Adaptive Copy Number Evolution in Malaria Parasites

Abstract

Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2–11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.

Genetic conflicts: the usual suspects and beyond

“…Such copy number alterations may provide the genetic fodder for many subsequent adaptations (Brennan et al, 2014). Similar copy number expansions have been observed in many 'slowly evolving' organisms including bacteria, yeast, Plasmodium and even mammalian cells (Bergthorsson et al, 2007;Demuth and Hahn, 2009;Dunham et al, 2002;Kugelberg et al, 2010;Nair et al, 2008;Pränting and Andersson, 2011;Singer et al, 2000;Stambuk et al, 2009;Sun et al, 2009). …”

Stepwise acquisition of pyrimethamine resistance in the malaria parasite

“…Consistent with the hypothesis of compensatory mutation, a copy-number polymorphism in the gene encoding GTPcyclohydrolase I, the first gene in the folate biosynthetic pathway, has recently been shown to be associated with the DHFR quadruple mutant in Thailand (9). Elsewhere in the genome, a selective sweep associated with pyrimethamine resistance has been reported across a region of chromosome 13 (39).…”

“…Both chloroquine resistance and pyrimethamine resistance are associated with multiple amino acid replacements in the target protein (5,6). Mutations in other parasite genes also may contribute quantitatively to increase the resistance phenotype or enhance parasite fitness (7)(8)(9).…”

Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales

“…Polymorphisms at a much larger genomic scale, such as gene duplications and deletions (Stranger et al 2007), also can alter the expression level of a particular protein (Nguyen et al 2006). The correlation between gene copy-number differences and changes in gene expression has been documented previously (Cheng et al 2005;Freeman et al 2006;Nair et al 2008), including in venoms (Margres et al 2015b), and venom protein families are believed to be the result of gene duplication and positive selection (Casewell et al 2011) via the birth-anddeath model of protein evolution (Fry et al 2008). The significant expression variation we detected therefore could be the result of variation in copy number, assuming that variation in copy number would affect low-expression (and presumably low-copy) genes more than high-expression, high-copy genes (e.g., the difference between 10 and 12 copies for a particular protein may not be significant, but the difference between 2 and 4 copies may be).…”