Michaela Petter

Targets of antibodies against Plasmodium falciparum –infected erythrocytes in malaria immunity

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.

Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter

Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

Absence of Erythrocyte Sequestration and Lack of Multicopy Gene Family Expression in Plasmodium falciparum from a Splenectomized Malaria Patient

Background To avoid spleen-dependent killing mechanisms parasite-infected erythrocytes (IE) of Plasmodium falciparum malaria patients have the capacity to bind to endothelial receptors. This binding also known as sequestration, is mediated by parasite proteins, which are targeted to the erythrocyte surface. Candidate proteins are those encoded by P. falciparum multicopy gene families, such as var, rif, stevor or PfMC-2TM. However, a direct in vivo proof of IE sequestration and expression of multicopy gene families is still lacking. Here, we report on the analysis of IE from a black African immigrant, who received the diagnosis of a malignant lymphoproliferative disorder and subsequently underwent splenectomy. Three weeks after surgery, the patient experienced clinical falciparum malaria with high parasitemia and circulating developmental parasite stages usually sequestered to the vascular endothelium such as late trophozoites, schizonts or immature gametocytes. Methodology/Principal Findings Initially, when isolated from the patient, the infected erythrocytes were incapable to bind to various endothelial receptors in vitro. Moreover, the parasites failed to express the multicopy gene families var, A-type rif and stevor but expression of B-type rif and PfMC-2TM genes were detected. In the course of in vitro cultivation, the parasites started to express all investigated multicopy gene families and concomitantly developed the ability to adhere to endothelial receptors such as CD36 and ICAM-1, respectively. Conclusion/Significance This case strongly supports the hypothesis that parasite surface proteins such as PfEMP1, A-type RIFIN or STEVOR are involved in interactions of infected erythrocytes with endothelial receptors mediating sequestration of mature asexual and immature sexual stages of P. falciparum. In contrast, multicopy gene families coding for B-type RIFIN and PfMC-2TM proteins may not be involved in sequestration, as these genes were transcribed in infected but not sequestered erythrocytes.

Diverse Expression Patterns of Subgroups of the rif Multigene Family during Plasmodium falciparum Gametocytogenesis

Background The maturation of Plasmodium falciparum gametocytes in the human host takes several days, during which the parasites need to efficiently evade the host immune system. Like asexual stage parasites, immature gametocytes can sequester at various sites in the human body, and only mature sexual stages are found in the circulation. Although the fundamental mechanisms of gametocyte immune evasion are still largely unknown, candidate molecules that may be involved include variant antigens encoded by multigene families in the P. falciparum genome, such as the PfEMP1, STEVOR and RIFIN proteins. While expression of the former two families in sexual stages has been investigated earlier, we report here RIFIN expression during gametocytogenesis. Methodology/Principal Findings Variants of two previously characterized RIFIN subfamilies (A- and B-type RIFINs) were found to be synthesized in gametocytes. Immunofluorescence experiments showed A-type RIFINs to be accumulated in a crescent-shaped pattern of discrete punctate structures at the infected erythrocyte membrane, while members of the B-type family were associated with the parasite. Transcription analysis demonstrated the existence of diverse transcriptional regulation patterns during sexual differentiation and indicated variant-specific regulation of B-type RIFINs, in contrast to group-specific regulation for A-type RIFINs. Phylogenetic analysis of 5′-upstream regions showed that the rif–gene family falls into five defined clusters, designated rups (rif upstream) A1, A2, AB, B and C. In trophozoites and early gametocytes, rif variants of the rupsA2-type were preferentially expressed. Conclusions/Significance In this work we demonstrate the expression dynamics of the rif-gene family during sexual differentiation and present indications for subgroup specific regulation patterns. Therefore, our data provide a first foundation and point to new directions for future investigations of the potential role of RIFINs in gametocyte immune evasion.

The Role of Bromodomain Proteins in Regulating Gene Expression

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.

H2A.Z and H2B.Z double‐variant nucleosomes define intergenic regions and dynamically occupy var gene promoters in the malaria parasite Plasmodium falciparum

Histone variants are important components of eukaryotic chromatin and can alter chromatin structure to confer specialized functions. H2B variant histones are rare in nature but have evolved independently in the phyla Apicomplexa and Trypanasomatida. Here, we investigate the apicomplexan‐specific Plasmodium falciparum histone variant Pf H2B.Z and show that within nucleosomes Pf H2B.Z dimerizes with the H2A variant Pf H2A.Z and that Pf H2B.Z and Pf H2A.Z occupancy correlates in the subset of genes examined. These double‐variant nucleosomes also carry common markers of euchromatin like H3K4me3 and histone acetylation. Pf H2B.Z levels are elevated in intergenic regions across the genome, except in the var multigene family, where Pf H2A.Z/Pf H2B.Z double‐variant nucleosomes are only enriched in the promoter of the single active var copy and this enrichment is developmentally regulated. Importantly, this pattern seems to be specific for var genes and does not apply to other heterochromatic gene families involved in red blood cell invasion which are also subject to clonal expression. Thus, Pf H2A.Z/Pf H2B.Z double‐variant nucleosomes appear to have a highly specific function in the regulation of P. falciparum virulence.

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion

BackgroundPlasmodium falciparum STEVOR proteins, encoded by the multicopy stevor gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation.MethodsImmunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process.ResultsAntigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown.ConclusionThe localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with invasion-related events has been shown.

A Plasmodium Falciparum Bromodomain Protein Regulates Invasion Gene Expression

During red-blood-cell-stage infection of Plasmodium falciparum, the parasite undergoes repeated rounds of replication, egress, and invasion. Erythrocyte invasion involves specific interactions between host cell receptors and parasite ligands and coordinated expression of genes specific to this step of the life cycle. We show that a parasite-specific bromodomain protein, PfBDP1, binds to chromatin at transcriptional start sites of invasion-related genes and directly controls their expression. Conditional PfBDP1 knockdown causes a dramatic defect in parasite invasion and growth and results in transcriptional downregulation of multiple invasion-related genes at a time point critical for invasion. Conversely, PfBDP1 overexpression enhances expression of these same invasion-related genes. PfBDP1 binds to acetylated histone H3 and a second bromodomain protein, PfBDP2, suggesting a potential mechanism for gene recognition and control. Collectively, these findings show that PfBDP1 critically coordinates expression of invasion genes and indicate that targeting PfBDP1 could be an invaluable tool in malaria eradication.

The role of chromatin in Plasmodium gene expression

The malaria parasite Plasmodium falciparum dynamically regulates transcription of the majority of its genes during its intraerythrocytic developmental cycle. Chromatin is an important contributor to this tight regulation of gene expression. P. falciparum appears to utilize most of the mechanisms of chromatin creation and modification found in other eukaryotes, although it occasionally uses them in surprising ways. Much of the P. falciparum genome is maintained in a euchromatic state, potentially permissive for transcription and heterochromatin appears to have a specialized role limited to silencing islands of genes involved in redundant host–parasite interactions. P. falciparum histones share canonical modifications with other eukaryotes but also have unique modifications of unknown function including hyperacetylations of two alternative histones possibly involved in gene regulation. Much of our knowledge of chromatin regulation of gene expression in P. falciparum derives from the study of virulence genes that are subject to chromatin regulatory mechanisms ranging from histone modifications and nucleosomal occupancy to non‐protein‐coding RNAs and subnuclear architecture. These mechanisms will be discussed along with other characterized components of P. falciparum chromatin.

Epigenetic regulation of the Plasmodium falciparum genome

Recent research has highlighted some unique aspects of chromatin biology in the malaria parasite Plasmodium falciparum. During its erythrocytic lifecycle P. falciparum maintains its genome primarily as unstructured euchromatin. Indeed there is no clear role for chromatin-mediated silencing of the majority of the developmentally expressed genes in P. falciparum. However discontinuous stretches of heterochromatin are critical for variegated expression of contingency genes that mediate key pathogenic processes in malaria. These range from invasion of erythrocytes and antigenic variation to solute transport and growth adaptation in response to environmental changes. Despite lack of structure within euchromatin the nucleus maintains functional compartments that regulate expression of many genes at the nuclear periphery, particularly genes with clonally variant expression. The typical components of the chromatin regulatory machinery are present in P. falciparum; however, some of these appear to have evolved novel species-specific functions, e.g. the dynamic regulation of histone variants at virulence gene promoters. The parasite also appears to have repeatedly acquired chromatin regulatory proteins through lateral transfer from endosymbionts and from the host. P. falciparum chromatin regulators have been successfully targeted with multiple drugs in laboratory studies; hopefully their functional divergence from human counterparts will allow the development of parasite-specific inhibitors.