Genome-wide Functional Analysis of Plasmodium Protein Phosphatases Reveals Key Regulators of Parasite Development and Differentiation

Abstract

SummaryReversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria.

CRISPR/Cas9 mediated sequential editing of genes critical for ookinete motility in Plasmodium yoelii

“…However, mCherry expression was completely lost in the oocysts at day 4 post infection. These results showed that P28::mCherry protein was expressed normally in this engineered parasite and is consistent with those observed using anti-P28 antibody staining [23–25]. …”

“…Plasmodium P28 protein is expressed abundantly in activated female gamete, zygote, ookinete, and young oocyst [23, 24]. Antibody-based immuno-detection of P28 protein expression was widely used in studies of parasite gametocyte activation and ookinete formation [25]. To tag the endogenous PyP28 gene with red fluorescence protein mCherry gene, we made two constructs based on the pYC and pYCm plasmids, respectively.…”

Functional characterization of Plasmodium berghei PSOP25 during ookinete development and as a malaria transmission-blocking vaccine candidate

“…with either 5 × 10 6 wildtype (WT) or Δ psop25 iRBCs. For each genotype, blood smears were used to monitor daily parasitemia, gametocytemia (mature gametocytes per 100 RBCs), and the gametocyte sex ratio (female: male ratio) [33]. To quantify male gamete exflagellation, 10 μl of P. berghei -infected blood collected from mouse tail vein on day 3 p.i.…”

The development of malaria parasites in the mosquito midgut

“…Also, depletion of the metallo‐dependent protein phosphatase PPM1 in P . berghei results in impaired exflagellation (Guttery et al ., 2014), while depletion of the Ca 2+ ‐dependent phosphatase Calcineurin affects male gametogenesis and subsequent fertilization in the rodent malaria parasite (Philip and Waters, 2015) (Fig. 1).…”

“…Further, deletion of the Shewanella‐like protein phosphatase (SHLP1), PPM2 or the kelch‐like domain‐containing phosphatase PPKL results in impaired P . berghei ookinete development, and phenotypes include impaired IMC and microneme formation (Guttery et al ., 2012b, 2014; Philip et al ., 2012; Patzewitz et al ., 2013) (Table 1). …”