# Deletion of the chloroquine resistance transporter gene confers reduced piperaquine susceptibility to the rodent malaria parasite Plasmodium berghei

## TL;DR

Deleting a gene in a rodent malaria parasite reduces its susceptibility to a malaria drug and affects its survival.

## Contribution

First evidence that deleting the PbCRT gene in Plasmodium berghei reduces piperaquine susceptibility.

## Key findings

- Deleting the PbCRT gene in Plasmodium berghei leads to reduced piperaquine susceptibility.
- PbCRT(−) parasites show compromised fitness in mice and during mosquito transmission.
- PbCRT gene deletion is a novel mechanism for drug resistance in rodent malaria parasites.

## Abstract

Malaria parasites acquire drug resistance through genetic changes, the mechanisms of which remain incompletely understood. Understanding the mechanisms of drug resistance is crucial for the development of effective treatments against malaria, and for this purpose, new genetic tools are needed. In a previous study, as a forward genetic tool, we developed the rodent malaria parasite Plasmodium berghei mutator (PbMut) line, which has a greatly increased rate of mutation accumulation and from which we isolated a mutant with reduced susceptibility to piperaquine (PPQ). We identified a mutation in the chloroquine resistance transporter (PbCRT N331I) as responsible for this phenotype. In the current study, we generated a marker-free PbMut to enable further genetic manipulation of the isolated mutants. Here, we screened again for PPQ-resistant mutants in marker-free PbMut and obtained a parasite population with reduced susceptibility to PPQ. Of five isolated clones, none had the mutation PbCRT N331I; rather, they possessed a nonsense mutation at amino acid 119 (PbCRT Y119*), which would truncate the protein before eight of its ten predicted transmembrane domains. The PbCRT orthologue in the human malaria parasite Plasmodium falciparum, PfCRT, is an essential membrane transporter. To address the essentiality of PbCRT, we successfully deleted the full PbCRT gene [PbCRT(−)] from wild-type parasites. PbCRT(−) parasites exhibited reduced susceptibility to PPQ, along with compromised fitness in mice and following transmission to mosquitoes. Taken together, our findings provide the first evidence that P. berghei can acquire reduced PPQ susceptibility through complete loss of PbCRT function.