Thomas Lavstsen

Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria

Cytoadhesion of infected red blood cells (iRBC) is mediated through parasite‐encoded, clonally variant surface antigens (VSA) and is a central process in the pathogenesis of Plasmodium falciparum malaria. Pregnancy‐associated malaria (PAM) has been linked to VSA‐mediated adhesion of iRBC to the glycosaminoglycan chondroitin sulphate A (CSA) in the placental intervillous space. Several studies have pointed to members of the PfEMP1 VSA family as mediators of CSA‐specific iRBC sequestration in the placenta. Here, we report marked upregulation of a single var gene in several P. falciparum parasite isolates after selection for adhesion to CSA in vitro. The gene belongs to a highly conserved and common var gene subfamily (var2csa). The var2csa genes are structurally distinct from all other var genes in the parasite genome in lacking both CIDR and DBL‐γ domains. These domains have previously been implicated in PfEMP1‐mediated adhesion to CD36 and CSA. We also show that var2csa was transcribed at higher levels in three placental parasite isolates compared with transcription in parasites from peripheral blood of two children with P. falciparum malaria. This var gene thus has the properties expected of a gene encoding the parasite adhesion molecule that initiates the pathology associated with PAM.

Evidence for the Involvement of VAR2CSA in Pregnancy-associated Malaria

In Plasmodium falciparum–endemic areas, pregnancy-associated malaria (PAM) is an important health problem. The condition is precipitated by accumulation of parasite-infected erythrocytes (IEs) in the placenta, and this process is mediated by parasite-encoded variant surface antigens (VSA) binding to chondroitin sulfate A (CSA). Parasites causing PAM express unique VSA types, VSAPAM, which can be serologically classified as sex specific and parity dependent. It is sex specific because men from malaria-endemic areas do not develop VSAPAM antibodies; it is parity dependent because women acquire anti-VSAPAM immunoglobulin (Ig) G as a function of parity. Previously, it was shown that transcription of var2csa is up-regulated in placental parasites and parasites selected for CSA binding. Here, we show the following: (a) that VAR2CSA is expressed on the surface of CSA-selected IEs; (b) that VAR2CSA is recognized by endemic plasma in a sex-specific and parity-dependent manner; (c) that high anti-VAR2CSA IgG levels can be found in pregnant women from both West and East Africa; and (d) that women with high plasma levels of anti-VAR2CSA IgG give birth to markedly heavier babies and have a much lower risk of delivering low birth weight children than women with low levels.

Plasmodium falciparum Associated with Severe Childhood Malaria Preferentially Expresses PfEMP1 Encoded by Group A var Genes

Parasite-encoded variant surface antigens (VSAs) like the var gene–encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in nonimmune patients tend to express a restricted subset of VSA (VSASM) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSAUM). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSAUM to in vitro–selected sublines expressing VSASM to identify PfEMP1 responsible for the VSASM phenotype. Expression of VSASM was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var genes, such as PFD1235w/MAL7P1.1, appear to be involved in the pathogenesis of severe disease and are thus attractive candidates for a vaccine against life-threatening P. falciparum malaria.

Severe malaria is associated with parasite binding to endothelial protein C receptor

Sequestration of Plasmodium falciparum-infected erythrocytes in host blood vessels is a key triggering event in the pathogenesis of severe childhood malaria, which is responsible for about one million deaths every year. Sequestration is mediated by specific interactions between members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins was unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology and the development of new malaria interventions.

Plasmodium falciparum Erythrocyte Membrane Protein 1 Diversity in Seven Genomes – Divide and Conquer

The var gene encoded hyper-variable Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates cytoadhesion of infected erythrocytes to human endothelium. Antibodies blocking cytoadhesion are important mediators of malaria immunity acquired by endemic populations. The development of a PfEMP1 based vaccine mimicking natural acquired immunity depends on a thorough understanding of the evolved PfEMP1 diversity, balancing antigenic variation against conserved receptor binding affinities. This study redefines and reclassifies the domains of PfEMP1 from seven genomes. Analysis of domains in 399 different PfEMP1 sequences allowed identification of several novel domain classes, and a high degree of PfEMP1 domain compositional order, including conserved domain cassettes not always associated with the established group A–E division of PfEMP1. A novel iterative homology block (HB) detection method was applied, allowing identification of 628 conserved minimal PfEMP1 building blocks, describing on average 83% of a PfEMP1 sequence. Using the HBs, similarities between domain classes were determined, and Duffy binding-like (DBL) domain subclasses were found in many cases to be hybrids of major domain classes. Related to this, a recombination hotspot was uncovered between DBL subdomains S2 and S3. The VarDom server is introduced, from which information on domain classes and homology blocks can be retrieved, and new sequences can be classified. Several conserved sequence elements were found, including: (1) residues conserved in all DBL domains predicted to interact and hold together the three DBL subdomains, (2) potential integrin binding sites in DBLα domains, (3) an acylation motif conserved in group A var genes suggesting N-terminal N-myristoylation, (4) PfEMP1 inter-domain regions proposed to be elastic disordered structures, and (5) several conserved predicted phosphorylation sites. Ideally, this comprehensive categorization of PfEMP1 will provide a platform for future studies on var/PfEMP1 expression and function.

Differential Expression of var Gene Groups Is Associated with Morbidity Caused by Plasmodium falciparum Infection in Tanzanian Children

ABSTRACT The var gene family of Plasmodium falciparum encodes the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is considered an important pathogenicity factor in P. falciparum infection because it mediates cytoadherence to host cell endothelial receptors. var genes can be grouped into three major groups, A, B, and C, and the conserved var genes, var1-4, according to sequence similarities in coding and noncoding upstream regions. Using real-time quantitative PCR in a study conducted in Tanzania, the var transcript abundances of the different var gene groups were compared among patients with severe, uncomplicated, and asymptomatic malaria. Transcripts of var group A and B genes were more abundant in patients with severe malaria than in patients with uncomplicated malaria. In general, the transcript abundances of var group A and B genes were higher for children with clinical malaria than for children with asymptomatic infections. The var group C and var1-like transcript abundances were similar between the three sample groups. A transcript abundance pattern similar to that for var group A was observed for var2csa and var3-like genes. These results suggest that substantial and systematic differences in var gene expression exist between different clinical presentations.

Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children

The clinical outcome of Plasmodium falciparum infections ranges from asymptomatic parasitemia to severe malaria syndromes associated with high mortality. The virulence of P. falciparum infections is associated with the type of P. falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the surface of infected erythrocytes to anchor these to the vascular lining. Although var2csa, the var gene encoding the PfEMP1 associated with placental malaria, was discovered in 2003, the identification of the var/PfEMP1 variants associated with severe malaria in children has remained elusive. To identify var/PfEMP1 variants associated with severe disease outcome, we compared var transcript levels in parasites from 88 children with severe malaria and 40 children admitted to the hospital with uncomplicated malaria. Transcript analysis was performed by RT-quantitative PCR using a set of 42 primer pairs amplifying var subtype-specific loci covering most var/PfEMP1 subtypes. In addition, we characterized the near-full-length sequence of the most prominently expressed var genes in three patients diagnosed with severe anemia and/or cerebral malaria. The combined analysis showed that severe malaria syndromes, including severe anemia and cerebral malaria, are associated with high transcript levels of PfEMP1 domain cassette 8-encoding var genes. Transcript levels of group A var genes, including genes encoding domain cassette 13, were also significantly higher in patients with severe syndromes compared with those with uncomplicated malaria. This study specifies the var/PfEMP1 types expressed in severe malaria in children, and thereby provides unique targets for future efforts to prevent and treat severe malaria infections.

PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum

The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of P. falciparum-infected red blood cells, is a critical virulence factor for malaria. Each parasite has 60 antigenically distinct var genes that each code for a different PfEMP1 protein. During infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune-evasion mechanism to avoid the host antibody response. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown. Here we show that knocking out the P. falciparum variant-silencing SET gene (here termed PfSETvs), which encodes an orthologue of Drosophila melanogaster ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on var genes, results in the transcription of virtually all var genes in the single parasite nuclei and their expression as proteins on the surface of individual infected red blood cells. PfSETvs-dependent H3K36me3 is present along the entire gene body, including the transcription start site, to silence var genes. With low occupancy of PfSETvs at both the transcription start site of var genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long noncoding RNA. These results uncover a previously unknown role of PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which orthologues of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1 proteins may also be applied to the development of a malaria vaccine.

Expression of Plasmodium falciparum erythrocyte membrane protein 1 in experimentally infected humans

BackgroundParasites causing severe malaria in non-immune patients express a restricted subset of variant surface antigens (VSA), which are better recognized by immune sera than VSA expressed during non-severe disease in semi-immune individuals. The most prominent VSA are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which is expressed on the surface of infected erythrocytes where it mediates binding to endothelial receptors. Thus, severe malaria may be caused by parasites expressing PfEMP1 variants that afford parasites optimal sequestration in immunologically naïve individuals and high effective multiplication rates.Methodsvar gene transcription was analysed using real time PCR and PfEMP1 expression by western blots as well as immune plasma recognition of parasite cultures established from non-immune volunteers shortly after infection with NF54 sporozoites.ResultsIn cultures representing the first generation of parasites after hepatic release, all var genes were transcribed, but GroupA var genes were transcribed at the lowest levels. In cultures established from second or third generation blood stage parasites of volunteers with high in vivo parasite multiplication rates, the var gene transcription pattern differed markedly from the transcription pattern of the cultures representing first generation parasites. This indicated that parasites expressing specific var genes, mainly belonging to group A and B, had expanded more effectively in vivo compared to parasites expressing other var genes. The differential expression of PfEMP1 was confirmed at the protein level by immunoblot analysis. In addition, serological typing showed that immune sera more often recognized second and third generation parasites than first generation parasites.ConclusionIn conclusion, the results presented here support the hypothesis that parasites causing severe malaria express a subset of PfEMP1, which bestows high parasite growth rates in individuals with limited pre-existing immunity.

Malaria's deadly grip: cytoadhesion of Plasmodium falciparum-infected erythrocytes.

Cytoadhesion of Plasmodium falciparum‐infected erythrocytes to host microvasculature is a key virulence determinant. Parasite binding is mediated by a large family of clonally variant adhesion proteins, termed P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by var genes and expressed at the infected erythrocyte surface. Although PfEMP1 proteins have extensively diverged under opposing selection pressure to maintain ligand binding while avoiding antibody‐mediated detection, recent work has revealed they can be classified into different groups based on chromosome location and domain composition. This grouping reflects functional specialization of PfEMP1 proteins for different human host and microvascular binding niches and appears to be maintained by gene recombination hierarchies. Inone extreme, a specific PfEMP1 variant is associated with placental binding and malaria during pregnancy, while other PfEMP1 subtypes appear to be specialized for infection of malaria naïve hosts. Here, we discuss recent findings on the origins and evolution of the var gene family, the structure–function of PfEMP1 proteins, and a distinct subset of PfEMP1 variants that have been associated with severe childhood malaria.