leukotrienes by parasitic larvae of Oesophagostomum dentatum and their role for the development of the larvae were studied. Larvae were cultured in vitro to the fourth stage (L4). Leukotriene B4 (LTB4) was detected in homogenates and in supernatant of larvae, with the homogenate-protein-based values steadily decreasing during development. The homogenate-protein-based values of peptidyl leukotrienes (pepLT) remained fairly stable in both homogenates and supernatants, whereas the wormcount-based pepLT values increased significantly. The addition of diethylcarbamazine (DEC) to the culture medium straight from the beginning of culturing (12.8 or 25.5 mmol/l) reversibly hampered growth and development to L4. Application of DEC at 12.8 mmol/l beginning on day 13 of in vitro cultivation exerted no significant effect on further development to L4. LTB4 appeared to counteract the inhibition of development by DEC. The results of this study indicate that endogenous LTs participate in regulation of the growth and development of O. dentatum.

Oesophagostomum dentatum: population dynamics and synthesis of prostanoids by histotropic stages cultured in vitro.

The dynamics of production and excretion of prostanoids by histotropic larvae of Oesophagostomum dentatum and the role of prostanoids for the development of O. dentatum were studied under in vitro conditions. In control cultures fourth stage larvae (L4) were first seen on Day 7 of in vitro cultivation. Their numbers steadily increased until Day 21 of in vitro cultivation. Thereafter, the numbers of L4 no further increased. The continuous presence of inhibitors of prostanoid synthesis such as acetylsalicyclic acid (ASA, 5.6 mmole/liter) or indomethacin (INDO, 1.4 mmole/liter) in the culture medium blocked the development to L4 almost completely. When ASA was given first on Day 13 of in vitro cultivation L4 counts still increased during the following 4 days. However, the numbers of larvae which reached the L4 stage in these cultures was reduced to 65% of that in control cultures. Resumption of development to L4 was seen after the withdrawal of INDO in cultures treated with this drug until Day 13 of in vitro cultivation. Larvae and supernatants were collected separately on Days 7, 14, 21, or 28 of in vitro cultivation and larvae were homogenized. The presence of prostaglandin (PG) E2, PGD2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (TX) B2 was assayed radioimmunologically in homogenates and supernatants. In the homogenates only PGE2 and PGD2 were regularly detected. The total amount of PGE2 and PGD2 in the homogenates significantly increased with the increase in L4 numbers, whereas the homogenate protein-based values did not. In addition to PGD2 and PGE2, PGF2 alpha and TXB2 were found in the supernatants. PGE2 accounted for about 50% of the prostanoids in the supernatants on Day 7 of in vitro cultivation but was generally absent thereafter. After 14, 21, and 28 days of in vitro cultivation TXB2 was the most prominent single prostanoid in the supernatants. PGD2 was found in the supernatants only after 7 and 28 days of in vitro cultivation. PGF2 alpha was present in decreasing amounts in the supernatants throughout the cultivation period. In conclusion, histotropic larvae of O. dentatum produce and excrete mediators of the prostanoid type which may serve as endogenous hormone-like growth factors.

Suppression of antigen- and mitogen-induced proliferation of bovine lymphocytes by excretory-secretory products of Oesophagostomum radiatum.

Excretory-secretory products (ESP) isolated from in vitro-grown stage-3 to -4 larvae of Oesophagostomum radiatum were found to inhibit both the in vitro antigen-specific proliferation of keyhole limpet hemocyanin- and ovalbumin-primed lymphocytes and the proliferation induced by the T-cell mitogen concanavalin A. As little as 50 ng of ESP protein per culture resulted in 50% reductions of subsequent proliferative responses. Antigen-induced responses were 100 to 1,000 times more sensitive to inhibition than were mitogen-induced responses. The inhibitory activity was found to affect the induction of proliferation as evidenced by the observation that complete inhibition was seen when ESP were added to cultures within the first 24 h. ESP were found to have no inhibitory activity when added 72 h after the initiation of the cultures. The inhibition was not a result of a direct action upon macrophages because pulsing of adherent cells with ESP had no more effect on a subsequent proliferative response than did a pulsing of the culture vessel itself. The inhibitory activity eluted from high-pressure liquid chromatography columns in the same fractions as protein standards with molecular weights of 25,000 to 35,000. Of special interest is the fact that this inhibitor of the expansion of immunoreactive clones of lymphocytes is found associated with the stages of parasites most intimately associated with host tissues, namely larval stages 3 and 4.

Nematoda: aerobic respiratory pathways of adult parasitic species.

Aerobic respiratory pathways have been compared in adult parasitic nematodes, including Trichostrongylus colubriformis, Nippostrongylus brasiliensis, Ostertagia ostertagi, Cooperia oncophora, Haemonchus contortus, Oesophagostomum venulosum, Chabertia ovina, Dictyocaulus filaria, Dictyocaulus viviparus, and Ascaridia galli. Respiration was measured in both whole worm or tissue homogenates and isolated mitochondrial fractions, and delineated into the mammalian type or alternative respiratory pathways on the basis of their inhibition by antimycin A. The alternative, antimycin A-insensitive respiratory pathway was of comparable activity in all parasitic nematodes studied, irrespective of the body diameter or habitat of the worm. The mammalian-type, antimycin A-sensitive respiratory pathway showed variations; the extent of this pathway correlated with both the body diameter and habitat of the worm, being greater in thinner worms and those worms whose habitat is supposedly more aerobic.

Oesophagostomum radiatum: glucose metabolism of larvae grown in vitro and adults grown in vivo.

Larval stages of Oesophagostomum radiatum grown in vitro and adults grown in vivo were incubated in complex media or in a simple salt solution containing radioactive glucose. Glucose disappearance and end product accumulation of third-stage larvae in a simple salt solution indicated that they excreted CO2 and acetic, propionic, and lactic acids. Larvae in third molt, fourth stage, and adults all excreted CO2, acetic, propionic, and lactic acids at twice the rate of third-stage larvae plus an additional product, methylbutyric acid. Carbon dioxide arose primarily from the 3 or 4 carbons of glucose. An anaerobic atmosphere (95% N2:5% CO2) had no apparent effect on metabolism. When incubation was done in complex media, isobutyric and 3-methylbutyric acids were seen as major excretion products (10 and 24%, respectively). However, these acids were quantitatively minor when incubations took place in simple salts-glucose medium (1 and 0-3%, respectively).

In vitro variation of glycogen content in three sheep nematodes.

In vitro variation of glycogen content under aerobic conditions was measured on fresh weight basis in 3 sheep nematodes inhabiting different niches; Haemonchus contortus, Oesophagostomum columbianum and Trichuris ovis. The parasites were saparated into species and then sexes and starved for varying periods of time up to 24 h in glucose-free physiological saline. The differences between females and males and among the species with respect to glycogen content and its rate of change with time are discussed.