The Systematic Functional Analysis of Plasmodium Protein Kinases Identifies Essential Regulators of Mosquito Transmission

Abstract

SummaryAlthough 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.

Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum

“…In comparison, a recent study of 66 ePKs of the rodent malaria parasite P. berghei identified 43 protein kinases that are refractory to deletion in the blood stage and 23 whose genes could be deleted 37 (Supplementary Table S1). Although there is overall a good level of agreement between the ePKs identified as essential in the mouse parasite and those we identify here to be have a crucial role in P. falciparum (Supplementary Table S1), there are nevertheless differences, which may underlie divergence between human and mouse malarial species resulting from a split estimated to have occurred 70 million years ago.…”

“…Although there is overall a good level of agreement between the ePKs identified as essential in the mouse parasite and those we identify here to be have a crucial role in P. falciparum (Supplementary Table S1), there are nevertheless differences, which may underlie divergence between human and mouse malarial species resulting from a split estimated to have occurred 70 million years ago. We emphasise here that formal demonstration that a given ePK gene is not essential for asexual schizogony requires analysis of clones from the populations displaying evidence for gene disruption or deletion; this has been achieved for the entire P. berghei kinome 37 but thus far only partially in P. falciparum.…”

The development of malaria parasites in the mosquito midgut

“…P . berghei parasites lacking the protein kinase PK7 or the cyclin G‐associated kinase GAK show severely reduced ookinete numbers (Tewari et al ., 2010). Further, deletion of the Shewanella‐like protein phosphatase (SHLP1), PPM2 or the kelch‐like domain‐containing phosphatase PPKL results in impaired P .…”

An evolutionary perspective on the kinome of malaria parasites

“…This revealed essential functions of a MAPK (Pbmap-2) and a CDPK (PbCDPK4) in male gametogenesis [29,46,47], of two NIMA-related kinases in meiosis in the mosquito vector [48,49], and of a CDK in asexual proliferation in erythrocytes [50]. This approach culminated in a kinome-wide study demonstrating that 23 P. berghei ePKs are redundant for asexual erythrocytic parasite development in mice and identifying phenotypes in sexual development for a number of these 23 ePKs [51]. A similar strategy in P. falciparum identified roles for an orphan kinase in proliferation rate linked to the number of progeny merozoites per schizont [36], for an eIF2a kinase in response to starvation stress, similar to GCN2, its closest homologue in yeast [52], and for CDPKs in motility during invasion [53] or egress of merozoites from the erythrocyte [54], to cite a few specific studies; more recently, a kinome-wide approach [19] identified 36 ePKs as refractory to gene disruption, and thus as likely crucial players in asexual proliferation in erythrocytes.…”