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 compound's 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.     

Plasmodial surface anion channel-independent phloridzin resistance in Plasmodium falciparum

The plasmodial surface anion channel (PSAC) is an unusual ion channel induced on the human red blood cell membrane after infection with the malaria parasite, Plasmodium falciparum. Because PSAC is permeant to small metabolic precursors essential for parasite growth and is present on red blood cells infected with geographically divergent parasite isolates, it may be an ideal target for future antimalarial development. Here, we used chemically induced mutagenesis and known PSAC antagonists that inhibit in vitro parasite growth to examine whether resistance mutations in PSAC can be readily induced. Stable mutants resistant to phloridzin were generated and selected within 3 weeks after treatment with 1-methyl-3-nitro-1-nitrosoguanidine. These mutants were evaluated with osmotic lysis and electrophysiological transport assays, which indicate that PSAC inhibition by phloridzin is complex with at least two different modes of inhibition. Mutants resistant to the growth inhibitory effects of phloridzin expressed PSAC activity indistinguishable from that on sensitive parasites, indicating selection of resistance via mutations in one or more other parasite targets. Failure to induce mutations in PSAC activity is consistent with a highly constrained channel protein less susceptible to resistance mutations; whether this protein is parasite- or host-encoded remains to be determined.     

Selective killing of the human malaria parasite Plasmodium falciparum by a benzylthiazolium dye

Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium. The most virulent form of the disease is caused by Plasmodium falciparum which infects hundreds of millions of people and is responsible for the deaths of 1-2 million individuals each year. An essential part of the parasitic process is the remodeling of the red blood cell membrane and its protein constituents to permit a higher flux of nutrients and waste products into or away from the intracellular parasite. Much of this increased permeability is due to a single type of broad specificity channel variously called the new permeation pathway (NPP), the nutrient channel, and the Plasmodial surface anion channel (PSAC). This channel is permeable to a range of low molecular weight solutes both charged and uncharged, with a strong preference for anions. Drugs such as furosemide that are known to block anion-selective channels inhibit PSAC. In this study, we have investigated a dye known as benzothiocarboxypurine, BCP, which had been studied as a possible diagnostic aid given its selective uptake by P. falciparum infected red cells. We found that the dye enters parasitized red cells via the furosemide-inhibitable PSAC, forms a brightly fluorescent complex with parasite nucleic acids, and is selectively toxic to infected cells. Our study describes an antimalarial agent that exploits the altered permeability of Plasmodium-infected red cells as a means to killing the parasite and highlights a chemical reagent that may prove useful in high throughput screening of compounds for inhibitors of the channel.     

Voltage-dependent inactivation of the plasmodial surface anion channel via a cleavable cytoplasmic component

Erythrocytes infected with malaria parasites have increased permeability to ions and various nutrient solutes, mediated by a parasite ion channel known as the plasmodial surface anion channel (PSAC). The parasite clag3 gene family encodes PSAC activity, but there may also be additional unidentified components of this channel. Consistent with a lack of clag3 homology to genes of other ion channels, PSAC has a number of unusual functional properties. Here, we report that PSAC exhibits an unusual form of voltage-dependent inactivation. Inactivation was readily detected in the whole-cell patch-clamp configuration after steps to negative membrane potentials. The fraction of current that inactivates, its kinetics, and the rate of recovery were all voltage-dependent, though with a modest effective valence (0.7±0.1 elementary charges). These properties were not affected by solution composition or charge carrier, suggesting inactivation intrinsic to the channel protein. Intriguingly, inactivation was absent in cell-attached recordings and took several minutes to appear after obtaining the whole-cell configuration, suggesting interactions with soluble cytosolic components. Inactivation could also be largely abolished by application of intracellular, but not extracellular protease. The findings implicate inactivation via a charged cytoplasmic channel domain. This domain may be tethered to one or more soluble intracellular components under physiological conditions.     

Unidirectional dominance of cytoplasmic inheritance in two genetic crosses of Plasmodium falciparum.

Malarial parasites have two highly conserved cytoplasmic DNA molecules: a 6-kb tandemly arrayed DNA that has characteristics of a mitochondrial genome, and a 35-kb circular DNA that encodes functions commonly found in chloroplasts. We examined the inheritance pattern of these elements in two genetic crosses of Plasmodium falciparum clones. Parent-specific oligonucleotide probes and single-strand conformation polymorphism analysis identified single nucleotide changes that distinguished the parental 6- and 35-kb DNA molecules in the progeny. In all 16 independent recombinant progeny of a cross between a Central American clone, HB3, and a Southeast Asian clone, Dd2, the 6- and 35-kb DNAs were inherited from the Dd2 parent. In all nine independent recombinant progeny of a cross between clone HB3 and a likely African clone, 3D7, the 6-kb DNA was inherited from the 3D7 parent. Inheritance of cytoplasmic genomes of the Dd2 and 3D7 parents was, therefore, dominant over that of the HB3 parent. Cytoplasmic DNA molecules were found almost exclusively in the female gametes of malarial parasites; hence, clone HB3 did not appear to have served as a maternal parent for the progeny of two crosses. Defective differentiation into male gametes by clone Dd2 is likely to be a reason for the cytoplasmic inheritance pattern seen in the HB3 x Dd2 cross. However, incompetence of male or female gametes is unlikely to explain the uniparental dominance in recombinant progeny of the HB3 x 3D7 cross, since both parents readily self-fertilized and completed the malaria life cycle on their own. Instead, the data suggest unidirectional parental incompatibility in cross-fertilization of these malarial parasites, where a usually cosexual parental clone can participate only as a male or as a female. Such an incompatibility may be speculated as indicating an early phase of reproductive isolation of P. falciparum clones from different geographical regions.     

Altered plasmodial surface anion channel activity and in vitro resistance to permeating antimalarial compounds

Erythrocytes infected with malaria parasites have increased permeability to various solutes. These changes may be mediated by an unusual small conductance ion channel known as the plasmodial surface anion channel (PSAC). While channel activity benefits the parasite by permitting nutrient acquisition, it can also be detrimental because water-soluble antimalarials may more readily access their parasite targets via this channel. Recently, two such toxins, blasticidin S and leupeptin, were used to select mutant parasites with altered PSAC activities, suggesting acquired resistance via reduced channel-mediated toxin uptake. Surprisingly, although these toxins have similar structures and charge, we now show that reduced permeability of one does not protect the intracellular parasite from the other. Leupeptin accumulation in the blasticidin S-resistant mutant was relatively preserved, consistent with retained in vitro susceptibility to leupeptin. Subsequent in vitro selection with both toxins generated a double mutant parasite having additional changes in PSAC, implicating an antimalarial resistance mechanism for water-soluble drugs requiring channel-mediated uptake at the erythrocyte membrane. Characterization of these mutants revealed a single conserved channel on each mutant, albeit with distinct gating properties. These findings are consistent with a shared channel that mediates uptake of ions, nutrients and toxins. This channel's gating and selectivity properties can be modified in response to in vitro selective pressure.     

Interchromosomal exchange of a large subtelomeric segment in a Plasmodium falciparum cross.

Duplications and interchromosomal transpositions of chromosome segments are implicated in the genetic variability of Plasmodium falciparum malaria parasites. One parasite clone, HB3, was shown to lack a subtelomeric region of chromosome 13 that normally carries a PfHRPIII gene. We show here that the chromosome 13 segment carrying PfHRPIII was replaced in HB3 by a duplicated terminal segment from chromosome 11. Mapping results indicate that the segment includes at least 100-200 kb of subtelomeric DNA and contains duplicated copies of the Pf332 and RESA-2 genes. We followed inheritance of this duplication in a genetic cross between the HB3 and another P.falciparum clone, Dd2, that is euploid for the Pf332, RESA-2 and PfHRPIII genes. Three types of progeny from the cross showed expected inheritance forms: a Dd2 euploid parent type, an HB3 aneuploid parent type, and a recombinant euploid type that carried PfHRPIII from Dd2 chromosome 13 and Pf332 from HB3 chromosome 11. However, a fourth euploid progeny type was also observed, in which the chromosome 13 segment from HB3 was transposed back to replace the terminus of chromosome 11. Three of 14 individual progeny were of this type. These findings suggest a mechanism of recombination from subtelomeric pairing and exchange between non-homologous chromosomes in meiosis.     

Epigenetic switches in clag3 genes mediate blasticidin S resistance in malaria parasites

Malaria parasites induce changes in the permeability of the infected erythrocyte membrane to numerous solutes, including toxic compounds. In Plasmodium falciparum, this is mainly mediated by PSAC, a broad‐selectivity channel that requires the product of parasite clag3 genes for its activity. The two paralogous clag3 genes, clag3.1 and clag3.2, can be silenced by epigenetic mechanisms and show mutually exclusive expression. Here we show that resistance to the antibiotic blasticidin S (BSD) is associated with switches in the expression of these genes that result in altered solute uptake. Low concentrations of the drug selected parasites that switched from clag3.2 to clag3.1 expression, implying that expression of one or the other clag3 gene confers different transport efficiency to PSAC for some solutes. Selection with higher BSD concentrations resulted in simultaneous silencing of both clag3 genes, which severely compromises PSAC formation as demonstrated by blocked uptake of other PSAC substrates. Changes in the expression of clag3 genes were not accompanied by large genetic rearrangements or mutations at the clag3 loci or elsewhere in the genome. These resultsdemonstrate that malaria parasites can become resistant to toxic compounds such as drugs by epigenetic switches in the expression of genes necessary for the formation of solute channels.     

Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors

Malaria parasites increase their host erythrocyte’s permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC) mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against in vitro parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel’s structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing.     

Variation among Plasmodium falciparum strains in their reliance on mitochondrial electron transport chain function

Previous studies demonstrated that Plasmodium falciparum strain D10 became highly resistant to the mitochondrial electron transport chain (mtETC) inhibitor atovaquone when the mtETC was decoupled from the pyrimidine biosynthesis pathway by expressing the fumarate-dependent (ubiquinone-independent) yeast dihydroorotate dehydrogenase (yDHODH) in parasites. To investigate the requirement for decoupled mtETC activity in P. falciparum with different genetic backgrounds, we integrated a single copy of the yDHODH gene into the genomes of D10attB, 3D7attB, Dd2attB, and HB3attB strains of the parasite. The yDHODH gene was equally expressed in all of the transgenic lines. All four yDHODH transgenic lines showed strong resistance to atovaquone in standard short-term growth inhibition assays. During longer term growth with atovaquone, D10attB-yDHODH and 3D7attB-yDHODH parasites remained fully resistant, but Dd2attB-yDHODH and HB3attB-yDHODH parasites lost their tolerance to the drug after 3 to 4 days of exposure. No differences were found, however, in growth responses among all of these strains to the Plasmodium-specific DHODH inhibitor DSM1 in either short- or long-term exposures. Thus, DSM1 works well as a selective agent in all parasite lines transfected with the yDHODH gene, whereas atovaquone works for some lines. We found that the ubiquinone analog decylubiquinone substantially reversed the atovaquone inhibition of Dd2attB-yDHODH and HB3attB-yDHODH transgenic parasites during extended growth. Thus, we conclude that there are strain-specific differences in the requirement for mtETC activity among P. falciparum strains, suggesting that, in erythrocytic stages of the parasite, ubiquinone-dependent dehydrogenase activities other than those of DHODH are dispensable in some strains but are essential in others.     

Proteolysis at a Specific Extracellular Residue Implicates Integral Membrane CLAG3 in Malaria Parasite Nutrient Channels

The plasmodial surface anion channel mediates uptake of nutrients and other solutes into erythrocytes infected with malaria parasites. The clag3 genes of P. falciparum determine this channel’s activity in human malaria, but how the encoded proteins contribute to transport is unknown. Here, we used proteases to examine the channel’s composition and function. While proteases with distinct specificities all cleaved within an extracellular domain of CLAG3, they produced differing degrees of transport inhibition. Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone. Inheritance of functional proteolysis in the HB3×Dd2 genetic cross, DNA transfection, and gene silencing experiments all pointed to the clag3 genes, providing independent evidence for a role of these genes. Protease protection assays with a Dd2-specific inhibitor and site-directed mutagenesis revealed that a variant L1115F residue on a CLAG3 extracellular loop contributes to inhibitor binding and accounts for differences in functional proteolysis. These findings indicate that surface-exposed CLAG3 is the relevant pool of this protein for channel function. They also suggest structural models for how exposed CLAG3 domains contribute to pore formation and parasite nutrient uptake.     

The plasmodial surface anion channel is functionally conserved in divergent malaria parasites

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 PSAC's 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.     

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.     

Two Distinct Mechanisms of Transport Through the Plasmodial Surface Anion Channel

The plasmodial surface anion channel (PSAC) is a voltage-dependent ion channel on erythrocytes infected with malaria parasites. To fulfill its presumed function in parasite nutrient acquisition, PSAC is permeant to a broad range of charged and uncharged solutes; it nevertheless excludes Na⁺ as required to maintain erythrocyte osmotic stability in plasma. Another surprising property of PSAC is its small single-channel conductance (<3 pS in isotonic Cl^?) in spite of broad permeability to bulky solutes. While exploring the mechanisms underlying these properties, we recently identified interactions between permeating solutes and PSAC inhibitors that suggest the channel has more than one route for passage of solutes. Here, we explored this possibility with 22 structurally diverse solutes and found that each could be classified into one of two categories based on effects on inhibitor affinity, the temperature dependence of these effects and a clear pattern of behavior in permeant solute mixtures. The clear separation of these solutes into two discrete categories suggests two distinct mechanisms of transport through this channel. In contrast to most other broad-permeability channels, selectivity in PSAC appears to be complex and cannot be adequately explained by simple models that invoke sieving through rigid, noninteracting pores.     

Specific inhibition of the plasmodial surface anion channel by dantrolene

The plasmodial surface anion channel (PSAC), induced on human erythrocytes by the malaria parasite Plasmodium falciparum, is an important target for antimalarial drug development because it may contribute to parasite nutrient acquisition. However, known antagonists of this channel are quite nonspecific, inhibiting many other channels and carriers. This lack of specificity not only complicates drug development but also raises doubts about the exact role of PSAC in the well-known parasite-induced permeability changes. We recently identified a family of new PSAC antagonists structurally related to dantrolene, an antagonist of muscle Ca++ release channels. Here, we explored the mechanism of dantrolene's actions on parasite-induced permeability changes. We found that dantrolene inhibits the increased permeabilities of sorbitol, two amino acids, an organic cation, and hypoxanthine, suggesting a common pathway shared by these diverse solutes. It also produced parallel reductions in PSAC single-channel and whole-cell Cl- currents. In contrast to its effect on parasite-induced permeabilities, dantrolene had no measurable effect on five other classes of anion channels, allaying concerns of poor specificity inherent to other known antagonists. Our studies indicate that dantrolene binds PSAC at an extracellular site distinct from the pore, where it inhibits the conformational changes required for channel gating. Its affinity for this site depends on ionic strength, implicating electrostatic interactions in dantrolene binding. In addition to the potential therapeutic applications of its derivatives, dantrolene's specificity and its defined mechanism of action on PSAC make it a useful tool for transport studies of infected erythrocytes.     

Long-range restriction maps of Plasmodium falciparum chromosomes: Crossingover and size variation among geographically distant isolates

Homologous chromosomes from the human malaria parasite Plasmodium falciparum exhibit striking size polymorphism from isolate to isolate. To examine the structural basis for these variations, we have determined full-length restriction maps of chromosome 4 from three P. falciparum clones. Two clones, HB3 and 3D7, are derived from geographically distant strains, while the third, XP5, is the product of an HB3/3D7 cross. The restriction maps show that, while the overall structure and organization of chromosome 4 from each clone are similar, large-scale variations occur within a few hundred kilobase pairs of the chromosome ends. An apparent crossover between the 3D7 and the HB3 parent chromosomes accounts for a chromosome of intermediate size in clone XP5. Similar restriction studies extended to other parasite chromosomes will ultimately yield a long-range physical map of the P. falciparum genome.     

Linkage Group Selection--a fast approach to the genetic analysis of malaria parasites

Genetic analysis of malaria parasites has shown that the mechanisms of inheritance in these organisms are classically Mendelian. In other words, alleles of genes at different loci recombine, and alleles at the same gene locus segregate, in the progeny of a genetic cross between two genetically distinct lines of malaria parasite. Importantly, such progeny are haploid in the first filial generation following genetic crossing. Consequently, genetic analysis, including linkage analysis, can be done directly upon the cloned cross progeny. Linkage analysis conducted upon the progeny of genetic crosses between malaria parasites can lead to the location of a single gene controlling a specific phenotype, as has been achieved to identify the gene for chloroquine resistance in Plasmodium falciparum. The work involved, however, is extremely labour intensive. It involves the generation of many hundreds, to a thousand or so, of independent recombinant clones from the cross progeny and the biological characterisation, and genetic typing for hundreds of molecular genetic markers of each such clone. We discuss here a fast-track method for identifying genes controlling specific phenotypes, e.g. drug resistance/sensitivity. It involves the mass screening with quantitative molecular genetic markers of the uncloned progeny of a genetic cross following its growth under a selection pressure representing the phenotype of interest. We have called the method Linkage Group Selection.     

The role of Plasmodium falciparum food vacuole plasmepsins

Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions.