Proteomic analysis of Oesophagostomum dentatum (Nematoda) during larval transition, and the effects of hydrolase inhibitors on development.

In this study, in vitro drug testing was combined with proteomic and bioinformatic analyses to identify and characterize proteins involved in larval development of Oesophagostomum dentatum, an economically important parasitic nematode. Four hydrolase inhibitors ο-phenanthroline, sodium fluoride, iodoacetamide and 1,2-epoxy-3-(pnitrophenoxy)-propane (EPNP) significantly inhibited (≥90%) larval development. Comparison of the proteomic profiles of the development-inhibited larvae with those of uninhibited control larvae using two-dimensional gel electrophoresis, and subsequent MALDI-TOF mass spectrometric analysis identified a down-regulation of 12 proteins inferred to be involved in various larval developmental processes, including post-embryonic development and growth. Furthermore, three proteins (i.e. intermediate filament protein B, tropomyosin and peptidyl-prolyl cis-trans isomerase) inferred to be involved in the moulting process were down-regulated in moulting- and development-inhibited O. dentatum larvae. This first proteomic map of O. dentatum larvae provides insights in the protein profile of larval development in this parasitic nematode, and significantly improves our understanding of the fundamental biology of its development. The results and the approach used might assist in developing new interventions against parasitic nematodes by blocking or disrupting their key biological pathways.

Sequence variability in three mitochondrial genes between the two pig nodule worms Oesophagostomum dentatum and O. quadrispinulatum.

In this study, sequence variation in three mitochondrial DNA regions, namely cytochrome c oxidase subunit (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), between Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical origins in Mainland China was examined, and their phylogenetic relationships were reconstructed. A partial of the cox1 (pcox1), nad1, and nad4 genes (pnad1 and pnad4) were amplified separately from individual nodule worms by PCR and were subjected to direct sequencing in order to define sequence variations. While the intraspecific sequence variations within each of the two species were 0.3-5.2% for pcox1, 0-4.9% for pnad1, and 0-7.1% for pnad4, the interspecific sequence differences were significantly higher, being 10.7-13.4% for pcox1, 11-14.6% for pnad1, and 14.9-18% for pnad4, respectively. There were a number of nucleotide positions in the pcox1, pnad1, and pnad4 sequences with no apparent intraspecific variation but distinct interspecific differences among those samples of Oesophagostomum spp. examined, which may be used as genetic makers for the identification and differentiation of the Oesophagostomum spp. Phylogenetic analyses using three inference methods, namely Bayesian inference, maximum likelihood, and maximum parsimony based on the combined sequences of pcox1, pnad1, and pnad4 revealed that the O. dentatum and O. quadrispinulatum form monophyletic groups, respectively. These findings demonstrated clearly the usefulness of the three mitochondrial sequences for studying systematics, population genetic structures, and the molecular ecology of Oesophagostomum spp.

Oesophagostomum dentatum: effect of glutathione S-transferase (GST) inhibitors on GST activity and larval development.

Sulphobromophthalein (SBP) inhibits isolated glutathione S-transferase of the porcine nodule worm Oesophagostomum dentatum (Od-GST) and reduces larval development in vitro. In this study possible inhibitory effects of various inhibitors were evaluated in an enzymatic (CDNB) assay with isolated Od-GST and in a larval development assay (LDA). Reversibility was tested in the LDA by removing the inhibitor from culture halfway through the cultivation period. SBP, indomethacin and ethacrynic acid inhibited both enzyme activity and larval development in a dose-dependent and reversible manner. HQL-79 also reduced larval development but had only a minor effect on the isolated enzyme. The phospholipase A(2) inhibitors dexamethasone and hydrocortisone had no major effect. High thermal stability of Od-GST was demonstrated with increasing activity between 4 and 50°C. Differences between Od-GST and GST of other organisms indicate structural and possibly functional peculiarities and highlight the potential of such enzymes as targets of intervention.

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.

Cytosolic glutathione S-transferases of Oesophagostomum dentatum.

Oesophagostomum dentatum stages were investigated for glutathione S-transferase (GST) expression at the protein and mRNA levels. GST activity was detected in all stages (infectious and parasitic stages including third- and fourth-stage larvae of different ages as well as males and females) and could be dose-dependently inhibited with sulfobromophthalein (SBP). Addition of SBP to in vitro larval cultures reversibly inhibited development from third- to fourth-stage larvae. Two glutathione-affinity purified proteins (23 and 25 kDa) were detected in lysates of exsheathed third-stage larvae by SDS-PAGE. PCR-primers were designed based on peptide sequences and conserved GST sequences of other nematodes for complete cDNA sequences (621 and 624 nt) of 2 isoforms, Od-GST1 and Od-GST2, with 72% nucleotide similarity and 75% for the deduced proteins. Genomic sequences consisted of 7 exons and 6 introns spanning 1296 bp for Od-GST1 and 1579 and 1606 bp for Od-GST2. Quantitative real-time-PCR revealed considerably elevated levels of Od-GST1 in the early parasitic stages and slightly reduced levels of Od-GST2 in male worms. Both Od-GSTs were most similar to GST of Ancylostoma caninum (nucleotides: 73 and 70%; amino acids: 80 and 73%). The first three exons (75 amino acids) corresponded to a synthetic prostaglandin D2 synthase (53% similarity). O. dentatum GSTs might be involved in intrinsic metabolic pathways which could play a role both in nematode physiology and in host-parasite interactions.

Ubiquitin-conjugating enzyme genes in Oesophagostomum dentatum.

Full-length genes representing different isoforms of the ubiquitin-conjugating enzyme UBC-2 were isolated from Oesophagostomum dentatum, cloned and sequenced. The alignment of their sequences (designated Od-ubc-2.1 to Od-ubc-2.3) revealed nucleotide variation at three positions within the predicted open reading frame of 444 bp. Substitutions were at positions 141 (A<-->G), 142 (A<-->G) and 296 (T<-->C). Both former substitutions resulted in amino acid changes from a glycine residue to an arginine residue, whereas the latter resulted in a change from isoleucine to threonine. Comparison of predicted OD-UBC-2 with UBC-2 (protein) homologues/orthologues from 12 other species representing nematodes, Drosophila melanogaster, Saccharomyces cerevisiae, mice and humans revealed identities between species varying from 77 to 100% at the amino acid level, and motifs associated with protein conformation and function were identified. While the function of a representative ubc-2 gene from O. dentatum could not be established in C. elegans, it is likely to play a key role in the catabolism of proteins and in the development of O. dentatum.

Molecular characterisation of a male-specific serine/threonine phosphatase from Oesophagostomum dentatum (Nematoda: Strongylida), and functional analysis of homologues in Caenorhabditis elegans.

A male-specifically expressed sequence tag was used as a probe to screen adult male Oesophagostomum dentatum (Nematoda; Strongylida) gene libraries. The cDNA clones isolated coded for a serine/threonine protein phosphatase with approximately 85% identity to two Caenorhabditis elegans proteins implicated in reproduction. However, the genomic structures for the two species were distinct, in that the O. dentatum gene contained seven introns, whereas the C. elegans homologues contained three (two of which were conserved between the two nematodes). The promoters of all three nematode genes contained two putative GATA motifs separated by six to seven nucleotides and located within 100 nucleotides of the predicted transcriptional start site. RNA interference (RNAi) experiments in C. elegans, targetting the two homologues, revealed a consistent reduction in the number of progeny produced by treated worms, indicating a functional role in reproduction. Expression of green fluorescent protein, directed by the putative promoters for the C. elegans phosphatase genes, was analysed in transgenic C. elegans. The present results suggest that there is a significant degree of conservation between O. dentatum and C. elegans in the features and function of the serine/threonine protein phosphatase characterised, which should have implications for detailed investigations into molecular reproductive processes of some parasitic nematodes.

Oesophagostomum dentatum: expression patterns of enzymes involved in eicosanoid production.

Cytosolic and membrane-bound proteins of various stages of Oesophagostomum dentatum, the nodular worm of pigs, were investigated for the presence of lipoxygenases (LOX) and cyclooxygenases (COX) using polyclonal and monoclonal antibodies. Putative 12-LOX and 15-LOX, but not 5-LOX, were detected in both fractions of all developmental stages in the expected size range of 75 kDa, with an isoelectric point of 6.0-6.5. The protein could be precipitated with 50% ammonium sulfate, as described for mammalian LOX. An antibody directed against both COX isoforms and one against mammalian COX-2 detected proteins of approximately 70 kDa with an isoelectric point of 6.0-6.5 in the membrane-bound fractions of third-stage larvae and adults, but not in the fourth-stage larvae. Anti-COX-1 or more specific anti-COX-2 antibodies failed to detect proteins. The constitutive LOX expression supports the assumption that the metabolites of this enzyme previously detected in O. dentatum serve intrinsic functions, while the production of anti-inflammatory COX-products in the invasive and luminal stages of the parasite implies a possible role in host-parasite interactions.

Sequence variations in the introns of the triosephosphate isomerase genes of Oesophagostomum dentatum and O. quadrispinulatum.

Degenerated primers were used to amplify DNA fragments of the triosephosphate isomerase (TPI) gene from complementary DNA (cDNA) and from genomic DNA of two species of porcine gastrointestinal nematodes, Oesophagostomum dentatum and O.quadrispinulatum. Polymerase chain reaction (PCR) fragments amplified from cDNA were 520 bp in size for both species, while genomic fragments were 1,035 bp for O. dentatum (GC-content: 45%) and 1,331 bp for O. quadrispinulatum (44%). Sequence analyses revealed blocks of high homology in the exons interrupted by more variable parts in the intron regions. Five exons were predicted from the genomic sequences in the conserved regions which corresponded to the respective cDNA sequences with 6% interspecific differences. The predicted protein sequences (161 amino acids) were 98% similar between the species and showed 71% similarity to the putative protein of Caenorhabditis elegans. As a housekeeping gene, TPI could be amplified from cDNA of both infectious third-stage larvae and adults. Interspecific variations in the non-coding regions allow the PCR-based differentiation of the two Oesophagostomum spp.

Characterization of Chabertia ovina by isoenzyme gel electrophoresis: comparative study with Oesophagostomum venulosum.

The glucose-6-phosphate dehydrogenase (G6PD, EC.1.1.1.49), glucose phosphate isomerase (GPI, EC.5.3.1.9), and malate dehydrogenase (MDH, EC.1.1.1.37) isoenzymatic patterns of Chabertia ovina were determined by starch-gel electrophoresis. The G6PD and GPI isoenzymatic patterns were characterized by the existence of three phenotypes: (1) a single and slow anodic band, (2) a single and fast anodic band, and (3) a large spot matching its migration with bands 1 and 2. These three phenotypes may be explained as the existence of only one gene locus for the G6PD and GPI in C. ovina. Allelic frequencies and the Hardy-Weinberg test were determined. This test indicated that the population was not in Hardy-Weinberg equilibrium. The MDH isoenzymatic pattern of C. ovina was characterized by the presence of two bands with anodic and cathodic migration. Furthermore, comparative isoenzyme studies were carried out between Oesophagostomum venulosum and C. ovina. The different G6PD, GPI, and MDH isoenzymatic patterns observed for the two species allowed us to distinguish them and, therefore, to use isoenzymatic patterns as a diagnostic tool to discriminate these species.

Characterization of porcine and ovine Oesophagostomum spp. by isoenzymatic patterns and restriction-fragment-length polymorphisms (RFLPs).

Four different morphological and biometrical populations of Oesophagostomum have been identified, using classical taxonomy methods, from Sus scrofa domestica (pigs): O. dentatum (Od), O. quadrispinulatum (Oq), O. granatensis (Og), and a fourth population, including individuals with morphological and biometric parameters overlapping these three species that were clasified as Oesophagostomum sp. The G6PD and MDH isoenzymatic patterns did not discriminate between the three species, while GPI showed a diagnostic isoenzymatic pattern for Oq. Og showed identical G6PD, GPI and MDH isoenzymatic pattern as Od. Furthermore, after rDNA amplification by polymerase chain reaction (PCR), the uncut PCR product showed that the ITS2 of these three species had a similar size of 320 base pairs (bp). Restriction-fragment-length polymorphisms (RFLP) were analyzed after digestion of the ITS2 with 13 different restriction enzymes. After electrophoretic separation of the digested PCR products, only one unique differentiating pattern of bands was observed for Od and Oq. This was when Sau3AI was used, while Og showed an identical band pattern to Od. Thus, our studies provided no evidence for the existence of Og and Od as differentiated populations. O. venulosum was isolated from sheep and goat; G6PD and MDH isoenzymatic patterns discriminated this species from porcine species of Oesophagostomum. The ITS2 region appeared as a different band of 380 bp from those observed for porcine Oesophagostomum species.

Genetic diversity in porcine Oesophagostomum dentatum and O. quadrispinulatum and their delineation by isoenzyme analysis.

The genetic diversity in eight strains of Oesophagostomum dentatum and O. quadrispinulatum was investigated by the electrophoresis study of ten enzyme systems. The loci Idh-2, Fbp, Sdh, and Pgm were found to be diagnostic between the species examined. Both the proportion of fixed allelic differences (26.3%) and the genetic distance coefficient (D = 0.54) are well above the range for differentiation of valid species. Isoenzyme patterns of susceptible and resistant lines of O. dentatum showed at polymorphic loci a reduced genetic heterogeneity in the latter group. No qualitative difference in terms of the presence/absence of alleles was observed among susceptible and resistant isolates with the enzymes studied. The detection of one possible hybrid indicates that introgression in O. dentatum and O. quadrispinuatum may occur.

Glucose 6-phosphate dehydrogenase: isoenzymatic pattern in Oesophagostomum venulosum, Trichuris ovis and T. suis.

In the present communication we have studied the isoenzymatic pattern activity of the glucose 6-phosphate dehydrogenase (G6PD) in Oesophagostomum venulosum, Trichuris ovis and T. suis, parasites of Capra hircus (goat), Ovis aries (sheep) and Sus scrofa domestica (pig) respectively, by polyacrylamide gel electrophoresis. Different phenotypes have been observed in the G6PD isoenzymatic pattern activity in males and females of Oesophagostomum venulosum. Furthermore, G6PD activity has been assayed in Trichuris ovis collected from Ovis aries and Capra hircus. No differences have been observed in the isoenzymatic patterns attending to the different hosts. All the individuals exhibited one single band or two bands; this suggests a monomeric condition for G6PD in T. ovis. In T. suis the enzyme G6PD appeared as a single electrophoretic band in about 85.7% of the individuals.