Haemonchus contortus: prokaryotic expression and enzyme activity of recombinant HcSTK, a serine/threonine protein kinase

Members of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily are important regulators of the cytoskeleton, and their characterization can provide insights into a number of critical processes relating to the development and survival of an organism. We previously investigated the mRNA expression for and organization of a gene (hcstk) representing HcSTK, an STK from the parasitic nematode Haemonchus contortus. In the present study, a recombinant form of HcSTK was expressed and characterized. Affinity-purified anti-HcSTK antibodies reacted with native HcSTK in protein homogenates extracted from third-stage larvae (L3) of H. contortus and were also used to immunolocalize the protein around the nuclei of ovarian and intestinal tissues of adult H. contortus. The enzyme activity of the recombinant HcSTK protein was also demonstrated. The findings show that recombinant HcSTK is a functional protein kinase, with activity directed to KXGS motifs, consistent with other members of the PAR-1/MARK STK subfamily.     

Prospects for exploring molecular developmental processes in Haemonchus contortus

Haemonchus contortus of small ruminants is a parasitic nematode of major socio-economic importance world-wide. While there is considerable knowledge of the morphological changes which take place during the life cycle of H. contortus, very little is understood about the molecular and biochemical processes which govern developmental changes in the parasite. Recent technological advances and the imminent genomic sequence for H. contortus provide unique opportunities to investigate the molecular basis of such processes in parasitic nematodes. This article reviews molecular and biochemical aspects of development in H. contortus, reports on some recent progress on signal transduction molecules in this parasite and emphasises the opportunities that new technologies and the free-living nematode, Caenorhabditis elegans, offer for investigating developmental aspects in H. contortus and related strongylid nematodes, also in relation to developing novel approaches for control.     

Evaluation of Caenorhabditis elegans glycoproteins as protective immunogens against Haemonchus contortus challenge in sheep

High levels of protection can be attained against Haemonchus contortus challenge infection in sheep using native antigens isolated from the gut of the adult parasite. However, vaccination with recombinant forms of these antigens, or components thereof, has disappointingly failed to generate similar levels of protection, suggesting that appropriate nematode glycosylation may be a prerequisite for protection. The free-living nematode, Caenorhabditis elegans is closely related to H. contortus and has been shown to share similar glycan moieties. In order to investigate the potentially protective role of these glycan moieties, a complex set of glycoproteins was isolated from C. elegans using ConA-lectin chromatography and their efficacy as immunogens against H. contortus challenge infection evaluated in sheep. Despite the generation of a high titre systemic IgG antibody response to the C. elegans glycoproteins and the ability of these antibodies to bind to the microvillar surface of the gut of H. contortus, no protection against challenge infection was observed. Serum antibodies to the C. elegans glycoproteins cross-reacted with the H. contortus host-protective antigen, H-gal-GP, by ELISA, although the level of cross-reactivity was not of a magnitude considered protective. Qualitative differences were also determined between the glycan epitopes of the C. elegans ConA-binding proteins and those of H-gal-GP, suggesting the presence of H. contortus-specific patterns of glycosylation.     

Genetic analysis of inbreeding of two strains of the parasitic nematode Haemonchus contortus

Haemonchus contortus is a sheep parasitic nematode that causes severe economic losses. Previous studies have indicated a high degree of genetic heterogeneity, which is hardly affected by selection for drug resistance. As a tool for the analysis of the population dynamics of H. contortus and its response to drug resistance, we designed a strategy to study the inbreeding of a benzimidazole-sensitive and a benzimidazole-resistant strain. After 15 generations, a theoretical inbreeding coefficient of 0.87 was achieved. The different stages of inbreeding were analysed using restriction fragment polymorphism, microsatellite variability and amplified fragment length polymorphism. Model-based clustering of the amplified fragment length polymorphism genotypes showed that the allele frequencies of the benzimidazole-resistant strain were stable during the last eight generations. In the sensitive strain a gradual shift of allele frequencies was observed, which led to a temporary increase of the genetic diversity around the eight generations.     

PAR-1 is required for morphogenesis of the Caenorhabditis elegans vulva

The Caenorhabditis elegans vulva provides a simple model for the genetic analysis of pattern formation and organ morphogenesis during metazoan development. We have discovered an essential role for the polarity protein PAR-1 in the development of the vulva. Postembryonic RNA interference of PAR-1 causes a protruding vulva phenotype. We found that depleting PAR-1 during the development of the vulva has no detectable effect on fate specification or precursor proliferation, but instead seems to specifically alter morphogenesis. Using an apical junction-associated GFP marker, we discovered that PAR-1 depletion causes a failure of the two mirror-symmetric halves of the vulva to join into a single, coherent organ. The cells that normally form the ventral vulval rings fail to make contact or adhere and consequently form incomplete toroids, and dorsal rings adopt variably abnormal morphologies. We also found that PAR-1 undergoes a redistribution from apical junctions to basolateral domains during morphogenesis. Despite a known role for PAR-1 in cell polarity, we have observed no detectable differences in the distribution of various markers of epithelial cell polarity. We propose that PAR-1 activity at the cell cortex is critical for mediating cell shape changes, cell surface composition, or cell signaling during vulval morphogenesis.     

MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termedMMK2. The predicted amino acid sequence ofMMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termedMMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness ofMMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeastMPK1 (SLT2) kinase byMMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.     

Thioredoxins of a parasitic nematode: comparison of the 16- and 12-kDA thioredoxins from Haemonchus contortus

Thioredoxins are a family of small proteins conserved through evolution, which are essential for the maintenance of cellular homeostasis. The "classic" thioredoxin, identified in most species, is a 12-kDa protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in nematodes a larger protein, 16 kDa, with a Cys-Pro-Pro-Cys (CPPC) active site was identified. We report that in the parasitic nematode Haemonchus contortus, both the 12-kDa (HcTrx1) and the 16-kDa (HcTrx3) species are expressed through the life cycle. However, the HcTrx3 is expressed at higher concentrations. Recombinant HcTrx1 and HcTrx3 were produced and both reduced insulin at a rate similar to that observed with ovine (host) and Escherichia coli thioredoxins and both were regenerated by a mammalian thioredoxin reductase, demonstrating that they have similar thioredoxin activity. Unlike mammalian thioredoxins, both proteins were able to reduce oxidised glutathione and hydrogen peroxide. This suggests essential roles for these proteins in response to oxidative stress and the host immune attack. Analysis of ivermectin-resistant H. contortus showed that expression of both genes were increased in a drug-resistant strain relative to a sensitive strain. Involvement in drug resistance identifies these thioredoxin proteins as potential drug targets for parasite control.     

Evaluation of a recombinant excretory secretory Haemonchus contortus protein for use in a diagnostic enzyme-linked immunosorbent assay

The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24 kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep.     

Molecular characterization of a novel RhoGAP, RRC-1 of the nematode Caenorhabditis elegans

The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPases in C. elegans.     

Haemonchus contortus: molecular cloning, sequencing, and expression analysis of the gene coding for the small subunit of ribonucleotide reductase

Gastro-intestinal (GI) parasites are of great agricultural importance, annually costing the livestock industry vast amounts in resources to control parasitism. One such GI parasite, Haemonchus contortus, is principally pathogenic to sheep; with the parasite's blood-feeding behaviour causing effects ranging from mild anaemia to mortality in young animals. Current means of control, which are dependent on repeated treatment with anthelmintic chemicals, have led to increasing drug resistance. Together with the growing concern over residual chemicals in the environment and food chain, this has led to attempts to better understand the biology of the parasite with the aim to develop alternate or supplementary means of control, including the development of molecular vaccines. As a first step towards the understanding of the synthesis of deoxyribonucleotides in H. contortus, and its potential application to therapeutic control of this economically important parasite, we report the cloning, sequencing, and mRNA expression analysis of the ribonucleotide reductase R2 gene.     

Involvement of HMG-12 and CAR-1 in the cdc-48.1 expression of Caenorhabditis elegans

Caenorhabditis elegans possesses two p97/VCP/Cdc48p homologues, named CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8), and their expression patterns and levels are differently regulated. To clarify the regulatory mechanisms of differential expression of two p97 proteins of C. elegans, we performed detailed deletion analysis of their promoter regions. We found that the promoter of cdc-48.1 contains two regions necessary for embryonic and for post-embryonic expression, while the promoter of cdc-48.2 contains the single region necessary for embryonic expression. In particular, two elements (Element A and Element B) and three conserved boxes (Box a, Box b and Box c) were essential for cdc-48.1 expression in embryos and at post-embryonic stages, respectively. By using South-Western blotting and MALDI-TOF MS analysis, we identified HMG-12 and CAR-1 as proteins that bind to Element A and Element B, respectively, from the embryonic nuclear extract. Importantly, we found the decreased expression of p97 in embryos prepared from hmg-12(RNAi) or car-1(RNAi) worms. These results indicate that both HMG-12 and CAR-1 play important roles in embryonic expression of cdc-48.1.     

Global patterns reveal strong population structure in Haemonchus contortus, a nematode parasite of domesticated ruminants

We have examined the global population genetic structure of Haemonchus contortus. The genetic variability was studied using both amplified fragment length polymorphism (AFLP) and nad4 sequences of the mitochondrial genome. To examine the performance and information content of the two different marker systems, comparative assessment of population genetic diversity was undertaken in 19 isolates of H. contortus, a parasitic nematode of small ruminants. A total of 150 individual adult worms representing 14 countries from all inhabited continents were analysed. Altogether 1,429 informative AFLP markers were generated using four different primer combinations. Also, the genetic variation was high, which agrees with results from previous AFLP studies of nematode parasites of livestock. The genetic structure was high, indicating limited gene flow between the different isolates and populations from each continent mostly formed monophyletic groups in the phylogenetic analysis. However, for isolates representing Australia, Greece and one laboratory strain that originated from South Africa (WRS), there was no clear genetic relationship between the isolates and the distance between their geographical origins. Basically the same pattern was observed for the mitochondrial marker, although the phylogenetic analysis was less resolved than for AFLP. In contrast with previous findings on the population genetic structure of H. contortus, the calculation of population structure gave high values (Nst=0.59). The strong structure was present also for the four Swedish isolates (Nst=0.16) representing a small geographical area.     

Hc-daf-2 encodes an insulin-like receptor kinase in the barber’s pole worm, Haemonchus contortus, and restores partial dauer regulation

Infective L3s (iL3s) of parasitic nematodes share common behavioural, morphological and developmental characteristics with the developmentally arrested (dauer) larvae of the free-living nematode Caenorhabditis elegans. It is proposed that similar molecular mechanisms regulate entry into or exit from the dauer stage in C. elegans, and the transition from free-living to parasitic forms of parasitic nematodes. In C. elegans, one of the key factors regulating the dauer transition is the insulin-like receptor (designated Ce-DAF-2) encoded by the gene Ce-daf-2. However, nothing is known about DAF-2 homologues in most parasitic nematodes. Here, using a PCR-based approach, we identified and characterised a gene (Hc-daf-2) and its inferred product (Hc-DAF-2) in Haemonchus contortus (a socioeconomically important parasitic nematode of ruminants). The sequence of Hc-DAF-2 displays significant sequence homology to insulin receptors (IR) in both vertebrates and invertebrates, and contains conserved structural domains. A sequence encoding an important proteolytic motif (RKRR) identified in the predicted peptide sequence of Hc-DAF-2 is consistent with that of the human IR, suggesting that it is involved in the formation of the IR complex. The Hc-daf-2 gene was transcribed in all life stages of H. contortus, with a significant up-regulation in the iL3 compared with other stages. To compare patterns of expression between Hc-daf-2 and Ce-daf-2, reporter constructs fusing the Ce-daf-2 or Hc-daf-2 promoter to sequence encoding GFP were microinjected into the N2 strain of C. elegans, and transgenic lines were established and examined. Both genes showed similar patterns of expression in amphidial (head) neurons, which relate to sensation and signal transduction. Further study by heterologous genetic complementation in a daf-2-deficient strain of C. elegans (CB1370) showed partial rescue of function by Hc-daf-2. Taken together, these findings provide a first insight into the roles of Hc-daf-2/Hc-DAF-2 in the biology and development of H. contortus, particularly in the transition to parasitism.     

Progression from mitotic catastrophe to germ cell death in Caenorhabditis elegans lis-1 mutants requires the spindle checkpoint

Deletion of the lissencephaly disease gene LIS-1 in humans causes an extreme disorganization of the brain associated with significant reduction in cortical neurons. Here we show that deletion or RNA interference (RNAi) of Caenorhabditis elegans lis-1 results in a reduction in germline nuclei and causes a variety of cellular, developmental, and neurological defects throughout development. Our analysis of the germline defects suggests that the reduction in nuclei number stems from dysfunctional mitotic spindles resulting in cell cycle arrest and eventually programmed cell death (apoptosis). Deletion of the spindle checkpoint gene mdf-1 blocks lis-1(lf)-induced cell cycle arrest and germline apoptosis, placing the spindle checkpoint pathway upstream of the programmed cell death pathway. These results suggest that apoptosis may contribute to the cell-sparse pathology of lissencephaly.     

Identification of HMG-5 as a double-stranded telomeric DNA-binding protein in the nematode Caenorhabditis elegans

Many protein components of telomeres, the multifunctional DNA-protein complexes at the ends of eukaryotic chromosomes, have been identified in diverse species ranging from yeast to humans. In Caenorhabditis elegans, CEH-37 has been identified by a yeast one hybrid screen to be a double-stranded telomere-binding protein. However, the role of CEH-37 in telomere function is unclear because a deletion mutation in this gene does not cause severe telomere defects. This observation raises the possibility of the presence of genetic redundancy. To identify additional double-stranded telomere-binding proteins in C. elegans, we used a different approach, namely, a proteomic approach. Affinity chromatography followed by Finnigan LCQ ion trap mass spectrometer analysis allowed us to identify several candidate proteins. We further characterized one of these, HMG-5, which is encoded by F45E4.9. HMG-5 bound to double-stranded telomere in vitro as shown by competition assays. At least two telomeric DNA repeats were needed for this binding. HMG-5 was expressed in the nuclei of the oocytes and all embryonic cells, but not in the hatched larvae or adults. HMG-5 mainly localized to the chromosomal ends, indicating that HMG-5 also binds to telomeres in vivo. These observations suggest that HMG-5 may participate, together with CEH-37, in early embryogenesis by acting at the telomeres.     

Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTN gene produces 1-3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis.     

Caenorhabditis elegans WASP-interacting protein homologue WIP-1 is involved in morphogenesis through maintenance of WSP-1 protein levels

Mammalian WASP and N-WASP are involved in reorganization of the actin cytoskeleton through activation of the Arp2/3 complex and in regulation of cell motility or cell shape changes. In the present study, we identified WASP-interacting protein homologue (WIP)-1 in Caenorhabditis elegans. WIP-1 contains the domains and sequences conserved among mammalian WIP family proteins. Yeast two-hybrid analysis detected a physical interaction between WIP-1 and WSP-1, the sole homologue of WASP/N-WASP in C. elegans. Western analysis of embryo lysates showed that RNA interference (RNAi) treatment for wip-1 decreased levels of WSP-1 protein, and wsp-1(RNAi) treatment decreased levels of WIP-1 protein. However, wsp-1 mRNA levels were not decreased in wip-1(RNAi)-treated embryos, and wip-1 mRNA levels were not decreased in wsp-1(RNAi)-treated embryos. Furthermore, disruption of WIP-1 by RNAi resulted in embryonic lethality with morphologic defects in hypodermal cell migration, a process known as ventral enclosure. This phenotype was similar to that observed in RNAi experiments for wsp-1. Immunostaining showed that WIP-1 was expressed by migrating hypodermal cells, as was WSP-1. This expression during ventral enclosure was reduced in wip-1(RNAi)-treated embryos and wsp-1(RNAi)-treated embryos. Our results suggest that C. elegans WIP-1 may function in hypodermal cell migration during ventral enclosure by maintaining levels of WSP-1.