Solute Restriction Reveals an Essential Role forclag3-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 ϫ 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.

An Epigenetic Antimalarial Resistance Mechanism Involving Parasite Genes Linked to Nutrient Uptake

“…2). We propose that silencing of individual clag3 genes in sensitive parasites serves to protect the silenced paralog from host immunity (51,52), but that the mechanism reported here functions to regulate host cell permeability in response to nutrient availability and drug pressure (53). For example, in vivo suppression of PSAC may contribute to development of dormant bloodstream parasites, as observed after drug exposure (54).…”

A CLAG3 Mutation in an Amphipathic Transmembrane Domain Alters Malaria Parasite Nutrient Channels and Confers Leupeptin Resistance

“…If potent PSAC inhibitors reduce the efficacy of the drug lead in growth inhibition experiments, then uptake via this channel would be implied. Because PSAC inhibitors also interfere with parasite growth (10,20), the relative contribution of each agent should be examined by isobologram analysis (44). A positive result of either of these tests should alert the discovery program to the risk of this additional resistance mechanism.…”

“…The clag multigene family, also conserved in and restricted to malaria parasites (8), has recently been linked to PSAC activity (9)(10)(11). Two paralogs on parasite chromosome 3, known as clag3.1 and clag3.2, appear to play a critical role, as identified by genetic mapping experiments with ISPA-28, an isolate-specific PSAC antagonist that blocks channels on the Dd2 laboratory clone but is ineffective against channels from other lines.…”

“…Alternatively, CLAG3 may have only a modulatory role, such as through enzymatic activation of quiescent channels in the host membrane (19). Distinguishing between these models is an important step in understanding the structural basis of solute and nutrient transport at the host membrane and in determining whether the channel can be targeted for antimalarial drug development (10,20).One way to address this uncertainty involves molecular studies with available PSAC mutants that have been generated through in vitro selection with toxins that require channel-mediated uptake (11,(21)(22)(23)(24). A leupeptin-resistant clone, HB3-leuR1, carries a nonsynonymous mutation in the clag3.2 gene.…”

“…Alternatively, CLAG3 may have only a modulatory role, such as through enzymatic activation of quiescent channels in the host membrane (19). Distinguishing between these models is an important step in understanding the structural basis of solute and nutrient transport at the host membrane and in determining whether the channel can be targeted for antimalarial drug development (10,20).…”