Atypical Mitogen-Activated Protein Kinase Phosphatase Implicated in Regulating Transition from Pre-S-Phase Asexual Intraerythrocytic Development of Plasmodium falciparum

Abstract

Intraerythrocytic development of the human malaria parasite Plasmodium falciparum appears as a continuous flow through growth and proliferation. To develop a greater understanding of the critical regulatory events, we utilized piggyBac insertional mutagenesis to randomly disrupt genes. Screening a collection of piggyBac mutants for slow growth, we isolated the attenuated parasite C9, which carried a single insertion disrupting the open reading frame (ORF) of PF3D7_1305500. This gene encodes a protein structurally similar to a mitogen-activated protein kinase (MAPK) phosphatase, except for two notable characteristics that alter the signature motif of the dual-specificity phosphatase domain, suggesting that it may be a low-activity phosphatase or pseudophosphatase. C9 parasites demonstrated a significantly lower growth rate with delayed entry into the S/M phase of the cell cycle, which follows the stage of maximum PF3D7_1305500 expression in intact parasites. Genetic complementation with the full-length PF3D7_1305500 rescued the wild-type phenotype of C9, validating the importance of the putative protein phosphatase PF3D7_1305500 as a regulator of pre-S-phase cell cycle progression in P. falciparum. Malaria caused by Plasmodium falciparum infection is the major type of severe malaria and results in hundreds of thousands of deaths each year and hundreds of millions of clinical illnesses (1, 2). Within the blood of infected individuals, asexual forms of the intraerythrocytic parasite grow rapidly through successive cycles of growth and proliferation. In each asexual generation, active entry into erythrocytes is followed by a growth phase culminating in dynamic release of erythrocyte-invading merozoites. The Plasmodium mitotic cycle is not fully understood and differs from the well-defined models established in yeast and mammalian cells (3). Observing the exact mitotic transitions during the cycle has been difficult due to variations in processes such as chromatid segregation, nuclear division, and spindle formation (4). However, this pattern of development has been observed in other members of the Apicomplexa, such as Toxoplasma gondii and Eimeria tenella, demonstrating the importance and conservation of this process across the phylum (5, 6).In eukaryotic systems, phosphorylation cascades are critical to cellular development and depend on the coordinated activity of kinases, which are in turn modulated by the activity of phosphatases. There is growing evidence that kinases are critical regulators of cell growth and development in Plasmodium species (7-9). Plasmodium kinases, for example, have been found to be involved with the initial invasion of host cells in addition to egress and differentiation (6, 10). Additional studies have identified kinases in phosphorylation cascades of the gametocyte-ookinete-oocyst transition in the mosquito midgut (11)(12)(13)(14)(15)(16)(17). In contrast, few studies have described the phosphatases involved in these processes, which would understandably function with kinases to coregul...

Phenotypic Screens Identify Parasite Genetic Factors Associated with Malarial Fever Response in Plasmodium falciparum piggyBac Mutants

“…To establish whether the heat shock phenotype was directly due to insertion in the PfMKP1 coding sequence, the PB-2 mutant was complemented with an episomal copy of the PfMKP1 gene with its native 5′ UTR and a 3′ three-hemagglutinin tag, followed by an HRP3 3′ UTR. Reverse transcription-PCR previously confirmed that the complemented line reintroduced the expression of PfMKP1 RNA (14), and PfMKP1 protein expression was confirmed in wild-type NF54 and the complemented mutant by Western blotting with a custom monoclonal antibody (anti-PfMKP1), while the PB-2 mutant showed no expression (Fig. 3A).…”

“…The complemented piggyBac mutant was made previously (14). The complemented piggyBac mutant was maintained in medium with 2 µg/ml blasticidin D to maintain the episome.…”

Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

“…3B). To rule out the possibility of additional, unknown piggyBac insertions in the genome, we aligned one file of the paired reads to the transposon sequence and the pair file to the genome (and vice versa) to see where each transposon-aligning paired read mapped (35). Paired ends were found to align only to the Maf1 locus or to regions of the genome used as regulatory sequences within the transposon itself (such as the calmodulin promoter or the HrpII 3′ untranscribed region [3′UTR]) (Table S1).…”

“…Paired ends were found to align only to the Maf1 locus or to regions of the genome used as regulatory sequences within the transposon itself (such as the calmodulin promoter or the HrpII 3′ untranscribed region [3′UTR]) (Table S1). Additionally, we analyzed the PB-11 genome for single nucleotide polymorphisms (SNPs) and indels within all annotated open reading frames in the genome but found none of significance compared to the parental line or a sister piggyBac insertion mutant sequenced previously (35) (Table S2). The latter results support the notion that any phenotype observed in the PB-11 mutant line is a consequence of the transposon insertion.…”

“…The analysis of the sequencing data largely followed the protocol described by Balu et al (35), who had previously sequenced the NF54 clone that was used as the parental line in the transposon screen which produced the PB-11 mutant (34). Two sets of paired-end reads from the parental NF54 clone (ERS038926 and ERS184445), produced by two different sequencing platforms, were obtained from the European Nucleotide Archive.…”