Generation of Antigenic Diversity in Plasmodium falciparum by Structured Rearrangement of Var Genes During Mitosis

Abstract

The most polymorphic gene family in P. falciparum is the ∼60 var genes distributed across parasite chromosomes, both in the subtelomeres and in internal regions. They encode hypervariable surface proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) that are critical for pathogenesis and immune evasion in Plasmodium falciparum. How var gene sequence diversity is generated is not currently completely understood. To address this, we constructed large clone trees and performed whole genome sequence analysis to study the generation of novel var gene sequences in asexually replicating parasites. While single nucleotide polymorphisms (SNPs) were scattered across the genome, structural variants (deletions, duplications, translocations) were focused in and around var genes, with considerable variation in frequency between strains. Analysis of more than 100 recombination events involving var exon 1 revealed that the average nucleotide sequence identity of two recombining exons was only 63% (range: 52.7–72.4%) yet the crossovers were error-free and occurred in such a way that the resulting sequence was in frame and domain architecture was preserved. Var exon 1, which encodes the immunologically exposed part of the protein, recombined in up to 0.2% of infected erythrocytes in vitro per life cycle. The high rate of var exon 1 recombination indicates that millions of new antigenic structures could potentially be generated each day in a single infected individual. We propose a model whereby var gene sequence polymorphism is mainly generated during the asexual part of the life cycle.

Author Summary

Malaria kills >600,000 people each year, with most deaths caused by Plasmodium falciparum. A family of proteins known as P. falciparum erythrocyte membrane protein 1, PfEMP1, is expressed on the surface of infected erythrocytes and plays an important role in pathogenesis. Each P. falciparum genome contains approximately 60 highly polymorphic var genes encoding the PfEMP1 proteins, and monoallelic expression with periodic switching results in immune evasion. Var gene polymorphism is thus critical to this survival strategy. We investigated how var gene diversity is generated by performing an in vitro evolution experiment, tracking var gene mutation in ‘real-time’ with whole genome sequencing. We found that genome structural variation is focused in and around var genes. These genetic rearrangements created new ‘chimeric’ var gene sequences during the mitotic part of the life cycle, and were consistent with processes of mitotic non-allelic homologous recombination. The recombinant var genes were always in frame and with conserved overall var gene architecture, and the recombination rate implies that many millions of rearranged var gene sequences are produced every 48-hour life cycle within infected individuals. In conclusion, we provide a detailed description of how new var gene sequences are continuously generated in the parasite genome, helping to explain long-term parasite survival within infected human hosts.