Plasmodium falciparum FIKK Kinase Members Target Distinct Components of the Erythrocyte Membrane

BACKGROUND: Modulation of infected host cells by intracellular pathogens is a prerequisite for successful establishment of infection. In the human malaria parasite Plasmodium falciparum, potential candidates for erythrocyte remodelling include the apicomplexan-specific FIKK kinase family (20 members), several of which have been demonstrated to be transported into the erythrocyte cytoplasm via Maurer's clefts. METHODOLOGY: In the current work, we have knocked out two members of this gene family (Pf fikk7.1 and Pf fikk12), whose products are localized at the inner face of the erythrocyte membrane. Both mutant parasite lines were viable and erythrocytes infected with these parasites showed no detectable alteration in their ability to adhere in vitro to endothelial receptors such as chondroitin sulfate A and CD36. However, we observed sizeable decreases in the rigidity of infected erythrocytes in both knockout lines. Mutant parasites were further analyzed using a phospho-proteomic approach, which revealed distinct phosphorylation profiles in ghost preparations of infected erythrocytes. Knockout parasites showed a significant reduction in the level of phosphorylation of a protein of approximately 80 kDa for FIKK12-KO in trophozoite stage and a large protein of about 300 kDa for FIKK7.1-KO in schizont stage. CONCLUSIONS: Our results suggest that FIKK members phosphorylate different membrane skeleton proteins of the infected erythrocyte in a stage-specific manner, inducing alterations in the mechanical properties of the parasite-infected red blood cell. This suggests that these host cell modifications may contribute to the parasites' survival in the circulation of the human host

Discovery of a new predominant cytosine DNA modification that is linked to gene expression in malaria parasites

International audienceDNA cytosine modifications are key epigenetic regulators of cellular processes in mammalian cells, with their misregulation leading to varied disease states. In the human malaria parasite Plasmodium falciparum, a unicellular eukaryotic pathogen, little is known about the predominant cytosine modifications, cytosine methylation (5mC) and hydroxymethylation (5hmC). Here, we report the first identification of a hydroxymethylcytosine-like (5hmC-like) modification in P. falciparum asexual blood stages using a suite of biochemical methods. In contrast to mammalian cells, we report 5hmC-like levels in the P. falciparum genome of 0.2–0.4%, which are significantly higher than the methylated cytosine (mC) levels of 0.01–0.05%. Immunoprecipitation of hydroxymethylated DNA followed by next generation sequencing (hmeDIP-seq) revealed that 5hmC-like modifications are enriched in gene bodies with minimal dynamic changes during asexual development. Moreover, levels of the 5hmC-like base in gene bodies positively correlated to transcript levels, with more than 2000 genes stably marked with this modification throughout asexual development. Our work highlights the existence of a new predominant cytosine DNA modification pathway in P. falciparum and opens up exciting avenues for gene regulation research and the development of antimalarial

PfAlbas constitute a new eukaryotic DNA/RNA-binding protein family in malaria parasites

In Plasmodium falciparum, perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Here we identify a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2, a TARE6-associated histone deacetylase linked to the process of antigenic variation. The nuclear location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation

clag9 Is Not Essential for PfEMP1 Surface Expression in Non-Cytoadherent Plasmodium falciparum Parasites with a Chromosome 9 Deletion

BACKGROUND: The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1. METHODOLOGY/PRINCIPAL FINDINGS: Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36. CONCLUSIONS/SIGNIFICANCE: Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications

De Novo Genome Assembly of a Plasmodium falciparum NF54 Clone Using Single-Molecule Real-Time Sequencing

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CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum

International audiencePlasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes—the intron—in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression.IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes—the intron—in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression

Discovery of a novel and conserved Plasmodium falciparum exported protein that is important for adhesion of PfEMP1 at the surface of infected erythrocytes

International audiencePlasmodium falciparum virulence is linked to its ability to sequester in post-capillary venules in the human host. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant surface antigen implicated in this process. Complete loss of parasite adhesion is linked to a large subtelomeric deletion on chromosome 9 in a number of laboratory strains such as D10 and T9-96. Similar to the cytoadherent reference line FCR3, D10 strain expresses PfEMP1 on the surface of parasitized erythrocytes, however without any detectable cytoadhesion. To investigate which of the deleted subtelomeric genes may be implicated in parasite adhesion, we selected 12 genes for D10 complementation studies that are predicted to code for proteins exported to the red blood cell. We identified a novel single copy gene (PF3D7_0936500) restricted to P. falciparum that restores adhesion to CD36, termed here virulence-associated protein 1 (Pfvap1). Protein knockdown and gene knockout experiments confirmed a role of PfVAP1 in the adhesion process in FCR3 parasites. PfVAP1 is co-exported with PfEMP1 into the host cell via vesicle-like structures called Maurer's clefts. This study identifies a novel highly conserved parasite molecule that contributes to parasite virulence possibly by assisting PfEMP1 to establish functional adhesion at the host cell surface

Strand-specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts in Plasmodium falciparum.

International audienceBACKGROUND: Advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts (NATs) in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. Likewise, the existence of NATs has been observed in Plasmodium but our understanding towards their genome-wide distribution remains incomplete due to the limited depth and uncertainties in the level of strand specificity of previous datasets. RESULTS: To gain insights into the genome-wide distribution of NATs in P. falciparum, we performed RNA-ligation based strand-specific RNA sequencing at unprecedented depth. Our data indicate that 78.3% of the genome is transcribed during blood-stage development. Moreover, our analysis reveals significant levels of antisense transcription from at least 24% of protein-coding genes and that while expression levels of NATs change during the intraerythrocytic developmental cycle (IDC), they do not correlate with the corresponding mRNA levels. Interestingly, antisense transcription is not evenly distributed across coding regions (CDSs) but strongly clustered towards the 3'-end of CDSs. Furthermore, for a significant subset of NATs, transcript levels correlate with mRNA levels of neighboring genes.Finally, we were able to identify the polyadenylation sites (PASs) for a subset of NATs, demonstrating that at least some NATs are polyadenylated. We also mapped the PASs of 3443 coding genes, yielding an average 3' untranslated region length of 523 bp. CONCLUSIONS: Our strand-specific analysis of the P. falciparum transcriptome expands and strengthens the existing body of evidence that antisense transcription is a substantial phenomenon in P. falciparum. For a subset of neighboring genes we find that sense and antisense transcript levels are intricately linked while other NATs appear to be regulated independently of mRNA transcription. Our deep strand-specific dataset will provide a valuable resource for the precise determination of expression levels as it separates sense from antisense transcript levels, which we find to often significantly differ. In addition, the extensive novel data on 3' UTR length will allow others to perform searches for regulatory motifs in the UTRs and help understand post-translational regulation in P. falciparum

An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum

Abstract Variegated surface antigen expression is key to chronic infection and pathogenesis of the human malaria parasite Plasmodium falciparum. This protozoan parasite expresses distinct surface molecules that are encoded by clonally variant gene families such as var, rif and stevor. The molecular mechanisms governing activation of individual members remain ill-defined. To investigate the molecular events of the initial transcriptional activation process we focused on a member of the apicomplexan ApiAP2 transcription factor family predicted to bind to the 5′ upstream regions of the var gene family, AP2-exp (PF3D7_1466400). Viable AP2-exp mutant parasites rely on expressing no less than a short truncated protein including the N-terminal AP2 DNA-binding domain. RNA-seq analysis in mutant parasites revealed transcriptional changes in a subset of exported proteins encoded by clonally variant gene families. Upregulation of RIFINs and STEVORs was validated at the protein levels. In addition, morphological alterations were observed on the surface of the host cells infected by the mutants. This work points to a complex regulatory network of clonally variant gene families in which transcription of a subset of members is regulated by the same transcription factor. In addition, we highlight the importance of the non-DNA binding AP2 domain in functional gene regulation