Conference Agenda

Combating malaria caused by Plasmodium falciparum requires strategies to mitigate drug resistance. The clinical candidates cipargamin and SJ733 target the Na⁺ pump PfATP4. A G358S mutation in PfATP4, identified in 68% of recrudescent cases in a cipargamin clinical trial, confers high-level resistance to both compounds while simultaneously increasing parasite sensitivity to PfATP4 inhibitors belonging to two distinct chemical classes (‘I’ and ‘II’). To investigate whether collateral sensitivity could be leveraged in preserving cipargamin efficacy, we exposed ‘hypermutator’ parasites to class I and II compounds in combination with a high (20× IC₅₀) concentration of cipargamin. The cipargamin/class I combination led to an L354V mutation in PfATP4, while for cipargamin/class II, no viable parasites emerged across 6 selections. However, exposure of PfATP4^G358S parasites to a class II compound drove the acquisition of an additional PfATP4 mutation (N355Y). The N355Y+G358S mutants exhibited > 2000-fold and 650-fold resistance to cipargamin and SJ733, respectively. The PfATP4-G358A mutation was also associated with treatment failure in the clinical trial. We show that this mutation confers 500-fold resistance to cipargamin, confirming its clinical significance. Together, these findings highlight that while multiple PfATP4 mutations can compromise cipargamin efficacy, combining certain PfATP4 inhibitors increases the barrier for resistance.

Resistance to the frontline antimalarial drug artemisinin is primarily mediated by mutations in the Plasmodium falciparum protein Kelch13 (K13), whose function has remained unclear. Parasites carrying mutant K13 ingest red blood cell haemoglobin more slowly. Because artemisinin is activated by haem released during parasite feeding, reduced haemoglobin uptake likely lowers intracellular levels of toxic artemisinin-derived species. How wild-type or mutant K13 contributes to this feeding process was unknown. Using multiple imaging approaches, we show that K13 localises to the collar that maintains the cytostome, a stable parasite invagination used for uptake of host cytosol. Three-dimensional electron microscopy reveals that mislocalised K13 abolishes formation of the electron-dense collar that stabilises the cytostomal neck and disrupts cytostome formation itself. Consistent with this, haemoglobin degradation products, including haem and haemozoin, are reduced when K13 is inactivated. Using expansion microscopy together with super-resolution and lattice light-sheet microscopy, we further show that new K13 collars form and segregate to daughter cells before division, but that this biogenesis is delayed in mutant parasites. These findings indicate that artemisinin resistance arises through defective cytostome formation, reduced endocytosis, and diminished drug activation in resistant parasites.