The Serine/Threonine Protein Phosphatase 6 Is Required for Efficient Gametogenesis in Sexual-Stage Plasmodium berghei

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 ?pbpp6 gametocytes. Phosphoproteomic analysis detected increased phosphorylation at the Ser508 site of guanylyl cyclase alpha (GCa), indicating that PbPP6 regulates cGMP-PKG-Ca2+ signaling through modulation of GCa activity during gametogenesis. Additionally, we observed altered expression of messenger ribonucleoproteins in the ?pbpp6 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. Overall design: 1 µg total RNA was extracted from purified gametocytes (The mice were treated with sulfadiazine (Sigma, 20mg/l in drinking water) at 3 dpi for 48 hrs to remove asexual stage parasites, and the activated gametocytes (25? for 15 min) were collected by using 48% Nycodenz) of ?pbpp6 and WT parasites using the Qiagen RNeasy kit (Qiagen, Dusseldorf, Germany) as per manufacturer's instructions. The quantity, purity and integrity of the RNA were evaluated by 1.5% agarose gel electrophoresis and on a NanodropTM OneC spectrophotometer (ThermoFisher). The mRNA-seq libraries were constructed with 2 µg RNA using KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina® (Catalog NO. DR08502, Wuhan Seqhealth Co., Ltd. China), which eliminates duplication bias in PCR and sequencing steps by using unique molecular identifiers (UMI) of 8 random bases to label the pre-amplified cDNA molecules (91). RNA-seq was performed from three biological replicates of the ?pbpp6 and WT parasites. The library products corresponding to 200-500 bps were enriched, quantified and finally sequenced on Hiseq X 10 sequencer (Illumina, San Diego, USA). Raw sequencing data were first filtered by Trimmomatic (version 0.36) to remove the low-quality reads, and were further treated with in-house scripts to eliminate duplication bias introduced in library preparation and sequencing. RNA-seq reads from each sample was aligned to the PbANKA01 of the P. berghei ANKA reference genome using the STAR RNA-seq alignment tool (92). HTSeq tool (v.0.6.1) was used to count transcripts for each gene (93). Cuffdiff v2.1 was used as the default method for normalization (94), while differential expression analysis was conducted using DESeq2 (v.1.28.1) in R (v.4.04) (20). The false discovery rate (FDR) was calculated to control for multiple hypothesis testing. Genes with an adjusted FDR below 0.1 and a minimal log2Fold change (log2FC) of 1 were considered to be significantly differentially expressed. Clustering and PCA were performed in R to estimate sampling distribution in each experiment. GO analysis for differentially expressed genes were performed in PlasmoDB (95), with the Benjamini adjusted P-value cutoff of 0.1to determine statistically significant enrichment.