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.     

Complex inheritance of the plasmodial surface anion channel in a Plasmodium falciparum genetic cross

Human erythrocytes infected with the malaria parasite Plasmodium falciparum have increased permeabilities to many solutes. The plasmodial surface anion channel (PSAC) may mediate these changes. Despite good understanding of the biochemical and biophysical properties, the genetic basis of PSAC activity remains unknown. Functional polymorphisms in laboratory isolates and two mutants generated by in vitro selection implicate a parasite-encoded channel, although parasite-induced modifications of endogenous channels have not been formally excluded. Here, we identified stable differences in furosemide efficacy against PSAC activity induced by HB3 and 3D7A parasites. This difference was apparent in both single PSAC patch-clamp recordings and in sorbitol-mediated osmotic lysis measurements, confirming that Cl^- and sorbitol are transported by a single-channel type. Examination of 19 progeny from a genetic cross between HB3 and 3D7A revealed complex inheritance with some cloned progeny exhibiting furosemide affinities outside the range of parental values. Isolates generated by selfing of the 3D7A clone also exhibited altered furosemide affinities, implicating changes in one or more alleles during meiosis or passage through a primate host. PSAC may be encoded by multiple parasite genes (e.g. a multi-gene family or multiple genes that encode distinct channel subunits) or a single polymorphic gene under strong selective pressure.     

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.     

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.     

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.     

Induction of Cycloheximide-Resistant Mutants in Saccharomyces cerevisiae with N-Methyl-N′-Nitro-N-Nitrosoguanidine and ICR-170

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cycloheximide-resistant mutations in Saccharomyces cerevisiae, but few, if any, resistant mutants are induced by the acridine mustard ICR-170. Cycloheximide sensitivity in yeast is associated with the ribosome, and treatment with the antibiotic at concentrations of 2 mug/ml results in complete inhibition of protein synthesis. Missense mutations induced by MNNG probably lead to the loss of cycloheximide binding sites on the ribosome, resulting in resistance to the antibiotic without altering the activity of the organelle in protein synthesis. ICR-170, however, induced primarily frameshift mutations that would alter ribosome structural integrity, resulting in cell death rather than resistance. ICR-170 and MNNG are both mutagenic in a system in which base-pair substitution and frameshift mutations can be detected. These results indicate that cycloheximide resistance in S. cerevisiae, like streptomycin and spectinomycin resistance in Escherichia coli, can be induced by base-pair substitution mutagens but not by frameshift mutagens such as ICR-170.     

L-Methionine SR-sulfoximine-resistant glutamine synthetase from mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium resistant to growth inhibition by the glutamine synthetase transition state analog, L-methionine SR-sulfoximine, were isolated and characterized. These mutants are glutamine bradytrophs and cannot use growth rate-limiting nitrogen sources. Although this phenotype resembles that of mutants with lesions in the regulatory gene for glutamine synthetase, glnG, these mutations do not lie in the glnG gene. Purification and characterization of the glutamine synthetase from one of the mutants and a control strain demonstrated that the mutant enzyme is defective in the reverse gamma-glutamyltransferase activity but has biosynthetic activity that is resistant to inhibition by L-methionine SR-sulfoximine. The mutant enzyme also has a 4.4-fold higher apparent Km for glutamate (0.2 mM versus 2.1 mM, respectively) and a 13.8-fold higher Km for NH3 (6.4 mM versus 0.46 mM) than the enzyme from the control. These data show that the glutamine synthetase protein has been altered by this mutation, designated as glnA982, and suggest that the L-methionine SR-sulfoximine resistance is conferred by a change in the NH3 binding domain of the enzyme.     

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.     

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.     

Galactokinase-deficient mutants of Tetrahymena thermophila: Selection and characterization

Summary We have isolated a series of mutants of Tetrahymena thermophila which are resistant to inhibition of growth by the galactose analog, 2-deoxygalactose. These mutants were obtained after mutagenesis with nitrosoguanidine and the induction of cytogamy to permit the recovery of recessive mutations induced in the germline micronucleus. Resistance to 2-deoxygalactose is correlated with a decreased rate of growth in galactose minimal medium and greatly reduced levels of galactokinase. The resistant phenotype of the mutants is apparently due to the galactokinase deficiency, which prevents the accumulation of toxic phosphorylated metabolites of 2-deoxygalactose. Genetic analyses reveal that the 2-deoxygalactose resistance alleles segregate as single Mendelian loci. The galactokinase-deficient strains described here represent the first mutants in this organism for which the biochemical basis of the mutant phenotype is known. These mutants, as well as others isolated similarly, should be of value in the elucidation of the mechanisms governing galactokinase gene regulation and in improving techniques of selection for other recessive mutations in Tetrahymena.     

Plasmodium falciparum regulatory subunit of cAMP-dependent PKA and anion channel conductance

Malaria symptoms occur during Plasmodium falciparum development into red blood cells. During this process, the parasites make substantial modifications to the host cell in order to facilitate nutrient uptake and aid in parasite metabolism. One significant alteration that is required for parasite development is the establishment of an anion channel, as part of the establishment of New Permeation Pathways (NPPs) in the red blood cell plasma membrane, and we have shown previously that one channel can be activated in uninfected cells by exogenous protein kinase A. Here, we present evidence that in P. falciparum-infected red blood cells, a cAMP pathway modulates anion conductance of the erythrocyte membrane. In patch-clamp experiments on infected erythrocytes, addition of recombinant PfPKA-R to the pipette in vitro, or overexpression of PfPKA-R in transgenic parasites lead to down-regulation of anion conductance. Moreover, this overexpressing PfPKA-R strain has a growth defect that can be restored by increasing the levels of intracellular cAMP. Our data demonstrate that the anion channel is indeed regulated by a cAMP-dependent pathway in P. falciparum-infected red blood cells. The discovery of a parasite regulatory pathway responsible for modulating anion channel activity in the membranes of P. falciparum-infected red blood cells represents an important insight into how parasites modify host cell permeation pathways. These findings may also provide an avenue for the development of new intervention strategies targeting this important anion channel and its regulation.     

The causal relationship between mutations in cAMP-dependent protein kinase and the loss of adrenocorticotropin-regulated adrenocortical functions.

The Y1 adrenocortical tumor cell mutants, Kin-7 and Kin-8, harbor point mutations in the regulatory subunit (RI) of the type 1 cAMP-dependent protein kinase (cAMPdPK) that render the enzyme resistant to activation by cAMP. These mutants also are resistant to many of the regulatory effects of ACTH and cAMP. In order to examine the causal relationships between the mutations in cAMPdPK and the resistance to ACTH and cAMP, the Kin mutants were transfected with expression vectors encoding wild type subunits of cAMPdPK in order to restore cAMP-responsive protein kinase activity. The transformants then were screened for the concomitant recovery of cellular responsiveness to ACTH and cAMP. In the mutant Kin-7, cAMP-responsive protein kinase activity was recovered after transfection with an expression vector encoding wild type mouse RI. Protein kinase activity in the mutant Kin-8 remained largely cAMP-resistant after transfection with the RI expression vector but could be rendered cAMP-responsive by transfection with an expression vector encoding the wild type catalytic subunit. The recovery of cAMP-responsive protein kinase activity was accompanied by the recovery of steroidogenic and morphological responses to ACTH and cAMP, suggesting that the cAMP-dependent signaling cascade plays an obligatory role in these actions of ACTH. The growth-regulatory effects of cAMP were not reversed with the recovery of cAMP-responsive protein kinase activity, suggesting that cAMP-resistant growth regulation results from second-site, adaptive mutations either in the original Kin mutant population or in the transformants. Studies on the conversion of 22(R)-hydroxycholesterol into steroid products in parent and mutant cells indicate that the Kin mutations reduce the steroidogenic capacity of the cell as well as inhibit the hormone- and cyclic nucleotide-dependent mobilization of substrate cholesterol.     

New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity.

In Escherichia coli K-12 mutants which had a new nalidixic acid resistance mutation at about 82 min on the chromosome map, cell growth was resistant to or hypersusceptible to nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, and novobiocin. Deoxyribonucleic acid gyrase activity as tested by supercoiling of lambda phage deoxyribonucleic acid inside the mutants was similarly resistant or hypersusceptible to the compounds. The drug concentrations required for gyrase inhibition were much higher than those for cell growth inhibition but similar to those for inhibition of lambda phage multiplication. Transduction analysis with lambda phages carrying the chromosomal fragment of the tnaA-gyrB region suggested that one of the mutations, nal-31, was located on the gyrB gene.     

Resistance of the melibiose carrier to inhibition by the phosphotransferase system due to substitutions of amino acid residues in the carrier of Salmonella typhimurium.

The melibiose carrier of Salmonella typhimurium is under the control of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). We isolated mutants of the melibiose carrier that showed resistance to inhibition via the PTS. Growth of the mutants on melibiose was not inhibited by 2-deoxyglucose, a non-metabolizable substrate of the PTS, although growth of the parent strain was inhibited. Transport activity of the melibiose carrier in the mutants was fairly resistant to inhibition by 2-deoxyglucose, although the activity in the parent was sensitive to inhibition. We cloned the mutated melB gene that encodes the melibiose carrier, determined the nucleotide sequences, and identified replaced nucleotides. The mutations resulted in substitutions of Asp-438 with Tyr, Arg-441 with Ser, or Ile-445 with Asn. All of these residues are in the COOH-terminal region of the carrier. The secondary structure of this region is predicted to be an alpha-helix, and the mutated residues were on the same side of the helix. This region showed sequence similarity to a region of the MalK protein, in which substitution of amino acid residues also resulted in PTS-resistant mutants. Thus the COOH-terminal portion of the melibiose carrier is important for the interaction of dephosphorylated IIIGlc, which is an entity causing reversible inactivation of the carrier.     

THE PHENOTYPIC AND FITNESS EFFECTS OF COLICIN RESISTANCE IN ESCHERICHIA COLI K-12

Previous studies indicate that most natural isolates of Escherichia coli are resistant to most or all colicins (antibiotics produced by E. coli) when assessed in the laboratory. Additionally, resistance to different colicin types appears to arise in a nonindependent manner. One possible mechanism to explain this nonindependence is pleiotropy: Multiple resistances are selected after exposure to a single colicin. This study, which was designed to address the role of pleiotropy in the generation of colicin resistance, revealed that 96% of colicin resistant mutants were resistant to two or more colicins. Mutational class was important because putative translocation mutants (Tol pathway mutants) resisted fewer colicins than putative receptor mutants. To determine whether colicin resistance is costly, the effects of colicin resistance mutations on maximal growth rate in a rich medium were also examined. Relative to the sensitive ancestor, translocation mutations lowered maximal growth rates by 17%, whereas putative receptor mutations did not significantly lower growth rates. Thus, when nutrients are abundant, the most advantageous forms of colicin resistance may not impose a cost. The ecological consequences of pleiotropic colicin resistance could involve population cycling between colicin sensitivity and resistance. Additionally, if the cost of resistance depends on the environment, ecological diversification could result.     

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.     

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.     

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.     

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.