Department of Tropical Medicine and Parasitology, Faculty of Medicine, Juntendo University, Tokyo, Japan.
Front Cell Infect Microbiol. 2021 Jun 16;11:672691. doi: 10.3389/fcimb.2021.672691. eCollection 2021.
Elucidation of the mechanisms of drug resistance in malaria parasites is crucial for combatting the emergence and spread of resistant parasites, which can be achieved by tracing resistance-associated mutations and providing useful information for drug development. Previously, we produced a novel genetic tool, a mutator (PbMut), whose base substitution rate is 36.5 times higher than that of wild-type parasites. Here, we report the isolation of a mutant with reduced susceptibility to piperaquine (PPQ) from PbMut under PPQ pressure by sequential nine-cycle screening and named it PbMut-PPQ-R-P9. The ED of PbMut-PPQ-R-P9 was 1.79 times higher than that of wild-type parasites, suggesting that its PPQ resistance is weak. In the 1 screen, recrudescence occurred in the mice infected with PbMut but not in those infected with wild-type parasites, suggesting earlier emergence of PPQ-resistant parasites from PbMut. Whole-genome sequence analysis of PbMut-PPQ-R-P9 clones revealed that eight nonsynonymous mutations were conserved in all clones, including N331I in , the gene encoding chloroquine resistance transporter (). The PbCRT(N331I) mutation already existed in the parasite population after the 2 screen and was predominant in the population after the 8 screen. An artificially inserted PbCRT(N331I) mutation gave rise to reduced PPQ susceptibility in genome-edited parasites (PbCRT-N331I). The PPQ susceptibility and growth rates of PbCRT-N331I parasites were significantly lower than those of PbMut-PPQ-R-P9, implying that additional mutations in the PbMut-PPQ-R9 parasites could compensate for the fitness cost of the PbCRT(N331I) mutation and contribute to reduced PPQ susceptibility. In summary, PbMut could serve as a novel genetic tool for predicting gene mutations responsible for drug resistance. Further study on PbMut-PPQ-R-P9 could identify genetic changes that compensate for fitness costs owing to drug resistance acquisition.
阐明疟原虫耐药机制对于对抗耐药寄生虫的出现和传播至关重要，这可以通过追踪耐药相关突变并为药物开发提供有用信息来实现。此前，我们开发了一种新型遗传工具——突变体（PbMut），其碱基替换率比野生型寄生虫高 36.5 倍。在这里，我们报告了在哌喹（PPQ）压力下，通过连续九轮筛选从 PbMut 中分离出一种对 PPQ 敏感性降低的突变体，并将其命名为 PbMut-PPQ-R-P9。PbMut-PPQ-R-P9 的 ED 值比野生型寄生虫高 1.79 倍，表明其对 PPQ 的耐药性较弱。在第 1 轮筛选中，感染 PbMut 的小鼠出现了复发，但感染野生型寄生虫的小鼠没有，这表明 PPQ 耐药寄生虫更早地从 PbMut 中出现。对 PbMut-PPQ-R-P9 克隆的全基因组序列分析表明，所有克隆中都保守了 8 个非同义突变，包括编码氯喹耐药转运蛋白（）的基因中的 N331I。在第 2 轮筛选后，PbCRT(N331I)突变已经存在于寄生虫群体中，并且在第 8 轮筛选后成为群体中的主要突变。人工插入的 PbCRT(N331I)突变导致基因组编辑寄生虫对 PPQ 的敏感性降低（PbCRT-N331I）。PbCRT-N331I 寄生虫的 PPQ 敏感性和生长速率明显低于 PbMut-PPQ-R-P9，这意味着 PbMut-PPQ-R9 寄生虫中的其他突变可以补偿 PbCRT(N331I)突变的适应成本，并有助于降低 PPQ 敏感性。总之，PbMut 可以作为一种新型遗传工具，用于预测与药物耐药性相关的基因突变。对 PbMut-PPQ-R-P9 的进一步研究可以确定补偿因耐药性获得而导致的适应成本的遗传变化。
