The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission

Abstract

Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified.

Figure 1

Phylogenetic Trees of Plasmodium ePK Domains (A) Protein kinases of P. berghei ANKA. Genes that were disrupted in this study are highlighted in yellow. Colored branches reflect associations with conserved ePK groups. Gray branches indicate orphan kinases and some conserved ePKs of uncertain association. Only the numeric part of systematic gene IDs is shown on the tree. Refer to Figure S1B for bootstrap values supporting individual nodes and see Table S1 for additional data. (B) Protein kinases of P. falciparum 3D7. Highlighted in blue are the features that distinguish this kinome from that of P. berghei (see also Table S2). Both panels are derived from the alignment and neighbor-joining tree shown in Figure S1.

Figure 2

Strategy for Gene Targeting and Genotyping (A) The typical strategy for gene deletion is illustrated, using the cdc2 homolog pk5 as an example. Also shown are primers P1–4 used for diagnostic PCR analysis, probes used for Southern hybridization, and diagnostic restriction sites. Refer to Figure S2 for all vector designs and genotyping data. (B) Pulsed-field gel analysis of 18 mutants confirms integration of targeting vectors into the expected chromosome. Chromosome blots were hybridized with a probe recognizing the tgdhfr/ts resistance cassette, which also hybridizes more weakly to the P. berghei dhfr 3′ UTR on chromosome 7. Only the first clone obtained for each mutant is shown. (C) Diagnostic PCR (left) and Southern hybridization (right) with target-specific probes showing disruption of pk5, srpk, and cdlk genes.

Figure 3

Phenotypic Screen of 23 ePK Mutants (A) Data from 43 transmission experiments are summarized. Mutants are arranged by life cycle stage and decreasing severity of the phenotype. Error bars show standard deviations from at least three independent experiments per mutant (also refer to Table S3). (B) Heat map representation of all phenotyping data. White space indicates the phenotype was not determined.

Figure 4

Characterization of an srpk Mutant (A) Fluorescence microscopy illustrating a P28-positive wild-type ookinete (upper row) and a P28-positive srpk macrogamete (lower row). The latter remained unfertilized, as indicated by weaker Hoechst labeling of nuclear DNA, consistent with the inability of the srpk mutant to produce microgametes. (B) Cross-fertilization experiments to examine the competence of srpk macrogametes to differentiate into ookinetes when fertilized by microgametes from a female defective mutant, such as nek-4. Crosses are not expected to restore ookinete conversion to wild-type levels, because around half of macrogametes in crosses are nek-4⁻ and therefore blocked in differentiation. Error bars show standard deviations of three biological replicates. (C) Average daily parasitemia in six BALB/c mice infected with 1 × 10³ wild-type or srpk parasites. The inset shows daily multiplication rates calculated as (parasitemia_{day n}/parasitemia_{day n− 1}). Error bars give standard errors of the mean. Growth curves are representative of three experiments with two independently generated knockout clones.

Figure 5

Oocyst Phenotypes (A) Representative images of wild-type and mutant oocysts on mosquito midguts dissected 21 days after feeding. (B) A representative cdpk6 oocyst compared to a previously characterized ecp1 cysteine protease mutant with egress phenotype. (C) Quantitative analysis of the oocyst granulation in a cdlk mutant. At each time point, 5–10 wild-type and cdlk infected midguts were dissected and labeled with SYTOX green, and oocysts were assigned to categories. Oocysts showing signs of sporoblast formation or sporozoites were counted as sporulated. Oocysts labeled with SYTOX green were scored as dead.

Figure 6

Schematic Illustration of Stage-Specific Kinase Gene Functions in P. berghei Mosquito Stages Cdpk4, map-2, and srpk are essential for male gamete formation. Nek-2 and nek-4 are individually essential for DNA replication that precedes meiosis in the zygote. In gak and pk7 mutants, zygotes largely fail to differentiate into ookinetes; a small number of oocysts are produced but fail to sporulate. Cdpk3 knockout ookinetes are defective in gliding motility and invasion of the mosquito midgut epithelium. Cdlk mutant oocysts fail late during development, at about the stage of sporoblast formation. Oocysts of parasites lacking kin, cdpk6, or PBANKA_040940 have fewer sporozoites in the salivary glands, which may be linked to their enhanced retention in the oocyst. Figure modified from Billingsley and Sinden (1997).

Figure 7

Target Prioritization among Small Gatekeeper Kinases of Plasmodium Subdomain V of all six P. berghei ePKs with small gatekeeper is aligned with the human tyrosine kinase Src and the cancer target Abl. P. falciparum homologs with small gatekeepers are shown on the tree, and redundancy in P. berghei is highlighted in red.