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Resistance

The exact mechanism of ivermectin resistance in nematode parasites is unknown.^[1] Mutations in three genes, avr-14, avr-15, and glc-1, are thought to be primarily responsible, with the UNC-7, UNC-9 and Dyf OSM-1 (dye filling defective) genes modulating it.^[2] The permeability of ivermectin is also thought to be reduced by mutations in OSM-1 by reducing cuticle permeability.^[2] In Caenorhabditis elegans, a mutation in the UNC-9 & OSM-1 gene also additively affects ivermectin sensitivity, however the mechanism of which OSM-1 acts is thought to be different from the other three GluCl genes, as worms without the OSM-1 gene were resistant but worms with the OSM-1 were even more resistant, the only exception being in organisms without all three GluCl mutations.^[2] An organism with the avr-15;osm-1 double mutation is significantly more resistant to ivermectin than the single mutant osm-1, however the osm-1 mutant is still more resistant than the avr-14;osm-1 mutant, suggesting that the osm-1 mutation preferentially reduces exposure in the extrapharyngeal nervous system. This is supported by the fact that neurons with sensory endings that are situated at the amphid that fill with dye are extrapharyngeal neurons, i.e. the dye filling defective gene that normally allows uptake of dyes in the environment has been inhibited somewhat by the mutation.^[2] Using reverse transcriptase PCR techniques, the full length cDNAs encoding GluClα3 and GluClβ were cloned from Cooperia oncophora, and statistical analysis in ivermectin-susceptible isolate and an ivermectin-resistant isolate suggests GluClα3 was primarily responsible, as no notable resistance was shown with the GluClβ gene. Usually, all three mutations (avr-14, avr-15, glc-1) must occur simultaneously in order for a parasite to become resistant, this is because severe loss of function mutations in avr-15 and glc-1 don't lead to resistance, possibly due to multiple GluCl genes independently contribute to ivermectin sensitivity, however a homozygous avr-14/15 mutation exhibits resistance.^[2]

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The IVS and IVR GluClα3 subunits differ at three amino acid positions, while the IVS and IVR GluClβ subunits differ at two amino acid positions. The aim of this study was to determine whether mutations in the IVR subunits affect agonist sensitivity. The subunits were expressed singly and in combination in Xenopus laevis oocytes. Electrophysiological whole-cell voltage-clamp recordings showed that mutations in the IVR GluClα3 caused a modest but significant threefold loss of sensitivity to glutamate, the natural ligand for GluCl receptors. As well, a significant decrease in sensitivity to the anthelmintics ivermectin and moxidectin was observed in the IVR GluClα3 receptor. Mutations in the IVR GluClβ subunit abolished glutamate sensitivity. Co-expressing the IVS GluClα3 and GluClβ subunits resulted in heteromeric channels that were more sensitive to glutamate than the respective homomeric channels, demonstrating co-assembly of the subunits. In contrast, the heteromeric IVR channels were less sensitive to glutamate than the homomeric IVR GluClα3 channels. The heteromeric IVS channels were significantly more sensitive to glutamate than the heteromeric IVR channels. Of the three amino acids distinguishing the IVS and IVR GluClα3 subunits, only one of them, L256F, accounted for the differences in response between the IVS and IVR GluClα3 homomeric channels.

Statistical analysis suggested an association between the C. oncophora GluClα3 gene and ivermectin resistance. No such association was seen with the GluClβ gene.

Preposed model to describe mechanisms of ivermectin sensitivity. Black arrows indicate the diffusion of ivermectin and the gray arrows indicate the flow of ivermectin-induced hyperpolarizing potential. Dyf gene mutations, which may reduce ivermectin permeability, act additively with all combinations of receptor mutants in the tier below. Ivermectin acts independently on each of the GluCl subunits to hyperpolarize cells: GluClα2 (AVR-15) acts in pharyngeal muscle, GluClα3 (AVR-14) acts in neurons. GluClα1 (GLC-1) is presumed to be neuronal but, because we do not know where gcl-1 is expressed, our model is not meant to imply that GluClα1 (GLC-1) and GluClα3 (AVR-14) are necessarily coexpressed or that they do or do not associate to form a channel. The effect of ivermectin on neurons expressing GluCls is not sufficient to kill the worms at low concentrations but requires that the ivermectin-induced hyperpolarization spread via gap junctions encoded by unc-7 and unc-9 to other excitable cells that are essential to the function of the worm. Our results indicate that the spread of hyperpolarization from the extrapharyngeal nervous system back to the pharynx is an important component of the gap-junction-mediated ivermectin sensitivity conferred by GluClα3 (AVR-14) and GluClα1 (GLC-1). The flow of hyperpolarizing potential from the extrapharyngeal neurons to the pharynx occurs via linking neurons such as I1 and RIP that may not themselves express GluCls.

To better understand the mechanism of synthetic ivermectin resistance, we cloned avr-14 by a combination of genetic mapping and identification of a candidate GluCl gene, gbr-2. gbr-2 encodes two alternatively spliced transcripts whose ORFs predict proteins that belong to the GluClα class of channel subunits (8). Two overlapping cosmids containing the gbr-2 gene, K07D2 and F56E2, as well as a minigene construct (see below) restored the ivermectin-sensitive phenotype when transformed into the avr-14; avr-15 double mutant (data not shown). We sequenced two alleles of avr-14 and found that one allele, ad1302, resulted from a missense V60E mutation in an exon common to both transcripts (Fig. 1A). This allele may be null for channel function; the mutated valine is conserved, even among vertebrate γ-aminobutyric acid type A and glycine receptors, and may play a role in subunit association (15). The second allele, ad1305, was a premature termination (nonsense) mutation that eliminates the last transmembrane domain of the channel encoded by transcript A and is likely to be null for the protein encoded by that transcript. We expressed RNAs corresponding to each of the two proteins encoded by gbr-2 in Xenopus oocytes. Oocytes injected with RNA encoding transcript A did not respond to ivermectin or glutamate. However, oocytes expressing transcript B gave a robust response to both ivermectin and glutamate (Fig. 1B) but did not respond to either of the chloride channel agonists γ-aminobutyric acid or glycine at 10 mM. As with the other characterized α-type subunits, the ivermectin response was only weakly desensitizing and was irreversible (5–7). We propose that the subunits encoded by avr-14/gbr-2 be called AVR-14A/GluClα3A and AVR-14B/GluClα3B.

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