Complex nutrient channel phenotypes despite Mendelian inheritance in a Plasmodium falciparum genetic cross

Author summary Malaria, a globally important infectious disease, is caused by parasites that invade and grow in circulating red blood cells to avoid host immune attack. Infected red blood cells have increased uptake of diverse nutrients, fueling parasite growth; this uptake is mediated by an ion channel that transports essential nutrients across the red blood cell membrane. Three proteins made by the parasite have been linked to this channel, but how they increase uptake is unknown. Here, we used mapping in a genetic cross of two strains of the virulent human malaria parasite to confirm a primary role of one protein known as CLAG3. We then used gene editing to produce a parasite that has reduced CLAG3 levels when a stabilizing chemical is removed; surprisingly, solute transport was minimally changed despite a 90% reduction in CLAG3. Gene editing was also used to make a parasite without any CLAG3. This knockout parasite had reduced nutrient uptake, but it still grew normally in media with high nutrient levels; it was unable to grow when nutrient levels were lowered to levels like those in the human bloodstream. The complex effects of channel inhibitors on these genetically modified parasites suggests that CLAG3 and the two other proteins interact with each other to form large protein clusters in the red blood cell membrane; these clusters may form the nutrient uptake pore. Our studies indicate that CLAG3 is required for parasite survival and growth in the bloodstream and that the channel it produces can be targeted to make new antimalarial drugs.