Isolation of Mutants With Reduced Susceptibility to Piperaquine From a Mutator of the Rodent Malaria Parasite Plasmodium berghei.
Isolation of Mutants With Reduced Susceptibility to Piperaquine From a Mutator of the Rodent Malaria Parasite Plasmodium berghei.
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DOI：
10.3389/fcimb.2021.672691
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发表时间：
2021
影响因子：
5.7
通讯作者：
Mita T
中科院分区：
文献类型：
--
作者：
Ikeda M;Hirai M;Tachibana SI;Mori T;Mita T
阐明疟疾寄生虫的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。在此之前，我们生产了一种新的遗传工具，伯氏疟原虫突变体（PbMut），其碱基替换率比野生型寄生虫高36.5倍。在此，我们报道了通过连续9个循环的筛选，在PPQ压力下从PbMut中分离出对哌喹（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 50是野生型寄生虫的1.79倍，表明其PPQ抗性较弱。在第一次筛选中，在感染PbMut的小鼠中发生复发，但在感染野生型寄生虫的小鼠中未发生复发，表明PbMut的PPQ抗性寄生虫较早出现。PbMut-PPQ-R-P9克隆的全基因组序列分析显示，8个非同义突变在所有克隆中是保守的，包括编码氯喹抗性转运蛋白（CRT）的基因PbCRT中的N331 I。PbCRT（N331 I）突变在第2次筛选后的寄生虫群体中已经存在，并且在第8次筛选后的群体中占优势。人工插入的PbCRT（N331 I）突变导致基因组编辑寄生虫（PbCRT-N331 I）的PPQ敏感性降低。PbCRT-N331 I寄生虫的PPQ敏感性和生长速率显著低于PbMut-PPQ-R-P9寄生虫，这意味着PbMut-PPQ-R9寄生虫中的其他突变可以补偿PbCRT（N331 I）突变的适应度成本，并有助于降低PPQ敏感性。总之，PbMut可以作为一种新的遗传工具，用于预测负责耐药性的基因突变。对PbMut-PPQ-R-P9的进一步研究可以鉴定出补偿由于获得耐药性而导致的适应性成本的遗传变化。
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 Plasmodium berghei 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 ED50 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 1st 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 PbCRT, the gene encoding chloroquine resistance transporter (CRT). The PbCRT(N331I) mutation already existed in the parasite population after the 2nd screen and was predominant in the population after the 8th 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.
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