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. 2000 Mar 14;97(6):2674-9.
doi: 10.1073/pnas.97.6.2674.

The Genetics of Ivermectin Resistance in Caenorhabditis Elegans

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Free PMC article

The Genetics of Ivermectin Resistance in Caenorhabditis Elegans

J A Dent et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The ability of organisms to evolve resistance threatens the effectiveness of every antibiotic drug. We show that in the nematode Caenorhabditis elegans, simultaneous mutation of three genes, avr-14, avr-15, and glc-1, encoding glutamate-gated chloride channel (GluCl) alpha-type subunits confers high-level resistance to the antiparasitic drug ivermectin. In contrast, mutating any two channel genes confers modest or no resistance. We propose a model in which ivermectin sensitivity in C. elegans is mediated by genes affecting parallel genetic pathways defined by the family of GluCl genes. The sensitivity of these pathways is further modulated by unc-7, unc-9, and the Dyf (dye filling defective) genes, which alter the structure of the nervous system. Our results suggest that the evolution of drug resistance can be slowed by targeting antibiotic drugs to several members of a multigene family.

Figures

Figure 1
Figure 1
avr-14 encodes a subunit of an ivermectin-sensitive GluCl. (A) The mutations in alternatively spliced transcripts of avr-14. The exons encoding the extracellular domain (black) are shared by both transcripts. The exons encoding the transmembrane domains (stippled) are produced by alternatively spliced exons (white in AVR-14A and gray in AVR-14B). The region surrounding the conserved valine (bold) that is mutated to a glutamate in the ad1302 allele (resulting from a T to A mutation in the second base of the V60 codon) is shown lined up with the corresponding region in other GluCl subunits and in the rat glycine and γ-aminobutyric acid (GABA) type A-β channel subunits (29, 30). The ad1305 mutation results from an A to T change in the first base of the R387 codon. (B) Electrophysiology of oocytes expressing AVR-14B/GluClα3B. Oocytes were injected with RNA transcribed from AVR-14B/GluClα3B and were voltage clamped; 10 mM glutamate or 10 μM ivermectin (in 1% DMSO) were applied during the periods of time indicated by the bars. (C) Expression of an avr-14 reporter construct. A GFP reporter construct was generated by fusing GFP in-frame to the sixth exon of avr-14. GFP fluorescence of an L4 worm is shown. The identified neuronal cell bodies (arrows) are indicated by their three-letter code. Arrowheads indicate ventral cord neurons. Multiple fluorescent neurons in the ring ganglia are indicated by the bracket. (Scale bar indicates 50 μm).
Figure 2
Figure 2
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.

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