About Adrian Wolstenholme
Anthelmintic Resistance in Parasitic Nematods
Parasitic nematodes (roundworms) are serious pathogens of humans and their domesticated animals. These infections can cause serious disease and economic losses, but for many years have been effectively controlled in livestock and companion animals by the use of anthelmintic drugs. Recently, however, resistance to these drugs in veterinary parasites has reached very serious levels in many parts of the world, including the Southeastern region of the United States. There is also the suggestion that resistance may be emerging in human parasites, which cause debilitating diseases, especially of the poor. We are interested in discovering more about how anthelmintic drugs act, how parasites become resistant to them and in the development of new techniques to detect and reverse drug resistance. There are three major classes of anthelmintic in current use, plus another three that have recently become available. The three major classes of anthelmintic are the benzimidazoles (such as albendazole), the macrocyclic lactones (like ivermectin) and the imadizothiazoles (levamisole) & tetrahydropyrimidines (pyrantel); the three recent classes are the cyclodepsipeptides, such as emodepside, the amino-acetonitrile derivatives (AADs), such as monepantel, and the nicotinic antagonist, derquantel.
The mechanism of action of the benzimidazoles is to prevent tubulin repolymerisation and resistance has been widely reported to be due to a Single Nucleotide Polymorphism (SNP) that causes a single amino-acid change in b-tubulin, F200Y. We are investigating the most reliable and quantitative method for detecting this SNP in Haemonchus contortus, a parasite of small ruminants. To date, we have produced a real-time PCR assay using locked nucleic acid (LNA) probes that can reliably detect resistance alleles at a frequency of 5 to 10% within a few hours. The results correlate well with in vivo tests, such as the egg-hatch assay. However, for most of the other drug classes the genetic basis of resistance has not been discovered for field isolates of parasites. We are using molecular methods to study how the macrocyclic lactones kill nematodes, and how the parasites become resistant to the drug. We are studying both gastro-intestinal parasites, such as Haemonchus contrtus and filarial parasites like Brugia malayi (one of the worms that cause lymphatic filariasis in people) and Dirofilaria immitis, the dog heartworm. Recently we have found intriguing indications that the drug may work in different ways to remove these parasites from the body. In particular, we have preliminary evidence for an interaction between ivermectin and the aspects of the host immune system playing an important role in the prevention of heartworm infections.
Transgenic C. elegans as a method for studying drug resistance genes of parasitic nematodes.
In this project we are trying to find out whether the model organism, C. elegans, is a useful way of studying parasite genes. C. elegans is easy to grow in the lab, and there are lots of useful mutant strains that are readily available, plus many fairly simple phenotypic assays. None of these things are available for parasitic nematodes, so can we express parasite genes in the model worm? Yes, we can, but the question is how useful the results that we obtain really are: many genes exist as families and we want to know how specific the effects are. We are using our favourite gene family, the ligand-gated ion channels, as a way of answering these questions. This family includes the targets for many anthelmintic drugs, so is of great relevance to our overall research.
The molecular basis of ivermectin resistance in parasitic nematodes.
Despite some intensive research, we still do not understand how parasitic nematodes become resistant to the macrocyclic lactones. We are taking several approaches to solving this problem; one of the most promising is by making a detailed comparison of the genes that are expressed in a very closely related pair of H. contortus isolates, one of which is susceptible to the drug; it was used to rapidly select a resistant population in only three populations. This close relationship should minimise the problems of genetic heterogeneity that have bedevilled work in this area.
Molecular helminthology: in particular the interactions of anthelmintic drugs with ion channels in the nervous systems of parasitic helminths, and the molecular basis of drug resistance.
- Wolstenholme AJ. Glutamate-gated chloride channels. J Biol Chem. 2012 Nov 23;287(48):40232-8. doi: 10.1074/jbc.R112.406280. Epub 2012 Oct 4. Review.
- Bennett HM, Lees K, Harper KM, Jones AK, Sattelle DB, Wonnacott S, Wolstenholme AJ. Xenopus laevis RIC-3 enhances the functional expression of the C. elegans homomeric nicotinic receptor, ACR-16, in Xenopus oocytes. J Neurochem. 2012 Dec;123(6):911-8.
- Bennett HM, Williamson SM, Walsh TK, Woods DJ, Wolstenholme AJ. ACR-26: a novel nicotinic receptor subunit of parasitic nematodes. Mol Biochem Parasitol. 2012 Jun;183(2):151-7. >br/>
- Wolstenholme AJ. Evolution. Surviving in a toxic world. Science. 2012 Feb 3;335(6068):545-6.
- Wolstenholme AJ, Kaplan RM. Resistance to macrocyclic lactones. Curr Pharm Biotechnol. 2012 May;13(6):873-87. Review.
- Williamson SM, Storey B, Howell S, Harper KM, Kaplan RM, Wolstenholme AJ. Candidate anthelmintic resistance-associated gene expression and sequence polymorphisms in a triple-resistant field isolate of Haemonchus contortus. Mol Biochem Parasitol. 2011 Dec;180(2):99-105.
- Glendinning SK, Buckingham SD, Sattelle DB, Wonnacott S, Wolstenholme AJ. Glutamate-gated chloride channels of Haemonchus contortus restore drug sensitivity to ivermectin resistant Caenorhabditis elegans. PLoS One. 2011;6(7):e22390. doi: 10.1371/journal.pone.0022390. Epub 2011 Jul 26.
- Williamson SM, Wolstenholme AJ. P-glycoproteins of Haemonchus contortus: development of real-time PCR assays for gene expression studies. J Helminthol. 2012 Jun;86(2):202-8.
- Wolstenholme AJ, Williamson SM, Reaves BJ. TRP channels in parasites. Adv Exp Med Biol. 2011;704:359-71.
- Wolstenholme AJ. Recent progress in understanding the interaction between avermectins and ligand-gated ion channels: putting the pests to sleep. Invert Neurosci. 2010 Nov;10(1):5-10.