A single point mutation in the Plasmodium falciparum 3′–5′ exonuclease does not alter piperaquine susceptibility

• Published in: Malaria Journal
• Main Authors: Nonlawat Boonyalai, Kirakarn Kirativanich, Chatchadaporn Thamnurak, Chantida Praditpol, Brian A. Vesely, Mariusz Wojnarski, John S. Griesenbeck, Norman C. Waters
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2022
• Subjects: Exonuclease; Gene editing; Malaria; Piperaquine resistance; Plasmodium falciparum; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1186/s12936-022-04148-z; https://doaj.org/article/1ceb154d913a401e96ce0e42e8665416

Abstract Background The rise in Plasmodium falciparum resistance to dihydroartemisinin–piperaquine (DHA–PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele. Methods Transgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility. Results The transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly. Conclusions Insertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and ...