Direct evidence for the adaptive role of copy number variation on antifolate susceptibility in Plasmodium falciparum

Abstract

Summary Resistance to antimalarials targeting the folate pathway is widespread. GTP-cyclohydrolase (gch1), the first enzyme in this pathway, exhibits extensive copy number variation (CNV) in parasite isolates from areas with a history of longstanding antifolate use. Increased CN of gch1 is associated with a greater number of point mutations in enzymes targeted by the antifolates, pyrimethamine and sulfadoxine. While these observations suggest that increases in gch1 CN are an adaptation to drug pressure, changes in CN have not been experimentally demonstrated to directly alter drug susceptibility. To determine if changes in gch1 expression alone modify pyrimethamine sensitivity, we manipulated gch1 CN in several parasite lines to test the effect on drug sensitivity. We report that increases in gch1 CN alter pyrimethamine resistance in most parasites lines. However we find evidence of a detrimental effect of very high levels of gch1 overexpression in parasite lines with high endogenous levels of gch1 expression, revealing the importance of maintaining balance in the folate pathway and implicating changes in gch1 expression in preserving proper metabolic flux. This work expands our understanding of parasite adaptation to drug pressure and provides a possible mechanism for how specific mutations become fixed within parasite populations.

DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

“…For example, changes in gene dosage have been linked to cancer and neurological disorders, such as autism (134). In P. falciparum, the fitness costs to parasite growth have been observed with amplifications of gch1 (106) and pfcrt (our unpublished results). However, in the context of selective or diverse environments, CNVs can prove advantageous in promoting adaptability, as in the case of in vitro culture growth and drug resistance.…”

“…A recent report (106) suggests that P. falciparum may be able to repair a DSB by an alternative EJ mechanism whereby a few bases are added to the broken ends, thereby providing microhomologies. The frequency of these events is very low, and larger deletions (e.g., possible SSA products) beyond the locus analyzed were not examined.…”

Characterizing the impact of sustained sulfadoxine/pyrimethamine use upon the Plasmodium falciparum population in Malawi

“…Given the hypothesized role for copy number variation (CNV) in gch1 -mediated SP resistance [35] and signals of selection observed, potential CNVs at this locus were investigated. Evidence of a promoter duplication was found (genomic region 973,804–974,240 bp; see Fig.…”

Indels, structural variation, and recombination drive genomic diversity in Plasmodium falciparum

“…Amplifications have also been found spanning gch1, conferring resistance to anti-folate drugs, in both HB3 and Dd2, although the amplifications are different in size and extent (Kidgell et al 2006;Heinberg et al 2013). The mdr1 amplification segregates in the progeny of HB3 × Dd2 (Wellems et al 1990), and there is evidence that meiotic recombination has occurred within the amplified region in two progeny clones (Samarakoon et al 2011a).…”