Pyrophosphate homeostasis in multiple subcellular compartments is essential in Plasmodium falciparum

Pyrophosphate is a byproduct of numerous cellular reactions that use ATP or other nucleoside triphosphates to synthesize DNA, RNA, protein, and various molecules. Its degradation into monophosphate is thus crucial for the survival and proliferation of all life forms. The human malaria parasite Plasmodium falciparum encodes two classes of pyrophosphatases to hydrolyze pyrophosphate. The first consists of P. falciparum proton pumping vacuolar pyrophosphatases (e.g., PfVP1), which localize to the parasite plasma membrane and work as proton pumps. The second includes P. falciparum soluble pyrophosphatases (PfsPPases), which have not been well characterized. Interestingly, the gene locus of PfsPPases encodes two isoforms, PfsPPase1 (PF3D7_0316300.1) and PfsPPase2 (PF3D7_0316300.2). PfsPPase2 contains a 51-amino acid organellar localization peptide that is absent in PfsPPase1. Here, we combine reverse genetics and biochemical approaches to identify the localization of PfsPPase1 and PfsPPase2 and elucidate their individual functions. We show that PfsPPases are essential for the asexual blood stages. While PfsPPase1 solely localizes to the cytoplasm, PfsPPase2 exhibits multiple localizations, including the mitochondrion, the apicoplast, and, to a lesser extent, the cytoplasm. Our data suggest that P. falciparum has taken a unique evolutionary trajectory in pyrophosphate metabolism by utilizing a leader sequence to direct sPPases to multiple organelles. This differs from model eukaryotes as they generally encode multiple sPPases at distinct genetic loci to facilitate pyrophosphate degradation in cytosolic and organellar compartments. The essentiality and divergence of PfsPPases also highlight them as promising targets for the development of novel antimalarial drugs.

Malaria kills over 600,000 people annually. Understanding parasite biology is critical for identifying prospective drug targets. Malaria parasites maintain pyrophosphate (PPi) homeostasis in at least three subcellular compartments-the cytoplasm, mitochondrion, and apicoplast, where PPi is generated through various reactions. While cytoplasmic PPi is known to be degraded by soluble pyrophosphatase, it remains unclear how PPi is metabolized in the organelles of malaria parasites. Here, we discovered that Plasmodium falciparum encodes two soluble pyrophosphatase isoforms from a single genetic locus. The longer isoform contains an N-terminal leader sequence that targets the enzymes into the mitochondrion and the apicoplast. This dual targeting mechanism of soluble pyrophosphatases has not been previously reported in any organisms. We show that both isoforms are essential for parasite growth and development. These findings highlight the critical role of organellar PPi degradation and identify soluble pyrophosphatases as promising antimalarial drug targets.