Malaria parasites transmitted by mosquito bite are remarkably efficient in establishing human infections. The infection process requires roughly 30 minutes and is highly complex as quiescent sporozoites injected with mosquito saliva must be rapidly activated in the skin, migrate through the body, and infect the liver. This process is poorly understood for Plasmodium vivax due to low infectivity in the in vitro models. To study this skin-to-liver-stage of malaria, we used quantitative bioassays coupled with transcriptomics to evaluate parasite changes linked with mammalian microenvironmental factors. Our in vitro phenotyping and RNA-seq analyses revealed key microenvironmental relationships with distinct biological functions. Most notable, preservation of sporozoite quiescence by exposure to insect-like factors coupled with strategic activation limits untimely activation of invasion-associated genes to dramatically increase hepatocyte invasion rates. We also report the first transcriptomic analysis of the P. vivax sporozoite interaction in salivary glands identifying 118 infection-related differentially-regulated Anopheles dirus genes. These results provide important new insights in malaria parasite biology and identify priority targets for antimalarial therapeutic interventions to block P. vivax infection.

Malaria parasites activate a broad-selectivity ion channel on their host erythrocyte membrane to obtain essential nutrients from the bloodstream. This conserved channel, known as the plasmodial surface anion channel (PSAC), has been linked to parasite clag3 genes in P. falciparum, but epigenetic switching between the two copies of this gene hinders clear understanding of how the encoded protein determines PSAC activity. Here, we used linkage analysis in a P. falciparum cross where one parent carries a single clag3 gene to overcome the effects of switching and confirm a primary role of the clag3 product with high confidence. Despite Mendelian inheritance, CLAG3 conditional knockdown revealed remarkably preserved nutrient and solute uptake. Even more surprisingly, transport remained sensitive to a CLAG3 isoform-specific inhibitor despite quantitative knockdown, indicating that low doses of the CLAG3 transgene are sufficient to confer block. We then produced a complete CLAG3 knockout line and found it exhibits an incomplete loss of transport activity, in contrast to rhoph2 and rhoph3, two PSAC-associated genes that cannot be disrupted because nutrient uptake is abolished in their absence. Although the CLAG3 knockout did not incur a fitness cost under standard nutrient-rich culture conditions, this parasite could not be propagated in a modified medium that more closely resembles human plasma. These studies implicate oligomerization of CLAG paralogs encoded by various chromosomes in channel formation. They also reveal that CLAG3 is dispensable under standard in vitro conditions but required for propagation under physiological conditions. 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.

Malaria parasites increase host erythrocyte permeability to ions and nutrients via a broad-selectivity channel known as the plasmodial surface anion channel (PSAC), linked to parasite-encoded CLAG3 and two associated proteins. These proteins lack the multiple transmembrane domains typically present in channel-forming proteins, raising doubts about their precise roles. Using the virulent human Plasmodium falciparum parasite, we report that CLAG3 undergoes self-association and that this protein’s expression determines channel phenotype quantitatively. We overcame epigenetic silencing of clag3 paralogs and engineered parasites that express two CLAG3 isoforms simultaneously. Stoichiometric expression of these isoforms yielded intermediate channel phenotypes, in agreement with observed trafficking of both proteins to the host membrane. Coimmunoprecipitation and surface labeling revealed formation of CLAG3 oligomers. In vitro selections applied to these transfectant lines yielded distinct mutants with correlated changes in channel activity. These findings support involvement of the identified oligomers in PSAC formation and parasite nutrient acquisition. IMPORTANCE Malaria parasites are globally important pathogens that evade host immunity by replicating within circulating erythrocytes. To facilitate intracellular growth, these parasites increase erythrocyte nutrient uptake through an unusual ion channel. The parasite CLAG3 protein is a key determinant of this channel, but its lack of homology to known ion channels has raised questions about possible mechanisms. Using a new method that allows simultaneous expression of two different CLAG3 proteins, we identify self-association of CLAG3. The two expressed isoforms faithfully traffic to and insert in the host membrane, while remaining associated with two unrelated parasite proteins. Both the channel phenotypes and molecular changes produced upon selections with a highly specific channel inhibitor are consistent with a multiprotein complex that forms the nutrient pore. These studies support direct involvement of the CLAG3 protein in channel formation and are relevant to antimalarial drug discovery projects targeting parasite nutrient acquisition.

Malaria parasites grow within circulating red blood cells and uptake nutrients through a pore on their host membrane. Here, we used gene editing to tag CLAG3 and RhopH2, two proteins linked to the nutrient pore, with fluorescent markers and tracked these proteins in living infected cells. After their synthesis in mature parasites, imaging showed that both proteins are packaged into membrane-bound rhoptries. When parasites ruptured their host cells and invaded new red blood cells, these proteins were detected within a vacuole around the parasite before they migrated and inserted in the surface membrane of the host cell. Using simultaneous labeling of CLAG3 and RhopH2, we determined that these proteins interact tightly during migration and after surface membrane insertion. Red blood cells infected with two parasites had twice the protein at their surface and a parallel increase in the number of nutrient pores. Our work suggests that these proteins directly facilitate parasite nutrient uptake from human plasma.