Forward genetics reveals microsporidian drug targets and recombination-based resistance

Microsporidia are a diverse group of intracellular fungal parasites that infect humans and many agriculturally important animals. Several microsporidia inhibitors have been characterized, but the targets of these inhibitors in microsporidia have not been confirmed, and the mechanisms by which microsporidia evolve resistance are unknown. Using the model organism Caenorhabditis elegans and its naturally occurring microsporidian parasite Nematocida parisii, we developed a technique to continually passage infected animals for multiple generations under increasing inhibitor concentrations. We obtained multiple independent N. parisii strains that are specifically resistant to the microsporidia inhibitors albendazole or dexrazoxane. The albendazole-resistant strains all contained either heterozygous or homozygous mutations in beta-tubulin, with several strains containing beta-tubulin variants that have been observed in other albendazole-resistant organisms. Strains containing homozygous variants are resistant to several albendazole analogs, whereas the heterozygous variant-containing strains are only resistant to albendazole. Several of the albendazole-resistant strains also contain mutations in alpha-tubulin. The dexrazoxane-resistant strains all contain heterozygous mutations in topoisomerase II and several of these mutations occur in the binding site of this inhibitor. By mapping heterozygosity of the resistant strains across the N. parisii genome, we observed loss of heterozygosity spanning the beta-tubulin locus in the albendazole-resistant strains containing homozygous beta-tubulin variants. Our study demonstrates the utility of forward genetics for identifying microsporidian drug targets. We also find that drug resistance arises through de novo heterozygous mutations that can subsequently become homozygous, likely via mitotic recombination.