The Plasmepsin-Piperaquine Paradox Persists

Abstract

Malaria is still a major health issue in many parts of the world, particularly in tropical and subtropical regions of Africa, Asia, and Latin America. Despite significant efforts to control and eliminate the disease, malaria remains a leading cause of illness and death, mainly due to the occurrence of drug-resistant parasites to the frontline antimalarials such as dihydroartemisinin-piperaquine (DHA-PPQ). Artemisinin resistance has been linked to kelch13 mutations, while decreased PPQ sensitivity has been associated with higher plasmepsin II and III gene copies and mutations in the chloroquine resistance transporter. In this study, we demonstrate the effective use of CRISPR/Cas9 technology to generate single knockouts (KO) of plasmepsin II and plasmepsin III, as well as a double KOs of both genes, in two isogenic lines of Cambodian parasites with varying numbers of plasmepsin gene copies. The deletion of plasmepsin II and/or III increased the parasites sensitivity to PPQ, evaluated by the area under the curve. We explored several hypotheses to understand how an increased plasmepsin gene copy number might influence parasite survival under high PPQ pressure. Our findings indicate that protease inhibitors have a minimal impact on parasite susceptibility to PPQ. Additionally, parasites with higher plasmepsin gene copy numbers did not exhibit significantly increased hemoglobin digestion, nor did they produce different amounts of free heme following PPQ treatment compared to wildtype parasites. Interestingly, hemoglobin digestion was slowed in parasites with plasmepsin II deletions. By treating parasites with digestive vacuole (DV) function modulators, we found that changes in DV pH potentially affect their response to PPQ. Our research highlights the crucial role of increased plasmepsin II and III gene copy numbers in modulating response to PPQ and begins to uncover the molecular and physiological mechanisms underlying PPQ resistance in Cambodian parasites. Author SummaryGlobal malaria control has plateaued, with drug-resistant Plasmodium falciparum posing a significant challenge. Artemisinin-based combination therapies (ACTs) are becoming less effective, especially in South-East Asia, where resistance to dihydroartemisinin-piperaquine (DHA-PPQ) is leading to treatment failures, notably in Cambodia. Genome-wide studies link artemisinin resistance to kelch13 mutations, while decreased PPQ sensitivity is tied to higher plasmepsin II and III gene copies and mutations in chloroquine resistance transporter. We previously showed a connection between increased plasmepsin gene copies and reduced PPQ sensitivity. In this study we try to understand the biological role of the plasmepsins in PPQ sensitivity. Therefore, we knocked out plasmepsin II and III genes in Cambodian strains using CRISPR/Cas9, and found increased PPQ sensitivity, confirming these genes roles in resistance. Plasmepsins are proteases that participate in the hemoglobin degradation cascade in the digestive vacuole of the parasites. Protease inhibitor experiments and hemoglobin digestion studies indicate that digestive vacuole pH fluctuations affect PPQ response, highlighting the need for further research into PPQ resistance mechanisms.