Susan E. Newton

Genomic and genetic research on bursate nematodes: significance, implications and prospects

Molecular genetic research on parasitic nematodes (order Strongylida) is of major significance for many fundamental and applied areas of medical and veterinary parasitology. The advent of gene technology has led to some progress for this group of nematodes, particularly in studying parasite systematics, drug resistance and population genetics, and in the development of diagnostic assays and the characterisation of potential vaccine and drug targets. This paper gives an account of the molecular biology and genetics of strongylid nematodes, mainly of veterinary socio-economic importance, indicates the implications of such research and gives a perspective on genome research for this important parasite group, in light of recent technological advances and knowledge of the genomes of other metazoan organisms.

Identification and cDNA cloning of two novel low molecular weight host-protective antigens from Taenia ovis oncospheres

Oncosphere antigens of Taenia ovis were solubilised in sodium dodecyl sulphate and separated by electrophoresis in polyacrylamide gels (SDS-PAGE). Antigen-containing gel fractions cut from the region covering 18-12 kDa were shown to be highly immunogenic in sheep challenge experiments. Specific antisera against 2 candidate antigens at 18 and 16 kDa were used to screen a cDNA library prepared from T. ovis oncosphere mRNA. Recombinant proteins selected with antibody to the 16 and 18 kDa native antigens were expressed as GST fusion proteins. Vaccination trials using either of the 2 fusion proteins To16.17-GST and To18-GST, revealed that each was capable of inducing high levels of immunity in sheep against challenge infection with T. ovis eggs. Antibodies induced by vaccination with the recombinant antigens reacted specifically with their respective 18 or 16 kDa native oncosphere antigens. There was no apparent homology between the T. ovis cDNA coding for To18 and To16.17, or with another host-protective antigen, To45W, described previously. These additional host-protective antigens should prove a valuable adjunct to To45W and permit the development of effective vaccination strategies.

Molecular aspects of sexual development and reproduction in nematodes and schistosomes

In contrast to the free-living nematode Caenorhabditis elegans, surprisingly little is known about the molecular aspects of reproduction in parasitic helminths. Investigations into such aspects would provide an improved understanding of the fundamentals of sexual differentiation, development, maturation and behaviour, as well as sex-specific genes and their expression. Such knowledge could lead to new means of parasite control by interfering with or disrupting one or more of these processes, which is particularly important given the emerging problems with genetic resistance in parasitic nematodes against anthelmintic drugs. This chapter brings together some relevant information on the sexual biology of C. elegans, summarizes studies of gender-specific expression in selected parasitic helminths of socio-economic significance, describes advanced molecular techniques for the analysis of gender-specific genes, and indicates the prospects for genomic research on reproductive processes and the implications thereof for controlling parasitic helminths.

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.

Analysis of developmentally regulated genes of the parasite Haemonchus contortus

Differential regulation of gene expression in the development of Haemonchus contortus was analysed using RNA arbitrarily-primed PCR. A study of third-stage larval and adult H. contortus revealed large differences between the two stages; 32 and 30% unique third-stage larval and adult RNA arbitrarily-primed PCR products, respectively. This finding is consistent with a high degree of differential gene expression between these developmental stages. A number of adult products were sequenced, revealing 11 molecules to be similar to deposits within sequence databases. Four other molecules that did not have significant similarity to sequences in the databases may represent developmentally regulated genes specific to H. contortus. Northern analysis of the putative adult-expressed molecules with homologues in the databases confirmed that four were expressed only in adults, while four were expressed in both stages, but had different sized transcripts. This may reflect differential splicing, or expression of closely related but different molecules at different life cycle stages. Two molecules were present in mRNA populations from both stages, suggesting these were false stage-associated molecules. No transcript was detected for one molecule by Northern analysis, probably due to low level of expression. In situ hybridisation analysis was used to localise expression of transcripts in the adult parasite, in particular, to gain some insight into the nature of those molecules with no known predicted function.

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.

HcSTK, a Caenorhabditis elegans PAR-1 homologue from the parasitic nematode, Haemonchus contortus

A putative serine/threonine protein kinase (HcSTK) from the parasitic nematode Haemonchus contortus was characterised at the mRNA and amino acid levels. HcSTK displays a high level of identity (85-93% in the catalytic domain) with proteins of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily, which represent signal transduction molecules involved in establishing and maintaining polarity in proliferating and differentiating cells. The transcript of hcstk is expressed in different developmental stages (second-, third-, fourth-stage larvae and adults) and various organs (muscle, intestine and reproductive) of H. contortus. In addition, there are several isoforms which appear to relate to a single gene. The expression profile of hcstk is similar to that of Caenorhabditis elegans PAR-1, and the level of sequence identity among members of the PAR-1/MARK STK subfamily, representing a range of species of vertebrates (e.g. humans and rodents), invertebrates (e.g. insects and C. elegans) and yeast, suggests that HcSTK may be involved in a conserved signal transduction pathway.

Haemonchus contortus: molecular characterisation of a small heat shock protein

A cDNA encoding a predicted small heat shock protein, HSP20, was isolated from the parasitic nematode Haemonchus contortus. This cDNA encoded a predicted protein of 156 amino acids, which had high sequence identity with other nematode small heat shock proteins. Southern blot analysis of genomic DNA suggested that in H. contortus HSP20 is encoded by a single copy gene. The HSP20 transcript and protein were expressed in the infective larvae (L3), early L4 and adult stages, but expression was not increased by heat shock treatment. In situ hybridisation analysis was used to localise expression of HSP20 mRNA in the adult parasite. Similar HSPs (heat shock protein) were detected by Western blotting in Ancylostoma caninum, Dictyocaulus viviparus, and Toxocara canis, but not in Trichostronglyus colubriformis. The conservation of HSP20 in several different nematode species may reflect its importance to parasites that require mammalian hosts as a part of their development. Index Descriptors and Abbreviations: Haemonchus contortus; nematode; small heat shock protein; L3, infective larvae; xL3, exsheathed L3; eL4, early L4; EST, expressed sequence tag; HSP20, heat shock protein 20; sHSP, small heat shock protein

The organization of a galectin gene from Teladorsagia circumcincta.

Galectins are a family of soluble beta-galactoside-binding lectins that are conserved amongst a broad range of organisms. We have previously isolated cDNA clones coding for galectins from the sheep gastrointestinal nematode parasites Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus colubriformis, revealing a high level of identity between these molecules. This subsequent study reports the organization of the T. circumcincta Tci-gal-1 galectin gene. The coding region is broken into eight exons covering 6.6 kbp, with introns ranging in size from 55 base pairs (bp) to 2.8 kbp. Comparisons with recently reported galectin structures from Caenorhabditis elegans reveal strong architectural similarity between galectins from the parasitic and free-living nematodes, but this structure is not conserved in mammalian galectins.

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.