Regulation of Plasmodium falciparum Development by Calcium-dependent Protein Kinase 7 (PfCDPK7)

Kumar, Praveen; Tripathi, Anuj; Ranjan, Ravikant; Halbert, Jean; Gilberger, Tim‐Wolf; Doerig, Christian; Sharma, Pushkar

doi:10.1074/jbc.m114.561670

Cited by 39 publications

(61 citation statements)

References 28 publications

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“…demonstrated that Pf CDPK7 binds to PI (4, 5) P2 and controls P . falciparum development in host erythrocytes [76]. These results suggest that Et CDPK4 is related to the invasion and survival of E .…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella

Wang¹,

Huang²,

Dong³

et al. 2016

PLoS ONE

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Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. The basis of cell invasion is not completely understood, but some key molecules of host cell invasion have been discovered. This paper investigated the characteristics of calcium-dependent protein kinase 4 (EtCDPK4), a critical molecule in E. tenella invasion of host cells. A full-length EtCDPK4 cDNA was identified from E. tenella using rapid amplification of cDNA ends. EtCDPK4 had an open reading frame of 1803 bp encoding a protein of 600 amino acids. Quantitative real-time PCR and western blotting were used to explore differences in EtCDPK4 transcription and translation in four developmental stages of E. tenella. EtCDPK4 was expressed at higher levels in sporozoites, but translation was higher in second-generation merozoites. In vitro invasion inhibition assays explored whether EtCDPK4 was involved in invasion of DF-1 cells by E. tenella sporozoites. Polyclonal antibodies against recombinant EtCDPK4 (rEtCDPK4) inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored EtCDPK4 distribution during parasite development after E. tenella sporozoite invasion of DF-1 cells in vitro. The results showed that EtCDPK4 might be important in sporozoite invasion and development. To analyze EtCDPK4 functional domains according to the structural characteristics of EtCDPK4 and study the kinase activity of rEtCDPK4, an in vitro phosphorylation system was established. We verified that rEtCDPK4 was a protein kinase that was completely dependent on Ca2+ for enzyme activity. Specific inhibitors of rEtCDPK4 activity were screened by kinase activity in vitro. Some specific inhibitors were applied to assays of DF-1 cell invasion by E. tenella sporozoites to confirm that the inhibitors functioned in vitro. W-7, H-7, H-89, and myristoylated peptide inhibited DF-1 invasion by E. tenella sporozoites. The experimental results showed that EtCDPK4 may be involved in E. tenella invasion of chicken cecal epithelial cells.

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Section: Discussionmentioning

confidence: 99%

Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella

Wang¹,

Huang²,

Dong³

et al. 2016

PLoS ONE

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“…Conditional knockout of TgCDPK7 had no impact on microneme secretion, but led to aberrant cell division [101]. Similarly, knockout of PfCDPK7 stalled the early stages of parasite development during the asexual cycle [102]. The biochemical consequences of these divergent domain structures remain unknown.…”

Section: Ca2+ Signaling and Its Biological Downstream Effects In Amentioning

confidence: 99%

The calcium signaling toolkit of the Apicomplexan parasites Toxoplasma gondii and Plasmodium spp

2015

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Apicomplexan parasites have complex life cycles, frequently split between different hosts and reliant on rapid responses as the parasites react to changing environmental conditions. Calcium ion (Ca2+) signaling is consequently essential for the cellular and developmental changes that support apicomplexan parasitism. Apicomplexan genomes reveal a rich repertoire of genes involved in calcium signaling, although many of the genes responsible for observed physiological changes remain unknown. There is evidence, for example, for the presence of a nifedipine-sensitive calcium entry mechanism in Toxoplasma, but the molecular components involved in Ca2+ entry in both Toxoplasma and Plasmodium, have not been identified. The major calcium stores are the endoplasmic reticulum (ER), the acidocalcisomes, and the plant-like vacuole in Toxoplasma, or the food vacuole in Plasmodium spp. Pharmacological evidence suggests that Ca2+ release from intracellular stores may be mediated by inositol 1,4,5-trisphosphate (IP3) or cyclic ADP ribose (cADPR) although there is no molecular evidence for the presence of receptors for these second messengers in the parasites. Several Ca2+-ATPases are present in apicomplexans and a putative mitochondrial Ca2+/H+ exchanger has been identified. Apicomplexan genomes contain numerous genes encoding Ca2+-binding proteins, with the notable expansion of calcium-dependent protein kinases (CDPKs), whose study has revealed novel roles in gliding motility, microneme secretion, host cell invasion and egress, and parasite differentiation. Microneme secretion has also been shown to depend on the C2 domain containing protein DOC2 in both Plasmodium spp. and Toxoplasma, providing further evidence for the complex transduction of Ca2+ signals in these organisms. The characterization of these pathways could lead to the discovery of novel drug targets and to a better understanding of the role of Ca2+ in these parasites.

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“…One exception is CDPK7, which has an unusual domain structure of several incomplete EF hands in an extended N-terminal domain that also has a PK domain (9). Regulated suppression of expression of TgCDPK7 affects cell division in T. gondii (28) and early stages in asexual development in P. falciparum (29), although the precise steps affected by this kinase have not yet been defined.…”

Section: -Isoleucine [I]-lysine [K]-lysine [K]) Kinases In Plasmodiummentioning

confidence: 99%

Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

2016

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Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially in Toxoplasma gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division in T. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPRassociated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes in T. gondii.

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Regulation of Plasmodium falciparum Development by Calcium-dependent Protein Kinase 7 (PfCDPK7)

Cited by 39 publications

References 28 publications

Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella

Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella

The calcium signaling toolkit of the Apicomplexan parasites Toxoplasma gondii and Plasmodium spp

Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

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