Molecular and cellular characterization of Plasmodium berghei PPM9 phosphatase, an enzyme dispensable during both asexual and sexual life cycle stages

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Protein phosphorylation is a key regulatory mechanism controlling many essential biological processes in Plasmodium berghei. While the parasite kinome has been extensively investigated, the phosphatome, particularly the metal-dependent PP2C phosphatases, remains poorly characterized. Among these enzymes, PPM9 has been proposed to play an important role in parasite development, although previous functional studies have produced conflicting conclusions regarding its essentiality. Here, we performed a comprehensive molecular and functional characterization of PbPPM9. Recombinant PbPPM9 exhibited intrinsic phosphatase activity in vitro that was strongly dependent on Mn²⁺ ions. Enzymatic activity was inhibited in a dose-dependent manner by EDTA, confirming the requirement of divalent metal cofactors. Structural modelling of the catalytic site further supported the coordination of Mn²⁺ ions within conserved residues typical of the PP2C family. Reverse-genetics analyses demonstrated that PbPPM9 is dispensable for parasite development. Targeted gene deletion produced viable parasites with no detectable defects in asexual blood-stage replication, sexual differentiation, or gametocyte formation. Moreover, knockout parasites completed the entire life cycle normally, including mosquito development, salivary gland colonization, hepatocyte infection, and subsequent erythrocytic proliferation in vivo. Together, these findings reveal that PbPPM9 is not essential for parasite growth or transmission, suggesting functional redundancy among PP2C phosphatases in Plasmodium.

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