Le Studium Research Fellow
(BSc Nottingham, PhD Edinburgh)
Parasitic nematodes pose a significant risk for both human and animal health.
Parasites are fascinating organisms that provide a unique opportunity to biologists. In my research I use the parasites Haemonchus contortus, Onchocerca volvulus and Heligmosomoides polygyrus (otherwise known as Nematospiroides dubius) to investigate the evolutionary development of drug resistance and the interaction between the genes an organism possesses and its appearance.
My background in population genetics holds that there are four main factors that affect the evolution of drug resistance:
- The initial frequency of resistance alleles.
- A higher frequency can greatly speed up the development of resistance.
- The number of different genes that affect the expression of resistance.
- In simple terms, if only one gene controls resistance then selection is relatively straightforward. If there are many genes it may take longer to assemble the right configuration of all the genes.
- The selection pressure exerted by drug application.
- If no drug is used there is no selection pressure. Similarly, if all individuals are killed there is no selection pressure since there are no survivors. Somewhere in between, the resistance alleles will have a selective advantage over susceptibility alleles.
- The relative dominance of resistance over susceptibility alleles.
- If a resistance allele is rare then it is found most often in heterozygotes. This becomes critical when one of the alleles is masked by the presence of the other. If resistance is recessive then heterozygotes appear susceptible and the rise of patent resistance can be significantly delayed.
Surveying allele frequencies in susceptible and derived resistant strains of parasite has shown that the frequency of resistance alleles can be as high as 25% (Beech et al, 1994). The number of genes that affect a trait can be affected by the method used to measure their effect. Allele frequency changes have been used to show a link between four different genes and ivermectin resistance (Blackhall et al, 1998a,b; Blackhall, 1999). I am currently focusing on the selection pressure exerted by the drug and relative dominance of different alleles involved in ivermectin resistance.
With advances in genome sequence databases, a major issue in biology today is to bridge the gap between information on the genetics of an organism and its biological characteristics, or phenotype. My research focuses on the use of a model host-parasite system to address how the genes an organism possesses influence its biological properties and the interaction between host and parasite.
Ivermectin is a member of the latest class in a series of anti-parasite drugs. Haemonchus contortus is a nematode that parasitizes the stomach of sheep throughout the world. Ivermectin resistance in H. contortus has developed in the field and we have shown that at least 4 genes appear to be involved in producing this resistance phenotype. There are several important aspects of this research.
- A biochemical and functional analysis of two of these genes, which play a fundamental role in the neurobiology of nematodes, allows a dissection of the function of the nematode nervous system.
- Genetic screens for mutations associated with drug resistance are a practical method for early detection and investigation of the evolutionary process that leads to drug resistance.
- P-glycoprotein is a generalized drug pump that seems to be involved in IVM resistance. We have found that mutations in this gene may be a general mechanism by which nematodes achieve resistance to a wide range of different anti-parasite drugs.
- We have now been able to correlate the genotype of individual parasites for these genes with their behaviour in the presence of IVM. In doing so we have demonstrated the effect of individual genetic changes on the biological phenotype of the organism.
The results we have obtained are being applied to filarial parasites that infect humans. In the future we will use this model system to dissect in detail the interaction between genes and phenotype in the nematode nervous system.
Investigating the biology of drug resistance involves the following steps:
- Identify genes involved in conferring resistance.
- Characterize resistant and susceptible forms of these genes.
- Identify specific changes responsible for resistance.
- Estimate the effect of each change on survival of the drug.
The first and last of these are being addressed using population genetics (see above). The second and third involve the biochemistry of the parasite nervous system.
Ivermectin affects the nervous system of nematodes by activating chloride channels which inhibit an action potential from being induced in muscle cells. These channels are composed, typically, of five subunits that make up a heteromeric channel. Two different channel subunits have been found associated with resistance to ivermectin: a glutamate-gated chloride channel subunit (HcGluCla) and a GABA-gated chloride channel subunit (HG1). Normally these channel subunits bind reversibly either glutamate, or GABA, which activates the channel. Ivermectin binds irreversibly, causing complete relaxation of the muscle and flaccid paralysis.
The sequences of alleles favoured by drug selection have been compared to alleles that are not favoured to reveal changes in the amino acid sequence. Mutations specific for the resistant form have been identified and we are characterizing the biochemical and functional differences caused by these mutations. One advantage of this kind of approach is that natural alleles collected from the field generally contain subtle mutations that alter, but do not destroy the activity of the channel. Experiments in Caenorhabditis elegans use mutagens and selection screen that identify null mutations. By combining the two approaches we can transfect the wild parasite alleles into null mutant C. elegans and characterize in great detail the functional aspects of these important nerve system proteins.