Department of Biology, University of Nebraska at Omaha, Omaha, Nebraska, USA.
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.
Antimicrob Agents Chemother. 2021 Feb 17;65(3). doi: 10.1128/AAC.01040-20.
is a globally distributed apicomplexan parasite and the causative agent of toxoplasmosis in humans. While pharmaceuticals exist to combat acute infection, they can produce serious adverse reactions, demonstrating a need for enhanced therapies. KG8 is a benzoquinone acyl hydrazone chemotype identified from a previous chemical screen for which we previously showed and efficacy against However, the genetic target and mechanism of action of KG8 remain unknown. To investigate potential targets, we generated resistant lines by chemical mutagenesis followed by selection. Whole-genome sequencing of resistant clones revealed a P207S mutation in the gene encoding rhoptry organelle protein 1 (ROP1) in addition to two lesser resistance-conferring mutations in the genes for rhoptry organelle protein 8 (ROP8) and a putative ADP/ATP carrier protein (TGGT1_237700). Expressing ROP1 in parental parasites was sufficient to confer significant (10.3-fold increased half-maximal effective concentration [EC]) KG8 resistance. After generating a library of mutants carrying hypermutated alleles followed by KG8 pressure, we sequenced the most resistant clonal isolate (>16.9-fold increased EC) and found independent recapitulation of the P207S mutation, along with three additional mutations in the same region. We also demonstrate that a knockout strain is insensitive to KG8. These data implicate ROP1 as a putative resistance gene of KG8. This work further identifies a compound that can be used in future studies to better understand ROP1 function and highlights this novel chemotype as a potential scaffold for the development of improved therapeutics.
刚地弓形虫是一种全球性分布的顶复门寄生虫，也是人类弓形虫病的病原体。虽然有药物可以治疗急性感染，但它们可能会产生严重的不良反应，这表明需要增强治疗方法。KG8 是一种苯醌酰腙化学型，是我们之前在针对 的化学筛选中发现的，我们之前证明它对 有 和 的功效。然而，KG8 的遗传靶标和作用机制仍然未知。为了研究潜在的靶点，我们通过化学诱变和 选择生成了耐药 系。耐药克隆的全基因组测序揭示了编码 rhoptry 细胞器蛋白 1 (ROP1) 的基因中的 P207S 突变，此外，ROP8 基因和假定的 ADP/ATP 载体蛋白 (TGGT1_237700) 中还有两个较小的耐药性赋予突变。在亲本寄生虫中表达 ROP1 足以赋予显著的 KG8 耐药性（增加的半最大有效浓度 [EC] 为 10.3 倍）。在生成携带超突变 等位基因的突变体文库并施加 KG8 压力后，我们对最耐药的克隆分离株（EC 增加了 16.9 倍以上）进行测序，发现 P207S 突变的独立重现，以及同一区域的另外三个突变。我们还证明 敲除株对 KG8 不敏感。这些数据表明 ROP1 是 KG8 的一个潜在耐药基因。这项工作进一步确定了一种可以在未来研究中用于更好地理解 ROP1 功能的化合物，并强调了这种新型化学型作为开发改进的 治疗方法的潜在支架。
