# Loss of serine/threonine protein phosphatase 6 severely impairs sexual stage development in malaria parasite Plasmodium berghei

## TL;DR

This study shows that a protein phosphatase called PbPP6 is crucial for the sexual development of malaria parasites, and its absence reduces transmission to mosquitoes.

## Contribution

The study identifies PbPP6 as a key regulator of cGMP-PKG-Ca2+ signaling in malaria gametogenesis through phosphoproteomic and transcriptomic analyses.

## Key findings

- Disruption of PbPP6 impairs male gametogenesis and reduces ookinete formation and mosquito transmission.
- PbPP6 regulates GCα activity by dephosphorylation, affecting cGMP-PKG-Ca2+ signaling and DNA replication in gametocytes.
- Altered mRNP expression in PbPP6-deficient parasites may impact post-fertilization development in mosquitoes.

## Abstract

Protein phosphorylation plays a critical role during the development of malaria parasites. Here, we performed a functional analysis of the Plasmodium berghei Ser/Thr protein phosphatase 6 (PbPP6), which is associated with the plasma membrane of macrogametes and ookinetes. Compared to wild-type P. berghei, the genetic disruption of pbpp6 (∆pbpp6) resulted in reduced asexual growth of the parasites and prolonged survival of infected mice. The ∆pbpp6 parasites showed impaired gametogenesis, particularly affecting male gametogenesis, which substantially decreased both ookinete formation and mosquito transmission. Transcriptomic analysis revealed an over 11-fold downregulation of nek3, a regulator of MAPK2 within the PKG-Ca2⁺ signaling cascade, foreshadowing pathway dysregulation that was further evidenced by significantly diminished intracellular cGMP levels, decreased cytosolic Ca2⁺ mobilization, and reduced DNA replication in activated Δpbpp6 gametocytes. Phosphoproteomic analysis detected increased phosphorylation at the Ser508 site of guanylyl cyclase alpha (GCα), indicating that PbPP6 regulates cGMP-PKG-Ca2+ signaling through modulation of GCα activity during gametogenesis. Additionally, we observed altered expression of messenger ribonucleoproteins in the Δpbpp6 parasites, which may affect the translational repression of stored mRNAs in female gametocytes and impact post-fertilization development in mosquitoes. Collectively, this study highlights the potential of targeting PP6 to disrupt malaria transmission.

Malaria transmission begins when Plasmodium gametocytes, ingested by a female Anopheles mosquito, activate and differentiate into gametes in the midgut. Through genetic and multi-omics analyses, we identified the protein phosphatase PbPP6 as a key regulator of the cGMP-PKG-Ca2⁺ signaling cascade, specifically via the dephosphorylation of GCα. Disruption of the pbpp6 gene impaired male gametogenesis and affected post-fertilization development, likely due to dysregulated mRNP-mediated translational repression. Our findings demonstrate that genetic disruption of PbPP6 drastically reduced parasite transmission, highlighting protein phosphatases as potential targets for blocking malaria transmission.