A blasticidin S-resistant Plasmodium falciparum mutant with a defective plasmodial surface anion channel

Abstract

Erythrocytes infected with malaria parasites exhibit marked increases in permeability to organic and inorganic solutes. The plasmodial surface anion channel (PSAC), an unusual voltagedependent ion channel induced on the host membrane after infection, may play a central role in these permeability changes. Here, we identified a functional PSAC mutant through in vitro selection with blasticidin S. Resistance to blasticidin S was generated during culture and correlated with significant reductions in permeability to multiple solutes, consistent with uptake via a common pathway. Single channel recordings revealed marked changes in PSAC gating with the addition of a subconductance state not present in wild-type channels. The channel's selectivity profile and pharmacology also were significantly altered. Eventual loss of the mutant phenotype upon removal of selective pressure and slower growth of mutant parasites suggest that PSAC serves an important role in intracellular parasite survival. These findings provide solid evidence for the uptake of diverse solutes via PSAC and implicate one or more parasite genes in expression of this channel.drug resistance ͉ malaria ͉ nutrient acquisition ͉ fitness cost T he malaria parasite, Plasmodium falciparum, is a highly successful intracellular pathogen. In the human host, replication in erythrocytes leads to an exponentially increasing number of parasites and accounts for much of the clinical syndrome of malaria. To facilitate its rapid intracellular growth, the parasite remodels the erythrocyte cytoplasm (1), adds virulence factors to the surface of infected cells (2), and increases erythrocyte permeability to many small solutes. Increased uptake of solutes may be mediated by the plasmodial surface anion channel (PSAC) (3). This unusual ion channel has a singlechannel conductance of 20 pS in molar Cl Ϫ solutions and fast-flickering voltage-dependent gating. PSAC also has an atypical selectivity profile for anions (SCN Ϫ Ͼ I Ϫ Ͼ Br Ϫ Ͼ Cl Ϫ ) and effectively excludes Na ϩ despite permeability to bulky cations (4). Various antagonists have quantitatively identical effects on PSAC's open probability (5-7) and uptake of organic solutes including sugars (8), amino acids (9), purines (10), organic cations (11), and some vitamins (12), suggesting that PSAC functions as a shared route for the increased uptake of these diverse solutes after infection.Nevertheless, PSAC's role in solute transport is debated because other ion channels have been observed by some workers (13-16) and because a single ion channel with broad permeability to diverse solutes but stringent exclusion of Na ϩ is unprecedented. It is also debated whether PSAC and other putative channels are parasite-encoded proteins or modified human proteins (5). Addressing these issues is an important hurdle for transport studies in malaria and for drug development programs that seek to target the parasite-induced permeability changes.Blasticidin S is a fungal toxin (molecular weight 422.2) that kills most prokaryotic and eukaryoti...

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

“…However, the 2C3 line exhibited a remarkably rapid adaptation as blasticidin S was unable to produce microscopic clearance of the culture and parasitemia recovered fully within 6 days. The fully susceptible FCB parasite did not recover within the time frame of this experiment, and instead required Ն2 months to acquire resistance (10). This difference between FCB and the revertant 2C3 daughter implicates genome-level changes in addition to the epigenetic mechanism studied here.…”

“…2 and 4). 2) Altered single channel gating and solute selectivity in these mutants is also inconsistent with the conservative prediction that transcriptional down-regulation yields a reduced number of otherwise unchanged channels (10,11). 3) Although FCB-br1 and FCB-2mut exhibit similar levels of clag gene silencing, these mutants have very different channel properties and responses to removal of selective pressure.…”

“…Mutant parasite lines resistant to either blasticidin S alone (FCB-br1) or to both blasticidin S and leupeptin (FCB2mut) were previously generated through in vitro selections applied to the wild-type FCB line (10,11); both are limiting dilution clones. The 2C3 clone was obtained by limiting dilution cloning of a blasticidin S-sensitive line resulting from cultivation of FCB-br1 without drug pressure.…”

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

“…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.…”

Chemical activation of a high-affinity glutamate transporter in human erythrocytes and its implications for malaria-parasite–induced glutamate uptake

“…At the same time, there is evidence for the involvement of parasite-encoded proteins. After earlier observations of significant parasite-strain-specific differences in the electrophysiologic characteristics of parasite-induced ion conductances 21,22 and the identification of parasite strains for which the infected erythrocyte has reduced permeability to various cytotoxic agents, 10,11,23 Desai and colleagues have demonstrated a role for parasite-encoded "clag3" proteins in the formation of parasite-induced channels. 24 Whether these proteins themselves form part of the channels is not yet clear.…”