Primers used in this study.

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 encoded by a multigene family conserved in Plasmodium spp. Surprisingly, an CLAG3 knockout parasite produced in P. falciparum exhibits incomplete reductions in PSAC activity, propagates normally in standard nutrient-rich media, but is unable to expand in modified media with more physiological levels of key nutrients. To explore these unexpected findings, we used in vitro selections on a CLAG3-null parasite and obtained a mutant capable of expansion under nutrient-limiting conditions. This growth was associated with restored solute uptake despite absence of CLAG3 protein. The mutant parasite expressed channels with characteristics of PSAC though with altered solute selectivity and lack of protease susceptibility, suggesting a modified channel and genome-level changes in the pathogen. Whole-genome sequencing revealed a dramatically increased clag2 copy number without other relevant changes. Quantitative PCR and DNA transfection confirmed increased production of the clag2 gene product. These findings implicate CLAG2 in direct formation of nutrient channels, suggest a new model that accounts for variable expansion of clag genes in Plasmodium spp., and uncover a dramatic genome plasticity available to malaria parasites.