Adrian J. Wolstenholme

The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery

BackgroundThe small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development and anthelmintic resistance research. Its remarkable propensity to develop resistance threatens the viability of the sheep industry in many regions of the world and provides a cautionary example of the effect of mass drug administration to control parasitic nematodes. Its phylogenetic position makes it particularly well placed for comparison with the free-living nematode Caenorhabditis elegans and the most economically important parasites of livestock and humans.ResultsHere we report the detailed analysis of a draft genome assembly and extensive transcriptomic dataset for H. contortus. This represents the first genome to be published for a strongylid nematode and the most extensive transcriptomic dataset for any parasitic nematode reported to date. We show a general pattern of conservation of genome structure and gene content between H. contortus and C. elegans, but also a dramatic expansion of important parasite gene families. We identify genes involved in parasite-specific pathways such as blood feeding, neurological function, and drug metabolism. In particular, we describe complete gene repertoires for known drug target families, providing the most comprehensive understanding yet of the action of several important anthelmintics. Also, we identify a set of genes enriched in the parasitic stages of the lifecycle and the parasite gut that provide a rich source of vaccine and drug target candidates.ConclusionsThe H. contortus genome and transcriptome provide an essential platform for postgenomic research in this and other important strongylid parasites.

The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets

The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9‐Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.—Godel, C., Kumar, S., Koutsovoulos, G., Ludin, P., Nilsson, D., Comandatore, F., Wrobel, N., Thompson, M., Schmid, C. D., Goto, S., Bringaud, F., Wolstenholme, A., Bandi, C., Epe, C., Kaminsky, R., Blaxter, M., Mäser, P. The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets. FASEB J. 26, 4650–4661 (2012). www.fasebj.org

An ivermectin-sensitive glutamate-gated chloride channel from the parasitic nematode Haemonchus contortus.

Nematode glutamate-gated chloride channels are targets of the macrocyclic lactones, the most important group of anthelmintics available. In Xenopus laevis oocytes, channels formed by the GluClα3B subunit from the parasite Haemonchus contortus were more sensitive to l-glutamate (EC50 = 27.6 ± 2.7 μM) than those formed by the homologous subunit from Caenorhabditis elegans (EC50 = 2.2 ± 0.12 mM). Ibotenate was a partial agonist (EC50 = 87.7 ± 3.5 μM). The H. contortus channels responded to low concentrations of ivermectin (estimated EC50 =∼0.1 ± 1.0 nM), opening slowly and irreversibly in a highly cooperative manner: the rate of channel opening was concentration-dependent. Responses to glutamate and ivermectin were inhibited by picrotoxinin and fipronil. Mutating an N-terminal domain amino acid, leucine 256, to phenylalanine increased the EC50 for l-glutamate to 92.2 ± 3.5 μM, and reduced the Hill number from 1.89 ± 0.35 to 1.09 ± 0.16. It increased the Kd for radiolabeled ivermectin binding from 0.35 ± 0.1 to 2.26 ± 0.78 nM. Two other mutations (E114G and V235A) had no effect on l-glutamate activation or ivermectin binding: one (T300S) produced no detectable channel activity, but ivermectin binding was similar to wild-type. The substitution of any aromatic amino acid for Leu256 had similar effects in the radioligand binding assay. Molecular modeling studies suggested that the GluCl subunits have a fold similar to that of other Cys-loop ligand-gated ion channels and that amino acid 256 was unlikely to play a direct role in ligand binding but may be involved in mediating the allosteric properties of the receptor.

Glutamate-gated Chloride Channels

Glutamate-gated chloride channels (GluCls) are found only in protostome invertebrate phyla but are closely related to mammalian glycine receptors. They have a number of roles in these animals, controlling locomotion and feeding and mediating sensory inputs into behavior. In nematodes and arthropods, they are targeted by the macrocyclic lactone family of anthelmintics and pesticides, making the GluCls of considerable medical and economic importance. Recently, the three-dimensional structure of a GluCl was solved, the first for any eukaryotic ligand-gated anion channel, revealing a macrocyclic lactone-binding site between the channel domains of adjacent subunits. This minireview will highlight some unique features of the GluCls and illustrate their contribution to our knowledge of the entire Cys loop ligand-gated ion channel superfamily.

Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions

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Candidate anthelmintic resistance-associated gene expression and sequence polymorphisms in a triple-resistant field isolate of Haemonchus contortus

An isolate of Haemonchus contortus, UGA/2004, highly resistant to benzimidazoles, levamisole, and ivermectin was isolated from sheep at the University of Georgia, and passaged through experimentally infected goats. We measured the expression of twenty-nine mRNAs encoding drug targets and P-glycoproteins (P-gps), comparing the results to a fully susceptible laboratory passaged isolate. Expression levels of some nicotinic acetylcholine receptor mRNAs were markedly different in UGA/2004. Levels of the Hco-acr-8b mRNA, encoding a truncated subunit, were very high in resistant L3, but undetectable in susceptible larvae, with expression of the full-length Hco-acr-8a mRNA also significant increased. Expression of Hco-unc-63 and Hco-unc-29.3 mRNAs was significantly reduced in the resistant larvae. Expression of the Hco-glc-3 and Hco-glc-5 mRNAs, encoding glutamate-gated chloride channel subunits, were slightly reduced in resistant larvae. We observed significant increases in the expression of the Hco-pgp-2 and Hco-pgp-9 mRNAs in the UGA/2004 larvae, consistent with previous reports; we also saw a decrease in the levels of Hco-pgp-1 mRNA. Treatment of the larvae with ivermectin and moxidectin in vitro produced variable and inconsistent changes in P-gp mRNA levels. The sequences of the β-tubulin isotype 1 mRNAs showed that the resistant larvae had a resistance-associated allele frequency of >95% at codon 200 and ∼40% and codon 167. No changes at codon 198 were present. The presence of the truncated acr-8b mRNA may be a reliable indicator of levamisole resistance, but complex changes in gene expression associated with macrocyclic lactone resistance make the identification of a single genetic marker for this resistance difficult.

Nematode parasite genes: what's in a name?

The central theme of Shakespeares Romeo and Juliet is that names are meaningless, artificial constructs, detached from any underlying reality. By contrast, we argue that a well chosen gene name can concisely convey a wealth of relevant biological information. A consistent nomenclature adds transparency that can have a real impact on our understanding of gene function. Currently, genes in parasitic nematodes are often named ad hoc, leading to confusion that can be resolved by adherence to a nomenclature standard adapted from Caenorhabditis elegans. We demonstrate this with ligand-gated ion-channels and propose that the flood of genome data and differences between parasites and the free living C. elegans will require modification of the standard.

Resistance to Macrocyclic Lactones

Resistance to the macrocyclic lactones (MLs) has been confirmed or suspected in many target organisms and is a serious problem in some. For some species, such as parasitic nematodes of small ruminants, ML resistance has become severe enough to threaten effective worm control worm control. Resistance is also a major concern in horse parasites and an emerging problem in cattle. Despite this, we have insufficient understanding of the mechanisms of ML resistance, especially in nematodes. Some insect and mite agricultural pests express higher levels of detoxifying enzymes, leading to cross-resistance to other pesticide classes. A major difficulty is in the identification of true resistance and distinguishing this from other causes of treatment or prophylaxis failure--some in vitro assays for ML resistance are available but more are badly needed. Changes in the way anthelmintic drugs are used in livestock farming have been proposed, based on the treatment of those animals that would benefit most, and schemes have been devised for identifying those animals, flocks and herds. The continued sustainable use of these invaluable drugs may depend on the adoption and improvement of such schemes, as resistance is likely to become an ever more serious problem.

Ion channels and receptor as targets for the control of parasitic nematodes

Many of the anthelmintic drugs in use today act on the nematode nervous system. Ion channel targets have some obvious advantages. They tend to act quickly, which means that they will clear many infections rapidly. They produce very obvious effects on the worms, typically paralyzing them, and these effects are suitable for use in rapid and high-throughput assays. Many of the ion channels and enzymes targeted can also be incorporated into such assays. The macrocyclic lactones bind to an allosteric site on glutamate-gated chloride channels, either directly activating the channel or enhancing the effect of the normal agonist, glutamate. Many old and new anthelmintics, including tribendimidine and the amino-acetonitrile derivatives, act as agonists at nicotinic acetylcholine receptors; derquantel is an antagonist at these receptors. Nematodes express many different types of nicotinic receptor and this diversity means that they are likely to remain important targets for the foreseeable future. Emodepside may have multiple effects, affecting both a potassium channel and a pre-synaptic G protein-coupled receptor; although few other current drugs act at such targets, this example indicates that they may be more important in the future. The nematode nervous system contains many other ion channels and receptors that have not so far been exploited in worm control but which should be explored in the development of effective new compounds.

Utilization of computer processed high definition video imaging for measuring motility of microscopic nematode stages on a quantitative scale: “The Worminator”

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