Spontaneous selection ofCryptosporidiumdrug resistance in a calf model of infection
Authors: Hasan Muhammad M. , Stebbins Erin E. , Choy Robert K.M. , Gillespie J. Robert , de Hostos Eugenio L. , Miller Peter , Mushtaq Aisha , Ranade Ranae M. , Teixeira José E. , Verlinde Christophe L. M. J. , Sateriale Adam , Zhang Zhongsheng , Osbourn Damon M. , Griggs David W. , Fan Erkang , Buckner Frederick S. , Huston Christopher D. 
Published: January 2021

AbstractThe intestinal protozoanCryptosporidiumis 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. In this study, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (CpMetRS) synthetase inhibitor2093and the spontaneous emergence of drug resistance. Dairy calves experimentally infected withCryptosporidium parvuminitially improved with2093treatment, but parasite shedding resumed in two of three calves on treatment day five. Parasites shed by each recrudescent calf had different amino acid alteringCpMetRSmutations, coding either an aspartate 243 to glutamate (D243E) or a threonine 246 to isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246ICpMetRS mutation using CRISPR/Cas9 grew normally but were highly2093resistant; the D243E and T246I mutant expressing parasites respectively had2093EC50Sof 613- or 128-fold that of transgenic parasites with wild-typeCpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the2093IC50by >170-fold. Structural modeling ofCpMetRS based on an inhibitor-boundTrypanosoma bruceiMetRS 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 emergingCryptosporidiumdrug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.ImportanceCryptosporidiumis a leading protozoan cause of diarrhea in young children with no reliable treatment. We report results of a dairy calf drug efficacy trial and the spontaneous emergence of drug resistance.Cryptosporidium parvuminfected calves initially improved with drug treatment, but infection relapsed in two animals. Parasites shed by each recrudescent calf had mutations in the gene encoding the drug target that altered its amino acid sequence. Recapitulation of the drug target mutations by CRISPR/Cas9 genome editing resulted in highly drug-resistant parasites, and recombinant mutant enzymes were resistant to inhibition. This is the first report of naturally emergingCryptosporidiumdrug resistance. There is a currently a great opportunity to impact public health with new drugs to treat cryptosporidiosis, and this report highlights the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence in order to realize their full potential.One-sentence summaryDrug-target point mutations mediating anticryptosporidial resistance spontaneously arose in the dairy calfC. parvuminfection model.