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This work was funded by grants from the Wellcome Trust (WT098051 and 079643/Z/06/Z) and the Medical Research Council (G0501670) to OB, a Wellcome Trust project grant to DB (WT094752), a Wellcome Trust Grant (WT093228) to TKS, a Marie Curie Fellowship (PIEF-GA-2008-220180) to SS, and a Marie Curie Fellowship (PIEF-GA-2009-253899) and an EMBO Long Term Fellowship (ALTF 45-2009) to MBr. C2 was synthesised and kindly provided by Katy Kettleborough and colleagues at MRC Technology through an MRC grant to DB (G10000779). Many critical events in the Plasmodium life cycle rely on the controlled release of Ca2+ from intracellular stores to activate stage-specific Ca2+-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca2+ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca2+ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca2+ effectors, PKG emerges as a unifying factor to control multiple cellular Ca2+ signals essential for malaria parasite development and transmission. Publisher PDF

RNA-seq comparison of gene expression in paired E64-treated and untreated P. falciparum 3D7 schizont preparations. (PDF 365â kb)

RNA-seq comparison of gene expression in paired E64-treated and untreated P. falciparum 3D7 schizont preparations. (PDF 365â kb)

Masking of P. falciparum genome annotation file to exclude polymorphic regions of genes. (PDF 546â kb)

Masking of P. falciparum genome annotation file to exclude polymorphic regions of genes. (PDF 546â kb)

Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. We adapted an RNA-seq protocol "DAFT-seq" to sequence the transcriptome of four P. vivax field isolates that were cultured for a short period ex vivo before using a density gradient for schizont enrichment. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples. This extensive data was used to define thousands of 5' and 3' untranslated regions, some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. This dataset has also significantly increased the known amount of heterogeneity between P. vivax schizont transcriptomes from individual patients. The majority of genes found to be differentially expressed between the isolates lack Plasmodium falciparum homologs and are predicted to be involved in host-parasite interactions, with an enrichment in reticulocyte binding proteins, merozoite surface proteins and exported proteins with unknown function. An improved understanding of the diversity within P. vivax transcriptomes will be essential for the prioritisation of novel vaccine targets.