Michael F. Duffy

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.

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.

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.

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.

Broad analysis reveals a consistent pattern of var gene transcription in Plasmodium falciparum repeatedly selected for a defined adhesion phenotype.

Transcription of the majority of the members of the Plasmodium falciparum var multigene family were analysed in two isolates by a quantitative approach. Both of these isolates had been repeatedly selected for adhesion to chondroitin sulphate A (CSA) and one had also been selected for adhesion to hyaluronic acid (HA). These adhesion phenotypes are expressed by many parasites isolated from placentae and are associated with malaria disease in pregnancy. Increased transcription of the var gene var2csa, or its homologue IT4 var4, was associated with the CSA and HA adhesion phenotypes in all parasites suggesting that it was the dominant, if not the only, var gene that encoded adhesion to CSA in these allogeneic isolates. Some var genes were consistently transcribed at higher levels than others regardless of expressed adhesion phenotypes suggesting a transcriptional hierarchy. Unspliced or partial transcripts were detected for most var genes tested. These atypical var gene transcripts may have implications for the regulation of var gene transcription.

Transcribed var Genes Associated with Placental Malaria in Malawian Women

ABSTRACT Determining the diversity of PfEMP1 sequences expressed by Plasmodium falciparum-infected erythrocytes isolated from placentas is important for attempts to develop a pregnancy-specific malaria vaccine. The DBLγ and var2csa DBL3x domains of PfEMP1 molecules are believed to mediate placental sequestration of infected erythrocytes, so the sequences encoding these domains were amplified from the cDNAs of placental parasites by using degenerate oligonucleotides. The levels of specific var cDNAs were then determined by quantitative reverse transcription-PCR. Homologues of var2csa DBL3x were the predominant sequences amplified from the cDNAs of most placental but not most childrens parasites. There was 56% identity between all placental var2csa sequences. Many different DBLγ domains were amplified from the cDNAs of placental and childrens isolates. var2csa transcripts were the most abundant var transcripts of those tested in 11 of 12 placental isolates and 1 of 6 childrens isolates. Gravidity did not affect the levels of var2csa transcripts. We concluded that placental malaria is frequently associated with transcription of var2csa but that other var genes are also expressed, and parasites expressing high levels of var2csa are not restricted to pregnant women. The diversity of var2csa sequences may be important for understanding immunity and for the development of vaccines for malaria during pregnancy.

Transcription of multiple var genes by individual, trophozoite-stage Plasmodium falciparum cells expressing a chondroitin sulphate A binding phenotype

In this study, we detected multiple var gene transcripts within single, mature trophozoite‐infected red blood cells (iRBCs) bound to chondroitin sulphate A (CSA). Several of the var detected had previously been demonstrated to encode Plasmodium falciparum erythrocyte membrane protein‐1 (PfEMP‐1) variants with domains that mediated iRBC adhesion to receptors other than CSA. Parasites expressing the CSA‐adherent phenotype transcribed far more of one var than of all others, but this gene was different from the two other var previously purported to encode adhesion to CSA. Previous work suggesting that only single var are transcribed by mature trophozoites needs re‐examination in the light of these data from single, infected cells.

Multiple var gene transcripts are expressed in Plasmodium falciparum infected erythrocytes selected for adhesion

Adherence of Plasmodium falciparum-infected erythrocytes to the post-capillary endothelium is an important characteristic of malaria infection. The adhesion is mediated predominantly by P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1), a clonally variant protein expressed on the surface of infected red blood cells that appears to be a target of protective immunity. A multi-membered var gene family encodes PfEMP1 and switching expression of different var genes conveys different antigenic and adhesive properties to infected red blood cells. Knowledge about transcriptional control of phenotypic expression, or the mechanisms that allow multiple binding specificities, is very limited. Here, we describe a series of phenotypic selection experiments, which resulted in the expression of different PfEMP1 and the detection of multiple full-length var gene transcripts in the mature trophozoite stage. However, a dominant form of PfEMP1 appeared to be expressed, which suggested that most var transcripts do not lead to a surface expressed PfEMP1 molecule. Parasites bound to specific receptors still expressed multiple full-length var genes and mature trophozoites selected for increased adhesion to a specific receptor retained the ability to bind to multiple receptors. Our findings suggest that a defined adhesive phenotype can be associated with expression of multiple var genes.

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.

Evaluation of the Antigenic Diversity of Placenta-Binding Plasmodium falciparum Variants and the Antibody Repertoire among Pregnant Women

ABSTRACT Pregnant women are infected by specific variants of Plasmodium falciparum that adhere and accumulate in the placenta. Using serological and molecular approaches, we assessed the global antigenic diversity of surface antigens expressed by placenta-binding isolates to better understand immunity to malaria in pregnancy and evolution of polymorphisms and to inform vaccine development. We found that placenta-binding isolates originating from all major regions where malaria occurs were commonly recognized by antibodies in different populations of pregnant women. There was substantial antigenic overlap and sharing of epitopes between isolates, including isolates from distant geographic locations, suggesting that there are limitations to antigenic diversity; however, differences between populations and isolates were also seen. Many women had cross-reactive antibodies and/or a broad repertoire of antibodies to different isolates. Studying VAR2CSA as the major antigen expressed by placenta-binding isolates, we identified antibody epitopes encoded by variable sequence blocks in the DBL3 domain. Analysis of global var2csa DBL3 sequences demonstrated that there was extensive sharing of variable blocks between Africa, Asia, Papua New Guinea, and Latin America, which likely contributes to the high level of antigenic overlap between different isolates. However, there was also evidence of geographic clustering of sequences and differences in VAR2CSA sequences between populations. The results indicate that there is limited antigenic diversity in placenta-binding isolates and may explain why immunity to malaria in pregnancy can be achieved after exposure during one pregnancy. Inclusion of a limited number of variants in a candidate vaccine may be sufficient for broad population coverage, but geographic considerations may also have to be included in vaccine design.