Global genetic diversity and evolution of var genes associated with placental and severe childhood malaria

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Abstract

In Plasmodium falciparum, var genes encode adhesive proteins that are transported to the surface of infected erythrocytes and act as major virulence determinants for infected erythrocyte binding and immune evasion. Var genes are highly diverse and can be classified into five major groups (UpsA, B, C, D, and E). Previous serological studies have suggested that the UpsA var group may contain common antigenic types that have important roles in severe childhood malaria. Here, our analysis found that UpsA vars are highly diverse between 22 world-wide parasite isolates, although they could be grouped into two broad clusters that may be separately recombining. By comparison, orthologs of the UpsA-linked Type 3 var and UpsE-linked var2csa were detected in nearly all parasite isolates, and a var2csa ortholog was also present in a chimpanzee malaria P. reichenowi that diverged from P. falciparum ∼5–7 million years ago. Although the specific function of Type 3 var genes is unknown, var2csa is a leading candidate for a pregnancy associated malaria vaccine. Compared to typical var genes, var2csa is unusually conserved but still had only 54–94% amino acid identity in extracellular binding regions. However, var2csa alleles have extensive gene mosaicism within polymorphic blocks that are shared between world-wide parasite isolates and recognizable in P. rechenowi suggesting a high rate of self–self recombination and an ancient and globally-related pool of var2csa polymorphism. These studies aid our understanding of the evolutionary mechanisms that shape var diversity and will be important to the development of vaccines against pregnancy associated malaria and severe malaria.

Introduction

During the blood stage development of Plasmodium falciparum, the parasite exports variant surface antigens to the infected erythrocyte (IE) surface that subvert immunity and mediate IE sequestration from blood circulation [1]. The best characterized antigens are the erythrocyte membrane protein 1 family (PfEMP1) encoded by var genes that function as adhesion molecules for host endothelial or erythrocyte receptors [2]. Every parasite strain encodes approximately 50–60 var genes, but in any given parasite only a single PfEMP1 protein is expressed at a time [3], [4], [5]. Switches in var gene expression correlate with changes in the binding and antigenic properties of IEs [6], [7], [8], which allow the parasite to establish persistent infections and sequester at different sites in the body [2]. From the 3D7 genome reference isolate, var genes can be classified into five broad categories (UpsA to UpsE) according to position on the chromosome and 5′ flanking sequence [3]. It is hypothesized that this genetic organization may help to restrict recombination within specific groups of genes and lead to their structural and functional specialization for binding at different sites in the body [9], [10], [11]. A critical issue in malaria pathogenesis is to determine if PfEMP1 variants causing disease have similar antigenic or structural characteristics that could provide the basis of a vaccine(s).

Although the overall diversity of var genes in the parasite population is immense [12], recent evidence suggests that PfEMP1 variants associated with disease may be partially restricted. For instance, pregnancy associated malaria (PAM) is associated with the expression of a var gene, called var2csa, which is unusually conserved across parasite isolates and binds a low sulfated form of chondroitin sulfate A (CSA) in the placenta [13], [14], [15], [16], [17], [18], [19]. Unlike typical var genes, most parasite isolates appear to have a var2csa ortholog. During pregnancy, women develop antibodies broadly reactive to placental isolates [20], [21], [22], suggesting it may be possible to vaccinate against PAM.

Similar to PAM, parasite variants associated with severe childhood malaria appear to have less antigenic diversity than those associated with mild infections, as indicated by their broader serological reactivity with semi-immune children's sera [23], [24], [25]. The adhesive phenotypes associated with severe childhood malaria are much less well defined than PAM, and the extent of PfEMP1 restriction remains to be characterized. However, serological investigations have suggested that the UpsA var group may contain common antigenic types that have an important role in severe childhood malaria [26]. Interestingly, both the UpsA var group and var2csa (UpsE) belong to a subset of var genes that is located at the chromosome ends and transcribed towards the telomere (Fig. 1) [3]. These findings raise the possibility that this subset of var genes may have an important role in severity of infection and, furthermore, that previously unsuspected mechanisms may maintain them at higher sequence conservation in the parasite population.

Here we studied genetic diversity of the UpsA var group and var2csa. Overall, UpsA var genes were highly diverse between parasite isolates with the exception of the Type 3 var gene, which is the smallest known var gene [3], and could be detected in all but one parasite isolate. Even more remarkably, a var2csa ortholog was detected in all P. falciparum isolates and is present in the chimpanzee malaria P. reichenowi. Sequence analysis showed extensive gene mosaicism in the var2csa orthologs that is occurring by recombination/gene conversion. Although polymorphism at var2csa is high compared to non-var genes in the genome, what is unexpected is the sequence similarity to the P. reichenowi ortholog, indicating that this gene, and the variation at this gene, has an ancient origin and has somehow been retained in the variant antigen repertoire for millions of years. These studies provide valuable insight into the evolutionary and genetic mechanisms that have shaped the variant antigen repertoire and its role in disease pathogenesis.

Section snippets

Parasite isolates

Genomic DNAs in this study were prepared from culture adapted parasite isolates that are available from the Malaria Research and Reference Reagent Resource Center at MR4/American Type Culture Collection (14 isolates) or were previously published [27].

Var primers

Degenerate UpsA type-specific primers were based upon sequence alignments of 3D7 var genes. Primers were designed to amplify an ∼1200 bp product between homology blocks in the upstream sequences (5′ UTR, −200 bp region) and downstream sequence of

UpsA-associated var sequences are highly diverse between parasite isolates

In the 3D7 parasite genome, three different upstream sequences (UpsA, D, and E) are associated with sub-telomeric var genes that are transcribed towards the telomere end (Fig. 1). 3D7 has a single UpsD var gene (PFE1640w pseudogene, or var1csa), a single UpsE var gene (var2csa, PFL0030c), and 9 UpsA var genes [3]. To study the genetic diversity of UpsA var sequences, we developed degenerate primers specific to the UpsA promoter and the DBL1 domain to amplify a portion of the coding sequence (

Discussion

Information about var genetic diversity is important for understanding malaria pathogenesis and the feasibility of designing disease interventions. Recent evidence suggests that var genes form separate recombination groups [10], [11]. Here we studied the UpsA, D, E group of var genes from a global collection of parasite isolates. Whereas it has been hypothesized that UpsA vars have a role in severe childhood malaria [26], our analysis shows that UpsA var sequences are highly diverse between

Acknowledgements

The authors thank the following agencies for supporting this work: Bill & Melinda Gates Foundation (JDS); National Institutes of Health (grant RO1 AI47953-01A1, JDS); MJ Murdock Charitable Trust (Seattle Biomedical Research Institute, support to JDS). We thank the scientists at the Wellcome Trust Sanger Institute for the P. reichenowi var1csa and var2csa sequences that were used in this analysis. P. reichenowi sequence data were produced by the Pathogen Sequencing Unit at the Wellcome Trust

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    Note: Nucloetide sequence data reported in this paper are available in the GenBank™ database under accession numbers DQ407935DQ408104.

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    Both authors contributed equally.

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