Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals extensive sequence variation and differential immune recognition

Haemonchus contortus is a nematode that infects small ruminants. It releases a variety of molecules, designated excretory/secretory products (ESP), into the host. Although the composition of ESP is largely unknown, it is a source of potential vaccine components because ESP are able to induce up to 90% protection in sheep. We used proteomic tools to analyze ESP proteins and determined the recognition of these individual proteins by hyperimmune sera. Following two-dimensional electrophoresis of ESP, matrix-assisted laser desorption ionization time-of-flight and liquid chromatography-tandem mass spectrometry were used for protein identification. Few sequences of H. contortus have been determined. Therefore, the data base of expressed sequence tags (dbEST) and a data base consisting of contigs from Haemonchus ESTs were also consulted for identification. Approximately 200 individual spots were observed in the two-dimensional gel. Comprehensive proteomics analysis, combined with bioinformatic search tools, identified 107 proteins in 102 spots. The data include known as well as novel proteins such as serine, metallo- and aspartyl proteases, in addition to H. contortus ESP components like Hc24, Hc40, Hc15, and apical gut GA1 proteins. Novel proteins were identified from matches with H. contortus ESTs displaying high similarity with proteins like cyclophilins, nucleoside diphosphate kinase, OV39 antigen, and undescribed homologues of Caenorhabditis elegans. Of special note is the finding of microsomal peptidase H11, a vaccine candidate previously regarded as a "hidden antigen" because it was not found in ESP. Extensive sequence variation is present in the abundant Hc15 proteins. The Hc15 isoforms are differentially recognized by hyperimmune sera, pointing to a possible specific role of Hc15 in the infectious process and/or in immune evasion. This concept and the identification of multiple novel immune-recognized components in ESP should assist future vaccine development strategies.     

An ivermectin-sensitive glutamate-gated chloride channel subunit from Dirofilaria immitis

Dirofilaria immitis is a filarial nematode that infects dogs and causes cardiopulmonary disease. The most effective way of controlling the infection is by chemoprophylaxis, using members of the avermectin/milbemycin (A/M) class of anthelmintics, which includes ivermectin; these drugs act at invertebrate glutamate-gated chloride channels (GluCl). We have cloned two cDNAs encoding D. immitis GluCl subunits and demonstrated that at least one may be an important molecular target for the A/Ms in vivo. The subunits are orthologues of the alternatively spliced GluClalpha3A and alpha3B subunits (encoded by the avr-14 gene) previously identified in Caenorhabditis elegans and in Haemonchus contortus. Although the alternative splicing of avr-14 is conserved across the species, the processing of the mature GluClalpha3A mRNA differs in D. immitis compared to C. elegans and H. contortus. Two-electrode voltage clamp recordings were made from Xenopus oocytes injected with subunit-specific cRNAs. The DiGluClalpha3B subunit formed channels that were gated by L-glutamate (1-100 mM) and ivermectin (1 microM). Oocytes injected with DiGluClalpha3A cRNA failed to respond to L-glutamate. The qualitative responses obtained were consistent with the pharmacology observed for the GluClalpha3 subunits from C. elegans and H. contortus.     

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.     

Analysis of FMRFamide-like peptide (FLP) diversity in phylum Nematoda

This study reports a series of systematic BLAST searches of nematode ESTs on the Genbank database, using search strings derived from known nematode FLPs (those encoded by Caenorhabditis elegans flp genes as well as those isolated from other nematodes including Ascaris suum), as well as query sequences representative of theoretical FLPs. Over 1000 putative FLP-encoding ESTs were identified from multiple nematode species. A total of 969 ESTs representing sequelogs of the 23 known C. elegans flp genes were identified in 32 species, from clades I, III, IV and V. Numerical analysis of EST numbers suggests that flp-1, flp-11 and flp-14 are amongst the most highly expressed flp genes. Speculative BLAST searches were performed using theoretical FLP C-termini as queries, in an attempt to identify putative novel FLP sequences in the EST database. These searches yielded eight multi-species sequelogs encoding FLPs with novel signatures that are believed to identify distinct flp genes. These novel genes encode 25 distinct previously unidentified FLPs, and raise the current total of known nematode flp genes to 31. Additionally, software-based analyses of the presence of signal peptides were performed, with signal peptides being identified on at least one member of each group of flp ESTs, further confirming their status as secreted peptides. The data reveal that nematode FLPs encompass the most complex neuropeptide family known within the metazoa. Moreover, individual FLPs and FLP motifs are highly conserved across the nematodes with little evidence for inter-clade or inter-lifestyle variation, supporting their fundamental role in free-living and parasitic species.     

Genetic variability following selection of Haemonchus contortus with anthelmintics

Genetic diversity in nematodes leads to variation in response to anthelmintics. Haemonchus contortus shows enormous genetic diversity, allowing anthelmintic resistance alleles to be rapidly selected. Anthelmintic resistance is now a widespread problem, especially in H. contortus. Here, I compare the genes involved in anthelmintic resistance in H. contortus with those that confer susceptibility or resistance on the free living nematode Caenorhabditis elegans. I also discuss the latest knowledge of genes associated with resistance to benzimidazoles, levamisole and the macrocyclic lactones and the need for DNA markers for anthelmintic resistance.     

Characterization of a hexokinase encoding cDNA of the parasitic nematode Hhaemonchus contortus

The nematode Haemonchus contortus is an important parasite of cattle and sheep. We describe here the cloning of a cDNA encoding a 53 kDa hexokinase (EC 2.7.1.1). The deduced protein shows 73% identity to a 50 kDa hexokinase deduced from a Caenorhabditis elegans cosmid. Alignment with mammalian hexokinases reveals two short amino acid insertions in the H. contortus hexokinase. Software tools for structural protein analysis (ExPASy server, Geneva) localize these insertions on the surface of the molecule, suggesting these surface changes as potential target sites for chemotherapeutic drugs.     

Testing the performance of automated annotation of ESTs with the Kegg Orthology (KO) database demonstrates lack of completeness of clusters

The KEGG Orthology (KO) database was tested as a source for automated annotation of expressed sequence tags (ESTs). We used a control experiment where every EST was assigned to its cognate protein, and an annotation experiment where the ESTs were annotated by proteins from other organisms. Analyzing the results, we could assign classes to the annotation: correct, changed and speculated. The correct annotation ranged from 57 (Caenorhabditis elegans) to 81% (Homo sapiens). In spite of the changed annotation being low (1 in H. sapiens to 9% in Arabidopsis thaliana), the speculation was very high (18 in H. sapiens to 38% in C. elegans). We propose eliminating part of the speculated annotation using the KEGG Genes database to enrich KO clusters, decreasing the speculation from 38 to 2% in C. elegans. Thus, the KO database still demands some effort for moving sequences from Kegg GENES to KO, to complement the annotation performance.     

Gene expression profiling of hereditary exencephaly in chickens

In this preliminary study, differentially expressed genes were investigated in cranial tissues from chickens with hereditary exencephaly using cDNA microarrays containing 1,152 genes and expressed sequence tags (ESTs). Genes showing twofold or greater differences at P < 0.05 between affected and normal cranial cells were considered to be candidates for hereditary exencephaly in chicken. Eighteen ESTs (11 known genes/homologues) were upregulated and 108 ESTs (51 known genes/homologues) were downregulated. The EST AL584231 (ROS006C9), orthologous to human MTHFD1, a known candidate gene for human neural tube defects (NTDs), was expressed at the same level both in normal and affected chicken cranial tissues. ESTs AL584253 (ROS006F7, thioredoxin reductase 1) and AL585511 (ROS024H9, thioredoxin), both involved in NTD pathogenic pathways in mice, were downregulated and had mean ratios of 0.41 and 0.04 for expression in affected vs. normal cells respectively. Expression differences of these two ESTs were confirmed by quantitative real-time polymerase chain reaction. These data indicate that ESTs AL584253 and AL585511 are candidates for hereditary exencephaly in chickens.     

Haemonchus contortus (Nematoda: Trichostrongylidae) is much more sensitive than Caenorhabditis elegans (Nematoda: Rhabditidae) to the ovicidal action of thiabendazole

When eggs of the trichostrongylid nematode Haemonchus contortus were exposed to thiabendazole, the concentration required to prevent hatching in 90% of the eggs (MIC90) was found to be 0.1 microg/ml (using 1% dimethylsulfoxide [DMSO] as solvent). In contrast, eggs of the free-living rhabditid nematode Caenorhabditis elegans hatched at normal rates at a concentration 200 times higher, i.e., 20 microg/ml, and showed only a partial inhibitory effect at a concentration 1,200 times higher, i.e., 120 microg/ml (in 3% DMSO). Because solubility limitations precluded the testing of higher concentrations of thiabendazole, a more soluble derivative, 5-([1-methylethoxy]carbonylamino)-2-(4-thiazloyl)1H-++ +benzimidazolyliminoacetic acid N,N-diethylethanamine salt, was tested against C. elegans eggs. The MIC90 was found to be 400 microg/ml, and although the derivative was not tested against H. contortus eggs, this finding further suggests that C. elegans eggs have an exceptionally low degree of benzimidazole sensitivity.     

A membrane associated glutamate binding protein from Caenorhabditis elegans and Haemonchus contortus

1. A glutamate binding protein has been identified in membrane preparations from the free living nematode, Caenorhabditis elegans, and from the parasitic nematode, Haemonchus contortus. 2. This putative glutamate receptor was solubilized with 30 mM octyl-B-glucoside and partially purified by anion exchange and gel filtration chromatography. 3. An 80-fold purification with recovery of 75% of the glutamate binding activity was achieved. 4. The soluble C. elegans binding protein displayed a Kd for glutamate of 0.1 microM, in close agreement with the findings for the membrane associated binding protein. 5. Quisqualate was capable of displacing glutamate from the soluble C. elegans receptor, again in agreement with previous findings for the membrane bound receptor. 6. The fact that a parasitic nematode, Haemonchus contortus, also possesses this putative glutamate receptor, strengthens the case for using C. elegans as a model system for the study of parasitic nematode neuromuscular physiology.     

Glutamate-gated chloride channels of Haemonchus contortus restore drug sensitivity to ivermectin resistant Caenorhabditis elegans

Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance.     

Ancylostoma caninum: the finger cell neurons mediate thermotactic behavior by infective larvae of the dog hookworm

Bhopale, V. M., Kupprion, E. K., Ashton, F. T., Boston, R., and Schad, G. A. 2001. Ancylostoma caninum: The finger cell neurons mediate thermotactic behavior by infective larvae of the dog hookworm. Experimental Parasitology 97, 70-76. In the amphids (anteriorly positioned, paired sensilla) of the free-living nematode Caenorhabditis elegans, the so-called finger cells (AFD), a pair of neurons, each of which ends in a cluster of microvilli-like projections, are known to be the primary thermoreceptors. A similar neuron pair in the amphids of the parasitic nematode Haemonchus contortus is also known to be thermoreceptive. The hookworm of dogs, Ancylostoma caninum, has apparent structural homologs of finger cells in its amphids. The neuroanatomy of the amphids of A. caninum and H. contortus is strikingly similar, and the amphidial cell bodies in the lateral ganglia of the latter nematode have been identified and mapped. When the lateral ganglia of first-stage larvae (L1) of A. caninum are examined with differential interference contrast microscopy, positional homologs of the recognized amphidial cell bodies in the lateral ganglia of H. contortus L1 are readily identified in A. caninum. The amphidial neurons in A. caninum were consequently given the same names as those of their apparent homologs in H. contortus. It was hypothesized that the finger cell neurons (AFD) might mediate thermotaxis by the skin-penetrating infective larvae (L3) of A. caninum. Laser microbeam ablation experiments with A. caninum were conducted, using the H. contortus L1 neuronal map as a guide. A. caninum L1 were anesthetized and the paired AFD class neurons were ablated. The larvae were then cultured to L3 and assayed for thermotaxis on a thermal gradient. L3 with ablated AFD-class neuron pairs showed significantly reduced thermotaxis compared to control groups. The thermoreceptive function of the AFD-class neurons associates this neuron pair with the host-finding process of the A. caninum infective larva and shows functional homology with the neurons of class AFD in C. elegans and in H. contortus.     

Silencing of essential genes by RNA interference in Haemonchus contortus

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the 'essential' genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.