Sanjay A. Desai

Malaria Parasite clag3 Genes Determine Channel-Mediated Nutrient Uptake by Infected Red Blood Cells

Development of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but its molecular basis is unknown. We report a parasite gene family responsible for PSAC activity. We used high-throughput screening for nutrient uptake inhibitors to identify a compound highly specific for channels from the Dd2 line of the human pathogen P. falciparum. Inheritance of this compounds affinity in a Dd2 × HB3 genetic cross maps to a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are encoded by two clag3 genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein increases host cell permeability to diverse solutes.

A Two-Compartment Model of Osmotic Lysis in Plasmodium falciparum-Infected Erythrocytes

We recently identified a voltage-dependent anion channel on the surface of human red blood cells (RBCs) infected with the malaria parasite, Plasmodium falciparum. This channel, the plasmodial erythrocyte surface anion channel (PESAC), likely accounts for the increased permeability of infected RBCs to various small solutes, as assessed quantitatively with radioisotope flux and patch-clamp studies. Whereas this increased permeability has also been studied by following osmotic lysis of infected cells in permeant solutes, these experiments have been limited to qualitative comparisons of lysis rates. To permit more quantitative examination of lysis rates, we have developed a mathematical model for osmotic fragility of infected cells based on diffusional uptake via PESAC and the two-compartment geometry of infected RBCs. This model, combined with a simple light scattering assay designed to track osmotic lysis precisely, produced permeability coefficients that match both previous isotope flux and patch-clamp estimates. Our model and light scattering assay also revealed Michaelian kinetics for inhibition of PESAC by furosemide, suggesting a 1:1 stoichiometry for their interaction.

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

Erythrocytes infected with malaria parasites exhibit marked increases in permeability to organic and inorganic solutes. The plasmodial surface anion channel (PSAC), an unusual voltage-dependent 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 channels 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.

Solute Restriction Reveals an Essential Role for clag3-Associated Channels in Malaria Parasite Nutrient Acquisition

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.

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

Background: Malaria parasites acquire antimalarial resistance through incompletely understood mechanisms. Results: Resistance to blasticidin S results from reversible silencing of parasite clag genes through histone modifications without DNA level changes. Conclusion: Sophisticated epigenetic control of clag genes permits regulated control of nutrient and antimalarial transport at the host membrane. Significance: This resistance mechanism allows rapid parasite adaptation to environmental pressures and is worrisome for drug discovery efforts. Acquired antimalarial drug resistance produces treatment failures and has led to periods of global disease resurgence. In Plasmodium falciparum, resistance is known to arise through genome-level changes such as mutations and gene duplications. We now report an epigenetic resistance mechanism involving genes responsible for the plasmodial surface anion channel, a nutrient channel that also transports ions and antimalarial compounds at the host erythrocyte membrane. Two blasticidin S-resistant lines exhibited markedly reduced expression of clag genes linked to channel activity, but had no genome-level changes. Silencing aborted production of the channel protein and was directly responsible for reduced uptake. Silencing affected clag paralogs on two chromosomes and was mediated by specific histone modifications, allowing a rapidly reversible drug resistance phenotype advantageous to the parasite. These findings implicate a novel epigenetic resistance mechanism that involves reduced host cell uptake and is a worrisome liability for water-soluble antimalarial drugs.

A Cell-Based High-Throughput Screen Validates the Plasmodial Surface Anion Channel As an Antimalarial Target

The plasmodial surface anion channel (PSAC) is an unusual small-conductance ion channel induced on erythrocytes infected with plasmodia, including parasites responsible for human malaria. Although broadly available inhibitors produce microscopic clearance of parasite cultures at high concentrations and suggest that PSAC is an antimalarial target, they have low affinity for the channel and may interfere with other parasite activities. To address these concerns, we developed a miniaturized assay for PSAC activity and carried out a high-throughput inhibitor screen. Approximately 70,000 compounds from synthetic and natural product libraries were screened, revealing inhibitors from multiple structural classes including two novel and potent heterocyclic scaffolds. Single-channel patch-clamp studies indicated that these compounds act directly on PSAC, further implicating a proposed role in transport of diverse solutes. A statistically significant correlation between channel inhibition and in vitro parasite killing by a family of compounds provided chemical validation of PSAC as a drug target. These new inhibitors should be important research tools and may be starting points for much-needed antimalarial drugs.

Babesia and plasmodia increase host erythrocyte permeability through distinct mechanisms

Human erythrocytes infected with Plasmodium falciparum have markedly increased permeability to diverse solutes, many of which may be mediated by an unusual small conductance ion channel, the plasmodial surface anion channel (PSAC). Because these increases may be essential for parasite survival in the bloodstream, an important question is whether other intraerythrocytic parasites induce similar ion channels. Here, we examined this question using human erythrocytes infected with Babesia divergens, a distantly related apicomplexan parasite that can cause severe disease in immunocompromised humans. Osmotic lysis experiments after enrichment of infected erythrocytes with a new method revealed that these parasites also increase host permeability to various organic solutes. These permeability changes differed significantly from those induced by P. falciparum in transport rates, selectivity profiles and temperature dependence. Cell‐attached and whole‐cell patch‐clamp experiments confirmed and extended these differences because neither PSAC‐like channels nor significant increases in whole‐cell anion conductance were seen after B. divergens infection. While both babesia and plasmodia increase host erythrocyte permeability to a diverse collection of organic solutes, they utilize fundamentally different mechanisms.

Ion and nutrient uptake by malaria parasite-infected erythrocytes.

Erythrocytes infected with malaria parasites have increased permeability to diverse organic and inorganic solutes. While these permeability changes have been known for decades, the molecular basis of transport was unknown and intensively debated. CLAG3, a parasite protein previously thought to function in cytoadherence, has recently been implicated in formation of the plasmodial surface anion channel (PSAC), an unusual small conductance ion channel that mediates uptake of most solutes. Consistent with transport studies, the clag genes are conserved in all plasmodia but are absent from other genera. The encoded protein is integral to the host membrane, as also predicted by electrophysiology. An important question is whether functional channels are formed by CLAG3 alone or through interactions with other proteins. In either case, gene identification should advance our understanding of parasite biology and may lead to new therapeutics.

The Plasmodial Surface Anion Channel Is Functionally Conserved in Divergent Malaria Parasites

ABSTRACT The plasmodial surface anion channel (PSAC), a novel ion channel induced on human erythrocytes infected with Plasmodium falciparum, mediates increased permeability to nutrients and presumably supports intracellular parasite growth. Isotope flux studies indicate that other malaria parasites also increase the permeability of their host erythrocytes, but the precise mechanisms are unknown. Channels similar to PSAC or alternative mechanisms, such as the upregulation of endogenous host transporters, might fulfill parasite nutrient demands. Here we evaluated these possibilities with rhesus monkey erythrocytes infected with Plasmodium knowlesi, a parasite phylogenetically distant from P. falciparum. Tracer flux and osmotic fragility studies revealed dramatically increased permeabilities paralleling changes seen after P. falciparum infection. Patch-clamp of P. knowlesi-infected rhesus erythrocytes revealed an anion channel with striking similarities to PSAC: its conductance, voltage-dependent gating, pharmacology, selectivity, and copy number per infected cell were nearly identical. Our findings implicate a family of unusual anion channels highly conserved on erythrocytes infected with various malaria parasites. Together with PSACs exposed location on the host cell surface and its central role in transport changes after infection, this conservation supports development of antimalarial drugs against the PSAC family.

Changes in the Plasmodial Surface Anion Channel Reduce Leupeptin Uptake and Can Confer Drug Resistance in Plasmodium falciparum-Infected Erythrocytes

ABSTRACT Cysteine protease inhibitors kill malaria parasites and are being pursued for development as antimalarial agents. Because they have multiple targets within bloodstream-stage parasites, workers have assumed that resistance to these inhibitors would not be acquired easily. In the present study, we used in vitro selection to generate a parasite resistant to growth inhibition by leupeptin, a broad-profile cysteine and serine protease inhibitor. Resistance was not associated with upregulation of cysteine protease activity, reduced leupeptin sensitivity of this activity, or expression level changes for putative cysteine or serine proteases in the parasite genome. Instead, it was associated with marked changes in the plasmodial surface anion channel (PSAC), an ion channel on infected erythrocytes that functions in nutrient and bulky organic solute uptake. Osmotic fragility measurements, electrophysiological recordings, and leupeptin uptake studies revealed selective reductions in organic solute permeability via PSAC, altered single-channel gating, and reduced inhibitor affinity. These changes yielded significantly reduced leupeptin uptake and could fully account for the acquired resistance. PSAC represents a novel route for the uptake of bulky hydrophilic compounds acting against intraerythrocytic parasite targets. Drug development based on such compounds should proceed cautiously in light of possible resistance development though the selection of PSAC mutants.