Structural requirements for the adherence of Plasmodium falciparum-infected erythrocytes to chondroitin sulfate proteoglycans of human placenta

Plasmodium falciparum infection during pregnancy results in the accumulation of infected red blood cells (IRBCs) in the placenta, leading to poor pregnancy outcome. In the preceding paper (Achur, R. N., Valiyaveettil, M., Alkhalil, A., Ockenhouse, C. F., and Gowda, D. C. (2000) J. Biol. Chem. 275, 40344-40356), we reported that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta mediate the IRBC adherence. In this study, we report the structural requirements for the adherence and the minimum chondroitin 4-sulfate (C4S) structural motif that supports IRBC adherence. Partially sulfated C4Ss with varying sulfate contents were prepared by solvolytic desulfation of a fully sulfated C4S. These and other nonmodified C4Ss, with different proportions of 4-, 6-, and nonsulfated disaccharide repeats, were analyzed for inhibition of IRBC adherence to the placental CSPG. C4Ss containing 30-50% 4-sulfated and 50-70% nonsulfated disaccharide repeats efficiently inhibited IRBC adherence; C6S had no inhibitory activity. Oligosaccharides of varying sizes were prepared by the partial depolymerization of C4Ss containing varying levels of 4-sulfation, and their ability to inhibit the IRBC adherence was studied. Oligosaccharides with six or more disaccharide repeats inhibited IRBC adherence to the same level as that of the intact C4Ss, indicating that a dodecasaccharide is the minimum structural motif required for optimal IRBC adherence. Of the C4S dodecasaccharides, only those with two or three sulfate groups per molecule showed maximum IRBC inhibition. These data define the structural requirements for the IRBC adherence to placental CSPGs with implications for the development of therapeutics for maternal malaria.     

In vitro screening of Fe2+‐chelating effect by a Fenton's reaction–luminol chemiluminescence system

In vitro screening of a Fe²⁺‐chelating effect using a Fenton's reaction–luminol chemiluminescence (CL) system is described. The luminescence between the reactive oxygen species generated by the Fenton's reaction and luminol was decreased on capturing Fe²⁺ using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 μL) at micromolar concentrations. After optimization of the screening conditions, the efficacies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10‐phenanthroline were examined. EC₅₀ values for these compounds (except 1,10‐phenanthroline) were in the range 3.20 ± 0.87 to 9.57 ± 0.64 μM (n = 3). Rapid measurement of the Fe²⁺‐chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Plasmodium falciparum cytoadherence to human placenta: evaluation of hyaluronic acid and chondroitin 4-sulfate for binding of infected erythrocytes.

Chondroitin 4-sulfate (C4S) is known to mediate the adherence of Plasmodium falciparum infected red blood cells (IRBCs) to human placenta. Recently, hyaluronic acid (HA) has also been reported to bind IRBCs, and HA has been suggested as an additional receptor for the sequestration of IRBCs in the placenta. In this study, we assessed the adherence of 3D7 parasite strain, which has been reported to bind both C4S and HA, using highly purified clinical grade rooster comb HA, Streptococcus HA, several preparations of human umbilical cord HA (hucHA), and bovine vitreous humor HA (bvhHA). While all hucHA preparations and bvhHA bound with moderate to high density to IRBCs, the rooster comb and bacterial HAs did not bind IRBCs. IRBCs binding to the hucHA and bvhHA could be abolished by pretreatment with testicular hyaluronidase but not with Streptomyces hyalurolyticus hyaluronidase, suggesting that IRBC binding to hucHA and bvhHA was due to chondroitin sulfate (CS) contaminants in HAs. Compositional analysis confirmed the presence of CS in both hucHA and bvhHA. The CSs present in these commercial hucHA and bvhHA samples were isolated, characterized, and studied for their ability to bind IRBCs. The data suggested that IRBC adherence to hucHA and bvhHA was mediated by the CS present in these samples. However, our data did not exclude the possibility of a minor population of distinct parasite subtype adhering to HA and further studies using pure HA conjugated to proteins or lipids and placental parasite isolates should clarify whether HA is an in vivo receptor for IRBC adherence.     

Characterization of proteoglycans of human placenta and identification of unique chondroitin sulfate proteoglycans of the intervillous spaces that mediate the adherence of Plasmodium falciparum-infected erythrocytes to the placenta

In pregnant women infected with Plasmodium falciparum, the infected red blood cells (IRBCs) selectively accumulate in the intervillous spaces of placenta, leading to poor fetal outcome and severe health complications in the mother. Although chondroitin 4-sulfate is known to mediate IRBC adherence to placenta, the natural receptor has not been identified. In the present study, the chondroitin sulfate proteoglycans (CSPGs) of human placenta were purified and structurally characterized, and adherence of IRBCs to these CSPGs investigated. The data indicate that the placenta contains three distinct types of CSPGs: significant quantities of uniquely low sulfated, extracellular CSPGs localized in the intervillous spaces, minor amounts of two cell-associated CSPGs, and major amounts of dermatan sulfate-like CSPGs of the fibrous tissue. Of the various CSPGs isolated from the placenta, the low sulfated CSPGs of the intervillous spaces most efficiently bind IRBCs. Based on IRBC adherence capacities and localization patterns of various CSPGs, we conclude that the CSPGs of the intervillous spaces are the receptors for placental IRBC adherence. The identification and characterization of these CSPGs provide a valuable tool for understanding the precise molecular interactions involved in placental IRBC adherence and for the development of therapeutic strategies for maternal malaria. In the accompanying paper (Alkhalil, A., Achur, R. N., Valiyaveettil, M., Ockenhouse, C. F., and Gowda, D. C. (2000) J. Biol. Chem. 275, 40357-40364), we report the structural requirements for the IRBC adherence.     

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.     

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.     

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.     

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.