Rapid antigen diversification through mitotic recombination in the human malaria parasite Plasmodium falciparum

Abstract

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity when var gene repertoires from different parasite isolates are compared. Previous studies indicated that this remarkable genome plasticity results from extensive ectopic recombination between var genes during mitotic replication; however, the molecular mechanisms that direct this process to antigen-encoding loci while the rest of the genome remains relatively stable were not determined. Using targeted DNA double-strand breaks (DSBs) and long-read whole-genome sequencing, we show that a single break within an antigen-encoding region of the genome can result in a cascade of recombination events leading to the generation of multiple chimeric var genes, a process that can greatly accelerate the generation of diversity within this family. We also found that recombinations did not occur randomly, but rather high-probability, specific recombination products were observed repeatedly. These results provide a molecular basis for previously described structured rearrangements that drive diversification of this highly polymorphic gene family.

Fig 1. Repair of DSBs within a subtelomeric region of chromosome 12 by HR.

(A) A map of the gene structure of the “left” end of chromosome 12. The annotation numbers for each var gene are shown, and the positions of the genes encoding acs7 and a member of the FIKK family (fikk12) are labelled. Scissors represent sites for cleavage by Cas9. Hashed lines represent telomeric repeats and the position of TAREs are shown. (B) A schematic representation of a plasmid containing hb1 and hb2 with significant sequence identity to regions on both sides of the site of a DSB target by Cas9. These blocks flank a selection cassette consisting of the dhfr selectable marker, a promoter from the cam gene, and a transcriptional terminator from the hrp gene. (C) The predicted result from HR-mediated repair. (D) Diagram showing the diagnostic PCRs used to confirm the products of HR and integration of the dhfr cassette into the genome. Diagrams 1–3 show integration into genes on chromosome 12, while 4 shows integration into an unlinked var gene on chromosome 6. (E) Gel electrophoresis of PCR products shown in D. acs7, acyl-CoA synthetase 7; cam, calmodulin; Cas9, CRISPR-associated protein-9 nuclease; dhfr, dihydrofolate reductase; DSB, double-strand break; FIKK, phenylalanine-isoleucine-lysine-lysine-motif–containing kinase; hb, homology block; HR, homologous recombination; hrp, histidine-rich protein 2; PCR, polymerase chain reaction; TARE, telomere-associated repeat element.

Fig 2. Telomere healing events resulting from DSBs within a subtelomeric region of chromosome 12.

(A) The subtelomeric region of the “left” end of chromosome 12 is shown (1), and the targeted site for a break by Cas9 is shown by scissors. This site is between var gene PF3D7_1200600 and the gene acs7. After induction of the DSB (2), an exonuclease (Exo1/DSB2) resects the DNA end. Telomerase then “heals” the end through the addition of telomere repeats (3), resulting in a stabilized chromosome end and a free DNA fragment. (B) An enlarged schematic of the chromosome region surrounding the acs7 gene. The sites of telomere healing in the clones D2, B10, and H10 are shown by vertical arrows, and the site of the targeted DSB is shown by scissors. The position of PCR primers used to detect DNA resection are shown as paired horizontal arrows. (C) PCR products from reactions using each primer pair shown in B for the parasite clones D2, B10, and H10. (D) Sequencing across the site of telomere healing shows the addition of telomere repeat sequences (underlined italics) to unique positions in each clone. acs7, acyl-CoA synthetase 7; Cas9, CRISPR-associated protein-9 nuclease; DSB, double-strand break; Exo, exonuclease; FIKK, phenylalanine-isoleucine-lysine-lysine-motif–containing kinase; PCR, polymerase chain reaction.

Fig 3. Detection of DNA fragments after truncation of the end of chromosome 12.

(A) Diagram of the “left” end of chromosome 12. The annotation numbers for var genes are shown. TAREs are shown as an open box, and telomere repeats are shown as hashed lines. The positions of specific PCR primers used to amplify each gene are shown as paired horizontal arrows. The site of the DSB induced by Cas9 expression is shown. (B) Products of PCR amplifications to detect each gene, demonstrating that several portions of the chromosome persist in the genome despite the truncation of this end of chromosome 12. acs7, acyl-CoA synthetase 7; Cas9, CRISPR-associated protein-9 nuclease; DSB, double-strand break; FIKK, phenylalanine-isoleucine-lysine-lysine-motif–containing kinase; PCR, polymerase chain reaction; TARE, telomere-associated repeat element.

Fig 4. Sequences of chimeric var genes identified by whole-genome sequencing and verified by PCR amplification.

(A) Top: schematic diagram of one end of chromosome 6 after recombination. Bottom: chimeric gene consisting of sequences from Pf3D7_1200400 (blue) and Pf3D7_0632500 (pink). (B) Top: schematic diagram of one end of chromosome 13 after recombination. Bottom: chimeric gene consisting of sequences from Pf3D7_0632800 (pink) and Pf3D7_1200100 (blue). Bases that are identical between the two genes are highlighted in yellow. PCR, polymerase chain reaction.

Fig 5. Sequential recombination events initiated by telomere healing that lead to the creation of chimeric var genes.

(A) Diagram of the “left” end of chromosome 12 after induction of a DSB between var gene PF3D7_1200600 and acs7. The annotation numbers for var genes are shown. TAREs are shown as an open box, and telomere repeats are shown as hashed lines. Note that new telomere repeats are now found directly downstream of acs7, creating a new end of chromosome 12 and liberating the remainder of the chromosome as a free DNA fragment. (B) The free DNA fragment derived from the end of chromosome 12 is shown aligned with the end of chromosome 6. Exonuclease resection of the free DNA end is shown by a Pac-Man symbol. Recombination between var genes PF3D7_1200400 and PF3D7_0632500 is shown with an X. Two products of recombination are shown, the new end of chromosome 6 including the chimeric var gene created by the recombination (right) and the new free DNA fragment (left). C) The free DNA fragment derived from the end of chromosome 6 is shown aligned with the end of chromosome 13. Exonuclease resection of the free DNA end is shown by a Pac-Man symbol. Recombination between var genes PF3D7_0632800 and PF3D7_1300100 is shown with an X. Two products of recombination are shown, the new end of chromosome 13 including the chimeric var gene created by the recombination (right) and the new free DNA fragment (left). acs7, acyl-CoA synthetase 7; Chrom, chromosome; FIKK, phenylalanine-isoleucine-lysine-lysine-motif–containing kinase; TARE, telomere-associated repeat gene.

Fig 6. Sequential recombination events between subtelomeric regions at additional genomic loci.

Previous work [18] identified a telomere healing event on chromosome 1 (A) that resulted in the translocation of the released subtelomeric fragment into a position near the end of chromosome 12 (B). Spontaneous sequential recombination events were also detected in subcloned parasite lines isolated by Claessens and colleagues [23] (C–D). Whole-genome sequencing of subcloned lines of 3D7 parasites identified multiple recombination events within var genes. For 3 subclones (named BLM-4k, WRN-1h, and BLM-P) closely spaced recombination events were suggestive of sequential cascades of recombination leading to multiple chimeric var genes. The putative order of events was inferred based on the position of the recombinations with reference to the central var gene within each cascade (pink). The initial recombination event for each clone occurred between 2 var genes near the chromosome ends (C), creating a chimeric var gene and releasing a free DNA fragment (D). Recombination of the free DNA fragment with a var gene at another chromosome site resulted in the creation of an additional chimeric var gene (E). Detection of these chimeric var genes was originally reported in supplemental data table S1 of reference [23]. Chrom, chromosome.