Global genetic diversity and evolution of var genes associated with placental and severe childhood 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
References (57)
- et al.
Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes
Cell
(1995) - et al.
Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes
Cell
(1995) - et al.
The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes
Cell
(1995) - et al.
Genetic diversity of the DBLalpha region in Plasmodium falciparum var genes among Asia-Pacific isolates
Mol Biochem Parasitol
(2002) - et al.
Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria
Lancet
(2004) - et al.
Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family
Mol Biochem Parasitol
(2000) - et al.
A study of var gene transcription in vitro using universal var gene primers
Mol Biochem Parasitol
(2000) - et al.
New tools to identify var sequence tags and clone full-length genes using type-specific primers to Duffy binding-like domains
Mol Biochem Parasitol
(2003) - et al.
The 3D7var5.2 (var COMMON) type var gene family is commonly expressed in non-placental Plasmodium falciparum malaria
Mol Biochem Parasitol
(2003) - et al.
Var gene diversity in Plasmodium falciparum is generated by frequent recombination events
Mol Biochem Parasitol
(2000)
The sick placenta-the role of malaria
Placenta
Protein glycosylation in the malaria parasite
Parasitol Today
Antigenic variation at the infected red cell surface in malaria
Annu Rev Microbiol
The pathogenic basis of malaria
Nature
Genome sequence of the human malaria parasite Plasmodium falciparum
Nature
Developmental selection of var gene expression in Plasmodium falciparum
Nature
Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum
EMBO J
Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome
Mol Microbiol
Evidence for the importance of genetic structuring to the structural and functional specialization of the Plasmodium falciparum var gene family
Mol Microbiol
Sub-grouping of Plasmodium falciparum 3D7 var genes based on sequence analysis of coding and non-coding regions
Malar J
Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta
Science
Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria
Nat Med
Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria
Mol Microbiol
Evidence for the involvement of VAR2CSA in pregnancy-associated malaria
J Exp Med
Identification of multiple chondroitin sulfate A (CSA)-binding domains in the var2CSA gene transcribed in CSA-binding parasites
J Infect Dis
Broad analysis reveals a consistent pattern of var gene transcription in Plasmodium falciparum repeatedly selected for a defined adhesion phenotype
Mol Microbiol
High Level of var2csa Transcription by Plasmodium falciparum Isolated from the Placenta
J Infect Dis
Maternal antibodies block malaria
Nature
Cited by (138)
Knobs, Adhesion, and Severe Falciparum Malaria
2023, Tropical Medicine and Infectious DiseaseThe human malaria parasite Plasmodium falciparum can sense environmental changes and respond by antigenic switching
2023, Proceedings of the National Academy of Sciences of the United States of America
- ☆
Note: Nucloetide sequence data reported in this paper are available in the GenBank™ database under accession numbers DQ407935–DQ408104.
- 1
Both authors contributed equally.