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Sequencing Study of Malaria Parasite Reveals Antigenic Diversity Created Through Structural Rearrangements

NEW YORK (GenomeWeb) – The malaria-causing parasite Plasmodium falciparum is constantly reshaping the genes that encode the antigens it presents on the surface of infected human red blood cells, a Wellcome Trust Sanger Institute team reported in PLOS Genetics today.

Sanger's Dominic Kwiatkowski and his colleagues grew various strains of P. falciparum on human red blood cells in the lab and sequenced the strains at various time points to track how the 60 or so var genes spread throughout the parasite genome evolved. From this, they found that structural variants crop up in these regions more commonly than in other genomic areas, and create new, chimeric var gene sequences and, in turn, new antigens.

They further estimated that about a million new surface proteins are produced by the parasite every two days within infected people.

"These genes are like decks of cards constantly being shuffled," said co-first author William Hamilton from the Sanger Institute in a statement. "The use of whole genome sequencing and the sheer number of samples we collected gave us a detailed picture of how the var gene repertoire changes continuously within red blood cells."

These constant changes to the var genes may partially explain, the researchers noted, how the parasite can evade immune response and survive for so long in human hosts, and may inform efforts to eradicate malaria.

For this study, Kwiatkowski and his colleagues developed large clone trees for four strains of P. falciparum — the reference strain 3D7, the Honduran strain HB3, and the drug-resistant strains Dd2 and W2 from Southeast Asia — letting them grow and divide in red blood cells in vitro. They then sequenced and compared the genomes of the strains every 20 to 30 replication cycles.

For the 3D7 strain, for instance, they sequenced the whole genomes of 37 subclones to find 20 de novo SNPs and 40 de novo structural variations. All 19 of the structural variations they found that affected coding regions were in var genes, suggesting to the researchers that structural variations occur frequently in P. falciparum, especially near those genes.

All of these 3D7 structural variations, they further noted, involved ectopic recombinations with other var genes, either on the same chromosomes or on a distant one.

By focusing on var gene exon 1, Kwiatkowski and his colleagues noted that they also observed var recombination events in the Dd2 and W2 strains, and calculated that in Dd2 about 0.2 percent of parasites in red blood cells undergo a var exon 1 recombination event leading to a new chimeric var gene every two days

These recombination events, the researchers reported, occur in such a way that the overall var gene architecture is preserved. All of the crossovers they saw were in frame, and 109 out of 110 cases involved recombining of regions belonging to the same domain class.

Still, they noted that the new gene sequences were quite diverse. Var genes didn't necessarily recombine with the var genes that were the most highly homologous with themselves, they said, but the crossover event itself does appear to take place in a region boasting high sequence similarity in which there is a smaller 4 basepair to 48 basepair stretch that is nearly identical.

"This process has presumably evolved to generate antigenic diversity in P. falciparum during the course of a single infection to evade the human immune response," the researchers wrote in their paper.

While previous reports had indicated that var recombination was likely to take place during meiosis, Kwiatkowski and his colleagues instead argued that these events mainly occur during mitosis. But the precise molecular mechanism guiding recombination and double-stranded break repair is unclear as P. falciparum lacks the non-homologous end-joining pathway and may not rely on homologous recombination-based repair as the parasite is haploid for most of its lifecycle, including the entire time it is in red blood cells.

The researchers noted that though ectopic recombination of var may not be of great importance during the early stages of infection, having "an almost limitless repertoire of antigenic variants" might later enable the parasite, through selection, to evade the immune response.

"When you consider that 200 million people across the world are infected with malaria and each of them is harboring parasites that are continually generating millions of antigenic variants, it becomes apparent why our fight against malaria is so challenging," said Kwiatkowski. "By learning the genetic tricks that the parasite uses to evade the human immune system, we will be in a much better position to eliminate this deadly disease."

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