AUTHOR=Ikeda Mie , Hirai Makoto , Tachibana Shin-Ichiro , Mori Toshiyuki , Mita Toshihiro 

TITLE=Isolation of Mutants With Reduced Susceptibility to Piperaquine From a Mutator of the Rodent Malaria Parasite Plasmodium berghei

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=11

YEAR=2021

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2021.672691

DOI=10.3389/fcimb.2021.672691

ISSN=2235-2988

ABSTRACT=<p>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 <italic>Plasmodium berghei</italic> 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<sub>50</sub> 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<sup>st</sup> 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 <italic>PbCRT</italic>, the gene encoding chloroquine resistance transporter (<italic>CRT</italic>). The PbCRT(N331I) mutation already existed in the parasite population after the 2<sup>nd</sup> screen and was predominant in the population after the 8<sup>th</sup> 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.</p>