Plasmodium falciparum utilizes pyrophosphate to fuel an essential proton pump in the ring stage and the transition to trophozoite stage.

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage. Author summary: Membrane-bound proton pumping pyrophosphatases (H⁺-PPases), also known as vacuolar pyrophosphatases (V-PPases), are single-subunit proton pumps powered by hydrolysis of pyrophosphate (PPi) rather than ATP. These ancient, ATP independent proton pumps have been evolutionarily conserved in bacteria, archaea, plants, and protists, but are absent in fungi and animals. While H⁺-PPases were discovered in Plasmodium spp. 20 years ago, their significance in malaria parasites has remained unclear. In this study, we have unveiled the pivotal roles of PfVP1, Plasmodium falciparum vacuolar pyrophosphatase 1, in the early phases of the asexual developmental cycle, including the ring stage and the ring-to-trophozoite transition. Through multiple approaches, we have confirmed that PfVP1 is a PPi hydrolyzing proton pump in P. falciparum, indicating, for the first time, that PPi serves as an energy source in malaria parasites. Our results further indicate that PfVP1 is an excellent antimalarial drug target due to its essential physiological roles and its absence in the human hosts. Antimalarials targeting the ring stage are highly desired, as most drugs have limited efficacy against this metabolically less active stage. [ABSTRACT FROM AUTHOR]