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Solute restriction reveals an essential role for clag3-associated channels in malaria parasite nutrient acquisition.

ABSTRACT: The plasmodial surface anion channel (PSAC) increases erythrocyte permeability to many solutes in malaria but has uncertain physiological significance. We used a PSAC inhibitor with different efficacies against channels from two Plasmodium falciparum parasite lines and found concordant effects on transport and in vitro parasite growth when external nutrient concentrations were reduced. Linkage analysis using this growth inhibition phenotype in the Dd2 × HB3 genetic cross mapped the clag3 genomic locus, consistent with a role for two clag3 genes in PSAC-mediated transport. Altered inhibitor efficacy, achieved through allelic exchange or expression switching between the clag3 genes, indicated that the inhibitor kills parasites through direct action on PSAC. In a parasite unable to undergo expression switching, the inhibitor selected for ectopic homologous recombination between the clag3 genes to increase the diversity of available channel isoforms. Broad-spectrum inhibitors, which presumably interact with conserved sites on the channel, also exhibited improved efficacy with nutrient restriction. These findings indicate that PSAC functions in nutrient acquisition for intracellular parasites. Although key questions regarding the channel and its biological role remain, antimalarial drug development targeting PSAC should be pursued.

SUBMITTER: Pillai AD

PROVIDER: S-EPMC3502622 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Solute restriction reveals an essential role for clag3-associated channels in malaria parasite nutrient acquisition.

Pillai Ajay D AD Nguitragool Wang W Lyko Brian B Dolinta Keithlee K Butler Michelle M MM Nguyen Son T ST Peet Norton P NP Bowlin Terry L TL Desai Sanjay A SA

Molecular pharmacology 20120904 6

The plasmodial surface anion channel (PSAC) increases erythrocyte permeability to many solutes in malaria but has uncertain physiological significance. We used a PSAC inhibitor with different efficacies against channels from two Plasmodium falciparum parasite lines and found concordant effects on transport and in vitro parasite growth when external nutrient concentrations were reduced. Linkage analysis using this growth inhibition phenotype in the Dd2 × HB3 genetic cross mapped the clag3 genomic ...[more]

PMID: 22949525

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Project description:Malaria parasites increase their host erythrocyte's permeability to various nutrients, fueling intracellular pathogen development and replication. The plasmodial surface anion channel (PSAC) mediates this uptake and is linked to the parasite-encoded RhopH complex, consisting of CLAG3, RhopH2, and RhopH3. While interactions between these subunits are well established, it is not clear whether they remain associated from their synthesis in developing merozoites through erythrocyte invasion and trafficking to the host membrane. Here, we explored protein-protein interactions between RhopH subunits using live-cell imaging and Förster resonance energy transfer (FRET) experiments. Using the green fluorescent protein (GFP) derivatives mCerulean and mVenus, we generated single- and double-tagged parasite lines for fluorescence measurements. While CLAG3-mCerulean served as an efficient FRET donor for RhopH2-mVenus within rhoptry organelles, mCerulean targeted to this organelle via a short signal sequence produced negligible FRET. Upon merozoite egress and reinvasion, these tagged RhopH subunits were deposited into the new host cell's parasitophorous vacuole; these proteins were then exported and trafficked to the erythrocyte membrane, where CLAG3 and RhopH2 remained fully associated. Fluorescence intensity measurements identified stoichiometric increases in exported RhopH protein when erythrocytes are infected with two parasites; whole-cell patch-clamp revealed a concomitant increase in PSAC functional copy number and a dose effect for RhopH contribution to ion and nutrient permeability. These studies establish live-cell FRET imaging in human malaria parasites, reveal that RhopH subunits traffic to their host membrane destination without dissociation, and suggest quantitative contribution to PSAC formation.IMPORTANCE 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.

Dataset Information

Solute restriction reveals an essential role for clag3-associated channels in malaria parasite nutrient acquisition.

Publications

Solute restriction reveals an essential role for clag3-associated channels in malaria parasite nutrient acquisition.

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