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The malaria parasite Plasmodium falciparum has evolved an unusual genome structure. The majority of the genome is relatively stable, with mutation rates similar to most eukaryotic species. However, some regions are very unstable with high recombination rates, driving the generation of new immune evasion-associated var genes. The molecular factors controlling the inconsistent stability of this genome are not known. Here we studied the roles of the two putative RecQ helicases in P. falciparum, PfBLM and PfWRN. When PfWRN was knocked down, recombination rates increased four-fold, generating chromosomal abnormalities, a high rate of chimeric var genes and many microindels, particularly in known ‘fragile sites’. This is the first identification of a gene involved in suppressing recombination and maintaining genome stability in Plasmodium. By contrast, no change in mutation rate appeared when the second RecQ helicase, PfBLM, was mutated. At the transcriptional level, however, both helicases evidently modulate the transcription of large cohorts of genes, with several hundred genes—including a large proportion of vars—showing deregulated expression in each RecQ mutant. Aberrant processing of stalled replication forks is a possible mechanism underlying elevated mutation rates and this was assessed by measuring DNA replication dynamics in the RecQ mutant lines. Replication forks moved slowly and stalled at elevated rates in both mutants, confirming that RecQ helicases are required for efficient DNA replication. Overall, this work identifies the Plasmodium RecQ helicases as major players in DNA replication, antigenic diversification and genome stability in the most lethal human malaria parasite, with important implications for genome evolution in this pathogen., Author summary Human malaria is caused by Plasmodium parasites, with most of the mortality (almost half a million deaths each year) being caused by one species, Plasmodium falciparum. This parasite has an unusual genome: it is exceptionally biased towards A and T nucleotides rather than G and C, and it contains specific areas rich in hypervariable virulence-associated genes which evolve very rapidly to produce new variants. This evolution is probably vital for the parasite to evade the human immune system and maintain chronic infections, but how it is controlled at a molecular level remains unknown. We have identified a helicase in the parasite with a huge influence on genome stability and the rate of genome evolution. It appears to function by unwinding various unusual DNA structures, and if this fails then the genome becomes unstable. In addition, the transcription of many genes whose DNA tends to form secondary structures is affected, and DNA replication is impeded. If this helicase was expressed variably in different parasite strains infecting humans, it could influence the parasites’ ability to grow and replicate efficiently, and also, crucially, its ability to evolve and thus evade the human immune system.

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

Additional file 1: Supplementary Figures. Figure S1. DAFT-seq schematic. Figure S2. PolyA+ vs Random hexamer priming of mRNA in the DAFT-seq protocol. Figure S3. Method for calling UTRs from DAFT-seq coverage data. Figure S4. Comparison of Chappell et al. 5’ UTRs with the Caro et al. study. Figure S5. Comparison of TSSs with the Adjalley et al. study. Figure S6. 5UTR-seq schematic. Figure S7. Method for calling 5’ UTRs with 5UTR-seq data. Figure S8. Alternative 5’ UTR sets. Figure S9. Homopolymer tracts. Figure S10. PacBio reads show the same features of the transcriptome as DAFT-seq data. Figure S11. Occupancy of covalent histone marks, histone variants, heterochromatin protein 1 (HP1) and the BDP1 chromatin reader at 3D7 TSSs. Figure S12. Nucleosome occupancy at 3D7 TSSs. Figure S13. Distinct temporal changes of TSS usage in the IDC (KAHRP). Figure S14. The TSS frequency and shape landscape for 3D7. Figure S15. TSS motifs in introns and exons. Figure S16. Sequence features of sharp and broad promoters. Figure S17. ApiAP2 motif occurrences relative to predicted TSSs and annotated translation start sites in the 3D7 strain. Figure S18. ApiAP2 motifs around the KAHRP gene. Figure S19. Exitrons in PacBio reads. Figure S20. Alternative splicing in PacBio reads. Figure S21. TSS associated non-coding RNAs. Figure S22. Long non-coding RNAs. Figure S23. Alignment of DAFT-seq data to microarray data. Figure S24. ApiAP2 expression in the 3 P. falciparum strains. Figure S25. AP2-G expression in the 3 P. falciparum strains. Figure S26. Amplitude change distributions of all genes in the 3 P. falciparum strains. Figure S27. Coverage-based UTRs for the 3 P. falciparum strains. Figure S28. Analysis of expression patterns of adjacent gene pairs for the 3 P. falciparum strains. Figure S29. A spliced non-coding RNA opposite GDV1.

The malaria parasite Plasmodium falciparum has evolved an unusual genome structure. The majority of the genome is relatively stable, with mutation rates similar to most eukaryotic species. However, some regions are very unstable with high recombination rates, driving the generation of new immune evasion-associated var genes. The molecular factors controlling the inconsistent stability of this genome are not known. Here we studied the roles of the two putative RecQ helicases in P. falciparum, PfBLM and PfWRN. When PfWRN was knocked down, recombination rates increased four-fold, generating chromosomal abnormalities, a high rate of chimeric var genes and many microindels, particularly in known 'fragile sites'. This is the first identification of a gene involved in suppressing recombination and maintaining genome stability in Plasmodium. By contrast, no change in mutation rate appeared when the second RecQ helicase, PfBLM, was mutated. At the transcriptional level, however, both helicases evidently modulate the transcription of large cohorts of genes, with several hundred genes-including a large proportion of vars-showing deregulated expression in each RecQ mutant. Aberrant processing of stalled replication forks is a possible mechanism underlying elevated mutation rates and this was assessed by measuring DNA replication dynamics in the RecQ mutant lines. Replication forks moved slowly and stalled at elevated rates in both mutants, confirming that RecQ helicases are required for efficient DNA replication. Overall, this work identifies the Plasmodium RecQ helicases as major players in DNA replication, antigenic diversification and genome stability in the most lethal human malaria parasite, with important implications for genome evolution in this pathogen.

Funder: Division of Intramural Research, National Institute of Allergy and Infectious Diseases; doi: http://dx.doi.org/10.13039/100006492, 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.

Background: Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-h blood stages of the major human malaria parasite Plasmodium falciparum have been described using cDNA microarrays and RNA-seq, but these assays have not always performed well within non-coding regions, where the AT-content is often 90–95%. Results: We developed a directional, amplification-free RNA-seq protocol (DAFT-seq) to reduce bias against AT-rich cDNA, which we have applied to three strains of P. falciparum (3D7, HB3 and IT). While strain-specific differences were detected, overall there is strong conservation between the transcriptional profiles. For the 3D7 reference strain, transcription was detected from 89% of the genome, with over 78% of the genome transcribed into mRNAs. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. These datasets allowed us to refine the 5′ and 3′ untranslated regions (UTRs), which can be variable, long (> 1000 nt), and often overlap those of adjacent transcripts. Conclusions: The approaches applied in this study allow a refined description of the transcriptional landscape of P. falciparum and demonstrate that very little of the densely packed P. falciparum genome is inactive or redundant. By capturing the 5′ and 3′ ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle that will be useful in guiding the definition of regulatory regions for use in future experimental gene expression studies.

Figure S3. Gene expression levels for eight genes newly detected as differentially expressed among clinical isolates (Table 1). Distributions of read counts (normalised to library size) for eight genes, showing data from each replicate culture preparation of each strain. (PDF 84â kb)

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

Additional file 1: Supplementary Figures. Figure S1. DAFT-seq schematic. Figure S2. PolyA+ vs Random hexamer priming of mRNA in the DAFT-seq protocol. Figure S3. Method for calling UTRs from DAFT-seq coverage data. Figure S4. Comparison of Chappell et al. 5’ UTRs with the Caro et al. study. Figure S5. Comparison of TSSs with the Adjalley et al. study. Figure S6. 5UTR-seq schematic. Figure S7. Method for calling 5’ UTRs with 5UTR-seq data. Figure S8. Alternative 5’ UTR sets. Figure S9. Homopolymer tracts. Figure S10. PacBio reads show the same features of the transcriptome as DAFT-seq data. Figure S11. Occupancy of covalent histone marks, histone variants, heterochromatin protein 1 (HP1) and the BDP1 chromatin reader at 3D7 TSSs. Figure S12. Nucleosome occupancy at 3D7 TSSs. Figure S13. Distinct temporal changes of TSS usage in the IDC (KAHRP). Figure S14. The TSS frequency and shape landscape for 3D7. Figure S15. TSS motifs in introns and exons. Figure S16. Sequence features of sharp and broad promoters. Figure S17. ApiAP2 motif occurrences relative to predicted TSSs and annotated translation start sites in the 3D7 strain. Figure S18. ApiAP2 motifs around the KAHRP gene. Figure S19. Exitrons in PacBio reads. Figure S20. Alternative splicing in PacBio reads. Figure S21. TSS associated non-coding RNAs. Figure S22. Long non-coding RNAs. Figure S23. Alignment of DAFT-seq data to microarray data. Figure S24. ApiAP2 expression in the 3 P. falciparum strains. Figure S25. AP2-G expression in the 3 P. falciparum strains. Figure S26. Amplitude change distributions of all genes in the 3 P. falciparum strains. Figure S27. Coverage-based UTRs for the 3 P. falciparum strains. Figure S28. Analysis of expression patterns of adjacent gene pairs for the 3 P. falciparum strains. Figure S29. A spliced non-coding RNA opposite GDV1.

BACKGROUND: Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-h blood stages of the major human malaria parasite Plasmodium falciparum have been described using cDNA microarrays and RNA-seq, but these assays have not always performed well within non-coding regions, where the AT-content is often 90-95%. RESULTS: We developed a directional, amplification-free RNA-seq protocol (DAFT-seq) to reduce bias against AT-rich cDNA, which we have applied to three strains of P. falciparum (3D7, HB3 and IT). While strain-specific differences were detected, overall there is strong conservation between the transcriptional profiles. For the 3D7 reference strain, transcription was detected from 89% of the genome, with over 78% of the genome transcribed into mRNAs. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. These datasets allowed us to refine the 5' and 3' untranslated regions (UTRs), which can be variable, long (> 1000 nt), and often overlap those of adjacent transcripts. CONCLUSIONS: The approaches applied in this study allow a refined description of the transcriptional landscape of P. falciparum and demonstrate that very little of the densely packed P. falciparum genome is inactive or redundant. By capturing the 5' and 3' ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle that will be useful in guiding the definition of regulatory regions for use in future experimental gene expression studies.

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

Figure S2. Normalised read counts for the ten most highly differentiated genes between cultured clinical isolates and laboratory-adapted clones. Individual sample replicates are plotted according to the time of overall peak transcriptome correlation with reference time course data (either 40 or 48 h). Replicates from clinical isolates are in red (those having peak correlation with 40 h are plotted), and replicates from laboratory isolates are in blue. (PDF 312 kb)

Figure S3. Differential expression of merozoite invasion-related genes among schizonts from different parasite cultures. Distributions of read counts (normalised to library size) for eight genes, for replicated laboratory-adapted and clinical isolate samples, showing data from each replicate culture preparation of each strain. (PDF 324â kb)

Figure S2. Normalised read counts for the ten most highly differentiated genes between cultured clinical isolates and laboratory-adapted clones. Individual sample replicates are plotted according to the time of overall peak transcriptome correlation with reference time course data (either 40 or 48 h). Replicates from clinical isolates are in red (those having peak correlation with 40 h are plotted), and replicates from laboratory isolates are in blue. (PDF 312 kb)

Figure S3. Gene expression levels for eight genes newly detected as differentially expressed among clinical isolates (Table 1). Distributions of read counts (normalised to library size) for eight genes, showing data from each replicate culture preparation of each strain. (PDF 84â kb)