Spontaneous Selection of Drug Resistance in a Calf Model of Infection

The intestinal protozoan is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last ∼10 years, with numerous lead compounds identified, including several tRNA synthetase inhibitors. Here, we report the results of a dairy calf efficacy trial of the methionyl-tRNA ( MetRS [MetRS]) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day 5. Parasites shed by each recrudescent calf had different amino acid-altering mutations in the gene encoding MetRS (), yielding either an aspartate 243-to-glutamate (D243E) or a threonine 246-to-isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I MetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant-expressing parasites, respectively, had 2093 half-maximal effective concentrations (ECs) that were 613- and 128-fold that of transgenic parasites with wild-type MetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC >170-fold. Structural modeling of MetRS based on an inhibitor-bound MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.