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 regulator's 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. The unicellular parasite Plasmodium falciparum is the cause of the most severe form of malaria and is responsible for 300 million infections and ∼2 million deaths a year. Infected erythrocytes clump and block capillaries in the peripheral circulation, the brain, and placenta and are a major contributor to the pathology of malaria. A parasite-derived protein displayed on the surface of the infected erythrocyte is responsible for erythrocyte clumping in capillaries. Although 60 subtelomeric var genes can encode different versions of this “sticky” capillary-binding protein, only one protein is expressed at a time, and switches in expression between these genes causes variation of this pathogenic molecule enabling the parasite to evade the immune system. Here we identify two chromatin-modifying proteins that cooperate to mediate silencing and mutual exclusive expression of var genes. These proteins are thus important virulence factors of the malaria-causing parasite.

Plasmodium falciparum is the major cause of malaria globally and is transmitted by mosquitoes. During parasitic development, P. falciparum–infected erythrocytes (P. falciparum–IEs) express multiple polymorphic proteins known as variant surface antigens (VSAs), including the P. falciparum erythrocyte membrane protein 1 (PfEMP1). VSA-specific antibodies are associated with protection from symptomatic and severe malaria. However, the importance of the different VSA targets of immunity to malaria remains unclear, which has impeded an understanding of malaria immunity and vaccine development. In this study, we developed assays using transgenic P. falciparum with modified PfEMP1 expression to quantify serum antibodies to VSAs among individuals exposed to malaria. We found that the majority of the human antibody response to the IE targets PfEMP1. Furthermore, our longitudinal studies showed that individuals with PfEMP1-specific antibodies had a significantly reduced risk of developing symptomatic malaria, whereas antibodies to other surface antigens were not associated with protective immunity. Using assays that measure antibody-mediated phagocytosis of IEs, an important mechanism in parasite clearance, we identified PfEMP1 as the major target of these functional antibodies. Taken together, these data demonstrate that PfEMP1 is a key target of humoral immunity. These findings advance our understanding of the targets and mediators of human immunity to malaria and have major implications for malaria vaccine development.

Histone modifications are important in regulating gene expression in eukaryotes. Of the numerous histone modifications which have been identified, acetylation is one of the best characterised and is generally associated with active genes. Histone acetylation can directly affect chromatin structure by neutralising charges on the histone tail, and can also function as a binding site for proteins which can directly or indirectly regulate transcription. Bromodomains specifically bind to acetylated lysine residues on histone tails, and bromodomain proteins play an important role in anchoring the complexes of which they are a part to acetylated chromatin. Bromodomain proteins are involved in a diverse range of functions, such as acetylating histones, remodeling chromatin, and recruiting other factors necessary for transcription. These proteins thus play a critical role in the regulation of transcription.

Increased tolerance of Plasmodium falciparum to front-line artemisinin antimalarials (ARTs) is associated with mutations in Kelch13 (K13), although the precise role of K13 remains unclear. Here, we show that K13 mutations result in decreased expression of this protein, while mislocalization of K13 mimics resistance-conferring mutations, pinpointing partial loss of function of K13 as the relevant molecular event. K13-GFP is associated with ∼170 nm diameter doughnut-shaped structures at the parasite periphery, consistent with the location and dimensions of cytostomes. Moreover, the hemoglobin-peptide profile of ring-stage parasites is reduced when K13 is mislocalized. We developed a pulse-SILAC approach to quantify protein turnover and observe less disruption to protein turnover following ART exposure when K13 is mislocalized. Our findings suggest that K13 regulates digestive vacuole biogenesis and the uptake/degradation of hemoglobin and that ART resistance is mediated by a decrease in heme-dependent drug activation, less proteotoxicity, and increased survival of parasite ring stages.