Artur Scherf

Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum.

Members of the Plasmodium falciparum var gene family encode clonally variant adhesins, which play an important role in the pathogenicity of tropical malaria. Here we employ a selective panning protocol to generate isogenic P.falciparum populations with defined adhesive phenotypes for CD36, ICAM‐1 and CSA, expressing single and distinct var gene variants. This technique has established the framework for examining var gene expression, its regulation and switching. It was found that var gene switching occurs in situ. Ubiquitous transcription of all var gene variants appears to occur in early ring stages. However, var gene expression is tightly regulated in trophozoites and is exerted through a silencing mechanism. Transcriptional control is mutually exclusive in parasites that express defined adhesive phenotypes. In situ var gene switching is apparently mediated at the level of transcriptional initiation, as demonstrated by nuclear run‐on analyses. Our results suggest that an epigenetic mechanism(s) is involved in var gene regulation.

Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum.

Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.

Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites.

Malaria parasites use antigenic variation to avoid immune clearance and increase the duration of infection in the human host. Variation at the surface of P. falciparum-infected erythrocytes is mediated by the differential control of a family of surface antigens encoded by var genes. Switching of var gene expression occurs in situ, mostly from telomere-associated loci, without detectable DNA alterations, suggesting that it is controlled by chromatin structure. We have identified chromatin modifications at telomeres that spread far into telomere-proximal regions, including var gene loci (>50 kb). One type of modification is mediated by a protein homologous to yeast Sir2 called PfSir2, which forms a chromosomal gradient of heterochromatin structure and histone hypoacetylation. Upon activation of a specific telomere-associated var gene, PfSir2 is removed from the promoter region and acetylation of histone occurs. Our data demonstrate that mutually exclusive transcription of var genes is linked to the dynamic remodeling of chromatin.

Heterochromatin silencing and locus repositioning linked to regulation of virulence genes in Plasmodium falciparum.

The malaria parasite Plasmodium falciparum undergoes antigenic variation to evade host immune responses through switching expression of variant surface proteins encoded by the var gene family. We demonstrate that both a subtelomeric transgene and var genes are subject to reversible gene silencing. Var gene silencing involves the SIR complex as gene disruption of PfSIR2 results in activation of this gene family. We also demonstrate that perinuclear gene activation involves chromatin alterations and repositioning into a location that may be permissive for transcription. Together, this implies that locus repositioning and heterochromatic silencing play important roles in the epigenetic regulation of virulence genes in P. falciparum.

Antigenic Variation in Plasmodium falciparum

The persistence of the human malaria parasite Plasmodium falciparum during blood stage proliferation in its host depends on the successive expression of variant molecules at the surface of infected erythrocytes. This variation is mediated by the differential control of a family of surface molecules termed PfEMP1 encoded by approximately 60 var genes. Each individual parasite expresses a single var gene at a time, maintaining all other members of the family in a transcriptionally silent state. PfEMP1/var enables parasitized erythrocytes to adhere within the microvasculature, resulting in severe disease. This review highlights key regulatory mechanisms thought to be critical for monoallelic expression of var genes. Antigenic variation is orchestrated by epigenetic factors including monoallelic var transcription at separate spatial domains at the nuclear periphery, differential histone marks on otherwise identical var genes, and var silencing mediated by telomeric heterochromatin. In addition, controversies surrounding var genetic elements in antigenic variation are discussed.

Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system

Genome manipulation in the malaria parasite Plasmodium falciparum remains largely intractable and improved genomic tools are needed to further understand pathogenesis and drug resistance. We demonstrated the CRISPR-Cas9 system for use in P. falciparum by disrupting chromosomal loci and generating marker-free, single-nucleotide substitutions with high efficiency. Additionally, an artemisinin-resistant strain was generated by introducing a previously implicated polymorphism, thus illustrating the value of efficient genome editing in malaria research.

Molecules on the surface of the Plasmodium falciparum infected erythrocyte and their role in malaria pathogenesis and immune evasion.

The surface of the erythrocyte undergoes a number of modifications during infection by Plasmodium falciparum. These modifications are critical for pathogenesis of severe disease and the acquisition of host immunity through their role in interactions between the host and the parasite and in antigenic variation. Our knowledge of the molecular basis for these processes has increased dramatically over the last few years, through a combination of genomic and biochemical studies. This review provides a summary of the molecules involved in cytoadherence and antigenic variation in P. falciparum.

Sir2 paralogues cooperate to regulate virulence genes and antigenic variation in Plasmodium falciparum.

Cytoadherance of Plasmodium falciparum-infected erythrocytes in the brain, organs and peripheral microvasculature is linked to morbidity and mortality associated with severe malaria. Parasite-derived P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) molecules displayed on the erythrocyte surface are responsible for cytoadherance and undergo antigenic variation in the course of an infection. Antigenic variation of PfEMP1 is achieved by in situ switching and mutually exclusive transcription of the var gene family, a process that is controlled by epigenetic mechanisms. Here we report characterisation of the P. falciparum silent information regulators A and B (PfSir2A and PfSir2B) and their involvement in mutual exclusion and silencing of the var gene repertoire. Analysis of P. falciparum parasites lacking either PfSir2A or PfSir2B shows that these NAD(+)-dependent histone deacetylases are required for silencing of different var gene subsets classified by their conserved promoter type. We also demonstrate that in the absence of either of these molecules mutually exclusive expression of var genes breaks down. We show that var gene silencing originates within the promoter and PfSir2 paralogues are involved in cis spreading of silenced chromatin into adjacent regions. Furthermore, parasites lacking PfSir2A but not PfSir2B have considerably longer telomeric repeats, demonstrating a role for this molecule in telomeric end protection. This work highlights the pivotal but distinct role for both PfSir2 paralogues in epigenetic silencing of P. falciparum virulence genes and the control of pathogenicity of malaria infection.

Genome-wide Analysis of Heterochromatin Associates Clonally Variant Gene Regulation with Perinuclear Repressive Centers in Malaria Parasites

Clonally variant gene families underlie phenotypic plasticity in Plasmodium falciparum, a process indispensable for survival of the pathogen in its human host. Differential transcription of one of these gene families in clonal parasite lineages has been associated with chromatin modifications. Here, we determine the genome-wide distribution in P. falciparum of a histone mark of heterochromatin, trimethylation of histone H3 lysine 9 (H3K9me3), using high-resolution ChIP-chip analysis. We show that H3K9me3 is specifically associated with clonally variant gene families, which are clustered on subtelomeric and some chromosome internal regions. High levels of H3K9me3 correlate with genes localized to the nuclear periphery, implying chromosome loop formation. Disruption of the histone deacetylase PfSir2 causes changes in H3K9me3 that are discontinuous along chromosomes and associated with disrupted monoallelic transcription. Our data point to the existence of perinuclear repressive centers associated with control of expression of malaria parasite genes involved in phenotypic variation and pathogenesis.

A single member of the Plasmodium falciparum var multigene family determines cytoadhesion to the placental receptor chondroitin sulphate A

In high‐transmission regions, protective clinical immunity to Plasmodium falciparum develops during the early years of life, limiting serious complications of malaria in young children. Pregnant women are an exception and are especially susceptible to severe P. falciparum infections resulting from the massive adhesion of parasitized erythrocytes to chondroitin sulphate A (CSA) present on placental syncytiotrophoblasts. Epidemiological studies strongly support the feasibility of an intervention strategy to protect pregnant women from disease. However, different parasite molecules have been associated with adhesion to CSA. In this work, we show that disruption of the var2csa gene of P. falciparum results in the inability of parasites to recover the CSA‐binding phenotype. This gene is a member of the var multigene family and was previously shown to be composed of domains that mediate binding to CSA. Our results show the central role of var2CSA in CSA adhesion and support var2CSA as a leading vaccine candidate aimed at protecting pregnant women and their fetuses.