A markedly increased clag2 copy number enables malaria parasite growth under nutrient stress by raising human erythrocyte permeability

To grow and replicate in erythrocytes, malaria parasites must increase the host cell's permeability to a broad range of nutrients. The plasmodial surface anion channel (PSAC) mediates this increased permeability and has been linked to CLAG3, a protein encodede by a multigene family conserved in Plasmodium spp. Surprisingly, a CLAG3 knockout parasite produced in P. falciparum exhibits incomplete reductions in PSAC activity, propagates normally in standard nutrient-rich media, and is unable to expand in modified media with more physiological levels of key nutrients. To explore these unexpected findings, we used the in vitro selections on a CLAG3-null parasite and obtained a mutant capable of expansion under nutrient-limiting conditions.This growth was associated with a largely restored PSAC-mediated solute uptake despite sustained absence of CLAG3 protein. The mutant parasite's channels exhibited altered selectivity for permeating solutes and reduced susceptibility to proteolysis, suggesting mutations or genome-level changes. Whole genome sequencing revealed a dramatically increased clag2 copy number without other changes. RT-PCR and DNA transfection confirmed increased production of the clag2 gene product. These findings implicate CLAG2 in direct formation of nutrient channels, account for variable expansion of clag genes in plasmodium spp., and uncover a dramatic genome plasticity available to malaria parasites.