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Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997.

Cover of C. elegans II

C. elegans II. 2nd edition.

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Section IIIAntinematode Compounds and Their Targets

The negative impact of nematodes on human and animal health and nutrition has resulted in an active search for effective nematicides. These drugs are often active against all nematodes rather than just the indicator species, and thus the search for improved compounds can use free-living species such as C. elegans. Although space does not permit consideration of all drugs and targets, two systems are described here where C. elegans research has had an impact on the understanding of drug action.

A. Benzimidazoles

The benzimidazoles (BZ; e.g., thiabendazole, benomyl, and mebendazole) disrupt microtubule assembly by binding to the nucleotide-binding site of specific isotypes of β-tubulin. They are potent nematicides in use worldwide for treatment of gut nematode infections of humans and domestic animals. Tissue-dwelling filarial parasites are unaffected. C. elegans is BZ-sensitive, and BZ-resistant lines have been obtained by mutagenesis (Driscoll et al. 1989). All of the BZ-resistant lines tested were mutant at a single locus ( ben-1 III) which encodes a β-tubulin. Although ben-1 mutants are wild type in the absence of drug, a number have gene deletions, suggesting that ben-1 is a dispensable gene. These observations have important implications for the generation of resistance in economically important species. If the BZ target is dispensable, the generation of resistance might be a relatively frequent event in natural populations.

Genetic resistance to BZ and derivatives is increasingly common in the field (Prichard 1994; Roos et al. 1995). In H. contortus, field resistance is associated with a rise in the population frequency of restriction fragment length polymorphisms (RFLPs) linked to particular β-tubulin isotype I alleles and a loss of heterozygosity (Roos et al. 1990). This correlates with a biochemical loss of high-affinity BZ-binding sites, and an increase in low-affinity sites, in tubulins prepared from parasites (Lacey and Gill 1994). Similar changes occur under in vitro selection regimes (Kwa et al. 1993; Roos et al. 1995). Comparison of an H. contortus β-tubulin isotype I gene (Hc tub-1 ) from European BZ-sensitive and BZ-resistant isolates revealed a substitution of phenylalanine (sensitive) for tyrosine (resistant) at residue 200, near a segment of the nucleotide-binding site (Kwa et al. 1994). An equivalent mutation has also been seen in BZ-resistant fungi. Thiabendazole-resistant ben-1 C. elegans can be rescued (to sensitivity) by transformation with the drug-sensitive allele of Hc tub-1 , although the rescued animals remain resistant to benomyl (Kwa et al. 1994, 1995; Roos et al. 1995).

C. elegans may be an effective test-bed for next-generation BZ derivative testing (Roos et al. 1995), but this assertion must be tempered by the realization that the level of susceptibility of C. elegans to BZ is on the order of that of resistant strongylid nematodes. The strongylids have additional high-affinity sites that bind BZ stably at 37°C, whereas C. elegans tubulin fractions bind BZ only at lower temperatures (Russell and Lacey 1991). Intestinal parasites (ascarids and strongylids) are also much more sensitive to BZ than are the tissue-dwelling filarids, and this may be related to the different routes of nutrition in these two groups. The gut of the intestinal parasites is well developed, and gut cell microtubule arrays may be the significant site of action of BZ in these species (Kohler and Bachmann 1981). The filarids have atrophied guts and obtain a significant portion of their nutrition transcuticularly (see below) (Chen and Howells 1981; Howells and Chen 1981).

B. The Avermectins

The avermectins (AVMs) are semisynthetic derivatives of fungally derived macrocyclic lactones (Putter et al. 1981) which are effective against many invertebrates including nematodes (Rohrer and Schaeffer 1995). They are widely used for veterinary treatment of gut parasites and treatment of human river blindness caused by the filarid Onchocerca volvulus (Bennett et al. 1988) and are also effective against plant-parasitic species (Sasser et al. 1982). AVMs appear to act through the inhibition of a glutamate-gated chloride channel present on muscle cells (Cully et al. 1994). These compounds inhibit pharyngeal pumping at ≥10−10 M and affect general motility at ≥10−8 M (Avery and Horvitz 1990; Holden-Dye and Walker 1990; Geary et al. 1993). Chloride channels of 10−11 to 10−10 M sensitivity have been described from isolated somatic muscle cells of Ascaris suum (Martin and Pennington 1989). At higher concentrations, inhibitory GABA responses are also blocked (Holden-Dye and Walker 1990). The effect of AVMs on gut parasites may be through a specific blockage of the pharyngeal pump rather than general paralysis.

C. elegans can readily be mutated to give an AVM-resistant phenotype, Avr (Novak 1992; C. Johnson, pers. comm.). The most common phenotype observed is that of low-level resistance. Such mutations are recessive and map to more than 20 loci. Detailed genetic mapping showed that avr-1 is allelic to a previously known locus, che-3 , and that avr-5 is allelic to osm-3 . The che-3 and osm-3 animals are defective in dye filling of amphid and plasmid neurons and chemotaxis and fail to avoid high osmolarity due to defects in their ciliated neurons (Perkins et al. 1986; Stravich et al. 1995). Examination of nematode strains carrying mutations at other avr loci showed that many of these also displayed amphid defects, such as the Dyf (dye-filling negative) phenotype (C. Johnson, pers. comm.). The avr-1 gene has been cloned and shown to encode a cytoplasmic dynein (W. Grant, pers. comm.), presumed to be active in amphidial neuronal transport processes. AVM-resistant parasitic nematodes, which can be readily generated by laboratory selection (Echevarria 1993), are becoming a serious threat in the real world (Prichard 1994). Like C. elegans Avr lines, AVM-resistant H. contortus have AVM-binding sites identical to those of wild type (Rohrer 1994). The anatomical correlation between the observed site of highest sensitivity to AVM in parasitic species (the pharyngeal musculature) and the site of action of avr mutants (amphid neurons) is interesting. Two models can be envisaged, one in which the amphid defects prevent access of the drug to a periamphidial essential site (C. Johnson, pers. comm.) and another in which the amphid neurons affected by the mutations inhibit the pharynx in wild-type animals in the presence of AVM, and genetic ablation of these neurons results in insensitivity to the drug (T. Geary, pers. comm.). It is reassuring to note that mutations conferring high-level or dominant resistance in C. elegans are rare (C. Johnson and P. Hunt, pers. comm.); hopefully, the same is true of parasitic species.

In gut parasites such as H. contortus, AVMs are adulticidal. However, adult filariae are relatively resistant, and the main effect is against microfilariae (L1s). Both adults and microfilaria have atrophied guts, and most nutritional exchange is across the cuticle (Chen and Howells 1981; Howells and Chen 1981). Gut-dwelling species also use this route (Ho et al. 1990, 1992; Geary et al. 1993), but the significance of transcuticular exchange in C. elegans and other free-living species is an open question. As AVMs have no lethal effect in vitro on microfilariae, their action may be through potentiation of a host attack or down-regulation of a parasite defense (as is also noted for another antifilarial drug, DEC [Maizels and Denham 1992]). The lack of activity against the macrofilariae (adults) of important human (e.g., Onchocerca, Brugia) and veterinary (e.g., Dirofilaria immitis, the dog heartworm) parasites is puzzling and is inspiring a search for both the mechanism of relative resistance in these stages and the development of active analogs.

Copyright © 1997, Cold Spring Harbor Laboratory Press.
Bookshelf ID: NBK20144

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