Comparative chemical genomics in species identifies the alkaline phosphatase PhoD as a determinant of antiparasitic resistance.

is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of parasites. The diversity of parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of spp. ( and ). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of . After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in confer resistance to MMV019266 in . We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in species.