PfVPS4, an ESCRT AAA-ATPase, is essential for asexual proliferation and gametocyte sexual conversion in Plasmodium falciparum

Abstract Background Malaria, caused by Plasmodium spp., remains a major global health threat. Among them, Plasmodium falciparum is the most pathogenic, and its asexual intraerythrocytic proliferation is the pathological basis. This process has enormous biosynthetic demands and highly relies on the coordinated function of the endomembrane and vesicular transport systems. The transition from asexual proliferation to sexual differentiation similarly involves remodeling of internal membrane complexes, membrane reshaping, and precise protein sorting. In eukaryotic cells, the Endosomal Sorting Complexes Required for Transport (ESCRT) complex is a core machinery for membrane remodeling and endosomal development. However, how the ESCRT system regulates the complex life cycle of Plasmodium, particularly during intraerythrocytic proliferation and sexual conversion, remains an important unresolved question. Methods In this study, using Plasmodium falciparum as a model system, we applied CRISPR-Cas9-mediated homologous recombination to achieve conditional knockdown of PfVPS4, the core ATPase of the ESCRT complex — vacuolar protein sorting-associated protein 4 (PfVPS4). Western blotting and immunofluorescence assays were used to assess PfVPS4 abundance and subcellular localization. Tightly synchronized cultures were used to evaluate its effects on parasite growth, merozoite numbers, and gametocyte conversion rate. In vitro protein purification, enzyme kinetics, and site-directed mutagenesis were performed to identify the impact of key residues on PfVPS4 ATPase activity and to validate the synergistic activation by its cofactor PfVta1. In addition, multiple sequence alignment and AlphaFold3 modeling were used to predict and display structural features before and after mutation of key sites. Results We successfully generated conditional knockdown lines in both Pf3D7 and PfNF54 parasite strains, enabling effective knockdown at different stages of the intraerythrocytic cycle and during gametocytogenesis. Knockdown of PfVPS4 led to an 84% reduction in asexual progeny parasite numbers, decreased merozoite numbers, and a 46% reduction in gametocyte conversion rate, without affecting subsequent gametocyte maturation. Biochemical assays showed that PfVPS4 ATPase activity is optimal at pH 7.5 and 37°C, and is dependent on Mg²⁺, with a Vmax of 2.23 ± 0.053 U/mg and a Km of 0.086 mM. Site-directed mutagenesis validated the essential role of the canonical catalytic residues (D213, E214) and the species-specific key residues (T161, I288) in maintaining enzymatic activity, and confirmed that the cofactor PfVta1 significantly enhances PfVPS4 activity. Conclusion PfVPS4 is essential for normal asexual blood-stage replication and efficient sexual conversion in Plasmodium falciparum. Its knockdown severely disrupts intraerythrocytic proliferative homeostasis and reduces gametocyte conversion, indicating that this protein has a broader role in coordinating parasite proliferation and transmission. Given its essentiality, species‑specific residues, and regulation by PfVta1, PfVPS4 and its complex are attractive antimalarial drug targets. Graphical Abstract