The role of Plasmodium falciparum var genes in malaria in pregnancy

Sequestration of Plasmodium falciparum-infected erythrocytes in the placenta is responsible for many of the harmful effects of malaria during pregnancy. Sequestration occurs as a result of parasite adhesion molecules expressed on the surface of infected erythrocytes binding to host receptors in the placenta such as chondroitin sulphate A (CSA). Identification of the parasite ligand(s) responsible for placental adhesion could lead to the development of a vaccine to induce antibodies to prevent placental sequestration. Such a vaccine would reduce the maternal anaemia and infant deaths that are associated with malaria in pregnancy. Current research indicates that the parasite ligands mediating placental adhesion may be members of the P. falciparum variant surface antigen family PfEMP1, encoded by var genes. Two relatively well-conserved subfamilies of var genes have been implicated in placental adhesion, however, their role remains controversial. This review examines the evidence for and against the involvement of var genes in placental adhesion, and considers whether the most appropriate vaccine candidates have yet been identified.     

Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes

The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum. We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family.     

Plasmodium falciparum heterochromatin protein 1 binds to tri-methylated histone 3 lysine 9 and is linked to mutually exclusive expression of var genes

Increasing experimental evidence shows a prominent role of histone modifications in the coordinated control of gene expression in the human malaria parasite Plasmodium falciparum. The search for the histone-mark-reading machinery that translates histone modifications into biological processes, such as formation of heterochromatin and antigenic variation is of foremost importance. In this work, we identified the first member of a histone modification specific recognition protein, an orthologue of heterochromatin protein 1 (PfHP1). Analysis of the PfHP1 amino-acid sequence revealed the presence of the two characteristic HP1 domains: a chromodomain (CD) and a chromo shadow domain (CSD). Recombinant CD binds to di- and tri-methylated lysine 9 from histone H3, but not to unmodified or methylated histone H3 in lysine 4. PfHP1 is able to interact with itself to form dimers, underlying its potential role in aggregating nucleosomes to form heterochromatin. Antibodies raised against PfHP1 detect this molecule in foci at the perinuclear region. ChIP analysis using anti-PfHP1 shows that this protein is linked to heterochromatin of subtelomeric non-coding repeat regions and monoallelic expression of the major virulence var gene family. This is the first report implicating an HP1 protein in the control of antigenic variation of a protozoan parasite.     

Time course of in vitro maturation of intra-erythrocytic malaria parasite: a comparison between Plasmodium falciparum and Plasmodium knowlesi

The schizont maturation assay for in vitro drug sensitivity tests has been a standard method employed in the global baseline assessment and monitoring of drug response in Plasmodium falciparum. This test is limited in its application to synchronous plasmodial infections because it evaluates the effect of drug on the maturation of parasite especially from ring to schizont stage and therefore synchronized P. falciparum cultures are required. On the other hand, P. knowlesi, a simian malaria parasite has a unique 24-h periodicity and maintains high natural synchronicity in monkeys. The present report presents the results of a comparative study on the course of in vitro maturation of sorbitol synchronized P. falciparum and naturally synchronous P. knowlesi. Ring stage parasites were incubated in RPMI medium supplemented with 10-15% pooled homologous serum in flat-bottomed 96-well micro plates using a candle jar at 37 degrees C. The results suggest that the ideal time for harvesting the micro-assay plates for in vitro drug sensitivity test for sorbitol-synchronized P. falciparum and naturally synchronous P. knowlesi are from 26 to 30 h and from 22 to 25 h, respectively. The advantages of using P. knowlesi in chemotherapeutic studies are discussed.     

Genetic diversity of expressed Plasmodium falciparum var genes from Tanzanian children with severe malaria

ABSTRACT: BACKGROUND: Severe malaria has been attributed to the expression of a restricted subset of the var multigene family, which encodes for Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 mediates cytoadherence and sequestration of infected erythrocytes into the post-capillary venules of the vital organs such as the brain, lung or placenta. Var genes are highly diverse and can be classified in three major groups (ups A, B and C) and two intermediate groups (B/A and B/C) based on the genomic location, gene orientation and upstream sequences. The genetic diversity of expressed var genes in relation to severity of disease in Tanzanian children was analysed. METHODS: Children with defined severe (SM) and asymptomatic malaria (AM) were recruited. Fulllength var mRNA was isolated and reversed transcribed into var cDNA. Subsequently, the DBL and N-terminal domains, and up-stream sequences were PCR amplified, cloned and sequenced. Sequences derived from SM and AM isolates were compared and analysed. RESULTS: The analysis confirmed that the var family is highly diverse in natural Plasmodium falciparum populations. Sequence diversity of amplified var DBL-1alpha and upstream regions showed minimal overlap among isolates, implying that the var gene repertoire is vast and most probably indefinite in endemic areas. var DBL-1alpha sequences from AM isolates were more diverse with more singletons found (p<0.05) than those from SM infections. Furthermore, few var DBL-1alpha sequences from SM patients were rare and restricted suggesting that certain PfEMP1 variants might induce severe disease. CONCLUSIONS: The genetic sequence diversity of var genes of P. falciparum isolates from Tanzanian children is large and its relationship to disease severity has been studied. Observed differences suggest that different var genes might have fundamentally different roles in the host-parasite interaction. Further research is required to examine clear disease-associations of var gene subsets in different geographical settings. The importance of very strict clinical definitions and appropriate large control groups needs to be emphasized for future studies on disease associations of PfEMP1.     

Decoding the language of var genes and Plasmodium falciparum sequestration

Sequestration and rosetting are key determinants of Plasmodium falciparum pathogenesis. They are mediated by a large family of variant proteins called P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 proteins are multispecific binding receptors that are transported to parasite-induced, 'knob-like' binding structures at the erythrocyte surface. To evade immunity and extend infections, parasites clonally vary their expressed PfEMP1. Thus, PfEMP1 are functionally selected for binding while immune selection acts to diversify the family. Here, we describe a new way to analyse PfEMP1 sequence that provides insight into domain function and protein architecture with potential implications for malaria disease.     

Variable SNP density in aspartyl-protease genes of the malaria parasite Plasmodium falciparum

An analysis of the diversity of the aspartyl proteases of Plasmodium falciparum, known as plasmepsins (PMs), was completed in view of their possible role as drug targets. DNA sequence polymorphisms were identified in nine pm genes including their non-coding (introns and 5' flanking) sequences. All genes contained at least one single nucleotide polymorphism (SNP). Extensive microsatellite diversity was observed predominantly in non-coding sequences. All but one non-synonymous polymorphism (a conservative substitution) were mapped to the surface of the predicted protein, contradicting a possible role in enzymatic activity. The distribution of SNPs was found to be non-random among pm genes, with pm6 and pm10 having significantly higher SNP densities, suggesting they were under selection. For pm6 the majority of the SNPs were in introns and some of these may contribute to splice site variation. SNPs were found at a high density in both the coding and non-coding sequences of pm10. Recombination was important in generating additional diversity at this locus. Although direct selection for pm10 mutations could not be ruled out, the presence of balancing selection and a high density of SNPs in non-coding sequence led us to propose that another gene under selection may be influencing the diversity in the region. By sequencing short DNA tags in a 200 kb region flanking pm10 we show that a cluster of antigen genes, known to be under diversifying selection, may contribute to the observed diversity. We discuss the importance of diversity and local selection effects when choosing drug targets for intervention strategies.