A var gene promoter controls allelic exclusion of virulence genes in Plasmodium falciparum malaria.

Mono-allelic expression of gene families is used by many organisms to mediate phenotypic variation of surface proteins. In the apicomplexan parasite Plasmodium falciparum, responsible for the severe form of malaria in humans, this is exemplified by antigenic variation of the highly polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1, encoded by the 60-member var gene family, represents a major virulence factor due to its central role in immune evasion and intravascular parasite sequestration. Mutually exclusive expression of PfEMP1 is controlled by epigenetic mechanisms involving chromatin modification and perinuclear var locus repositioning. Here we show that a var promoter mediates the nucleation and spreading of stably inherited silenced chromatin. Transcriptional activation of this promoter occurs at the nuclear periphery in association with chromosome-end clusters. Additionally, the var promoter sequence is sufficient to infiltrate a transgene into the allelic exclusion programme of var gene expression, as transcriptional activation of this transgene results in silencing of endogenous var gene transcription. These results show that a var promoter is sufficient for epigenetic silencing and mono-allelic transcription of this virulence gene family, and are fundamental for our understanding of antigenic variation in P. falciparum. Furthermore, the PfEMP1 knockdown parasites obtained in this study will be important tools to increase our understanding of P. falciparum-mediated virulence and immune evasion.

VAR2CSA is the principal ligand for chondroitin sulfate A in two allogeneic isolates of Plasmodium falciparum.

Malaria during pregnancy causes serious disease that is associated with sequestration in the placenta of Plasmodium falciparum infected erythrocytes that adhere to several host receptors, including chondroitin sulfate A (CSA). The principal CSA binding ligand associated with placental sequestration is the P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var2csa gene. We disrupted the var2csa gene in two allogeneic parasites and ablated CSA binding. However, in one parasite line we were able to re-select for adhesion to bovine trachea CSA associated with transcription of two var genes, var-CS2 and varP. Parasites transcribing parts of var-CS2 and varP were present in the placentae of some infected women but the mutant parasites that transcribed var-CS2 and varP were recognized by sera from men and pregnant women independent of parity. This work raises the possibility that the PfEMP1 molecules encoded by var-CS2 and varP may be minor contributors to placental malaria but also confirms the importance of the immunodominant, conserved var2csa PfEMP1s in pregnancy associated malaria and strengthens the case for var2csa as a pregnancy-specific malaria vaccine.

Alterations in local chromatin environment are involved in silencing and activation of subtelomeric var genes in Plasmodium falciparum.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, undergoes antigenic variation and plays an important role in chronic infection and severe malaria. Only a single var gene is transcribed per parasite, and epigenetic control mechanisms are fundamental in this strategy of mutually exclusive transcription. We show that subtelomeric upsB var gene promoters carried on episomes are silenced by default, and that promoter activation is sufficient to silence all other family members. However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation. Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression. Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family.

Evidence that Plasmodium falciparum chromosome end clusters are cross-linked by protein and are the sites of both virulence gene silencing and activation.

The malaria parasite Plasmodium falciparum undergoes antigenic variation through allelic exclusion and variant expression of surface proteins encoded by the var gene family. Regulation of var genes is under epigenetic control and involves reversible silencing and activation that requires the physical repositioning of a var locus into a transcriptionally permissive zone of the nuclear periphery. P. falciparum chromosome ends appear to aggregate into large perinuclear clusters which house both subtelomeric and chromosome central var genes. In this study we further define the composition of telomeric clusters using fluorescent in situ hybridization, and provide evidence that chromosome end clusters are formed by cross-linking protein. In addition, we demonstrate that a subtelomeric reporter gene and a var gene remain within clusters regardless of their transcriptional status. Our findings support a model whereby a highly localized structure dedicated to the activation of a single var gene can be housed within a gene dense chromosome end cluster that is otherwise transcriptionally silent.

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.