Widespread genomic heterogeneity at the type II NAD(P)H dehydrogenase locus predisposes Cryptosporidium to clofazimine resistance.

The parasite Cryptosporidium is a leading cause of life-threatening diarrheal disease, and effective treatment is not available. The discovery of potent anti-Cryptosporidium activity of clofazimine, offered the opportunity to repurpose a drug already used to treat leprosy and tuberculosis. However, clofazimine failed in a human trial, which was attributed to poor bioavailability. Here, we observed differential susceptibility among parasite isolates which we exploit to map the mode of action to type II NADH dehydrogenase (NDH2) in an unbiased genetic cross. Targeted genetic ablation of NDH2 resulted in profound clofazimine resistance, and biochemical studies demonstrated NDH2 mediated electron transfer to clofazimine. Through genomic analyses we uncovered heterogeneity at the NDH2 locus for C. parvum and C. hominis and widespread carriage of a conserved attenuated allele across multiple continents. This heterogeneity allows parasites genomically linked through frequent sexual recombination to adjust to changing NDH2 requirements and predisposes Cryptosporidium to evade clofazimine treatment.