The intestinal protozoan Cryptosporidium 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. In this study, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (CpMetRS) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day five. Parasites shed by each recrudescent calf had different amino acid altering CpMetRS mutations, 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 T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant expressing parasites respectively had 2093 EC₅₀s of 613- or 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC₅₀ by >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei 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 Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.

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在小牛感染模型中自发选择隐孢子虫的耐药性

肠道原生动物隐孢子虫是腹泻病和幼儿死亡的主要原因。目前尚无针对隐孢子虫病的完全有效的治疗方法，在过去约10年中，它已被鉴定出包括多种tRNA合成酶抑制剂在内的众多先导化合物，引起了人们对抗隐孢子虫病发展的兴趣。在这项研究中，我们报告了甲硫氨酰-tRNA（CpMetRS）合成酶抑制剂2093和自发出现的耐药性的小牛犊功效试验的结果。实验上感染小球隐孢子虫的乳牛最初在2093年治疗后有所改善，但在治疗的第5天，三只小牛中有两只恢复了寄生虫脱落。每只小牛皮脱落的寄生虫具有不同的氨基酸改变CpMetRS突变，编码天冬氨酸243为谷氨酸（D243E）或苏氨酸246为异亮氨酸（T246I）突变。使用CRISPR / Cas9改造为D243E或T246I CpMetRS突变的转基因寄生虫正常生长，但对2093的耐药性很高；D243E和T246I突变体寄生虫分别具有2093 EC ₅₀ s，是野生型CpMetRS的转基因寄生虫的EC ₅₀ s的613或128倍。在使用重组酶的研究中，D243E和T246I突变使2093 IC ₅₀移位了> 170倍。基于抑制剂结合的布鲁氏锥虫的CpMetRS的结构建模MetRS晶体结构表明，抗性突变可重新定位附近的疏水残基，从而在不影响底物结合的情况下干扰化合物的结合。这是自然产生的隐孢子虫耐药性的第一份报告，强调需要解决潜在的抗隐孢子虫耐药性并建立限制其发生的策略。