High Guanidinium Permeability Reveals Dehydration-Dependent Ion Selectivity in the Plasmodial Surface Anion Channel

Malaria parasites grow within vertebrate erythrocytes and increase host cell permeability to access nutrients from plasma. This increase is mediated by the plasmodial surface anion channel (PSAC), an unusual ion channel linked to the conserved clag gene family. Although PSAC recognizes and transports a broad range of uncharged and charged solutes, it must efficiently exclude the small Na+ ion to maintain infected cell osmotic stability. Here, we examine possible mechanisms for this remarkable solute selectivity. We identify guanidinium as an organic cation with high permeability into human erythrocytes infected with Plasmodium falciparum, but negligible uptake by uninfected cells. Transport characteristics and pharmacology indicate that this uptake is specifically mediated by PSAC. The rank order of organic and inorganic cation permeabilities suggests cation dehydration as the rate-limiting step in transport through the channel. The high guanidinium permeability of infected cells also allows rapid and stringent synchronization of parasite cultures, as required for molecular and cellular studies of this pathogen. These studies provide important insights into how nutrients and ions are transported via PSAC, an established target for antimalarial drug development

A potential role for plasma uric acid in the endothelial pathology of Plasmodium falciparum malaria.

BACKGROUND: Inflammatory cytokinemia and systemic activation of the microvascular endothelium are central to the pathogenesis of Plasmodium falciparum malaria. Recently, 'parasite-derived' uric acid (UA) was shown to activate human immune cells in vitro, and plasma UA levels were associated with inflammatory cytokine levels and disease severity in Malian children with malaria. Since UA is associated with endothelial inflammation in non-malaria diseases, we hypothesized that elevated UA levels contribute to the endothelial pathology of P. falciparum malaria. METHODOLOGY/PRINCIPAL FINDINGS: We measured levels of UA and soluble forms of intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-Selectin), thrombomodulin (sTM), tissue factor (sTF) and vascular endothelial growth factor (VEGF) in the plasma of Malian children aged 0.5-17 years with uncomplicated malaria (UM, n = 487) and non-cerebral severe malaria (NCSM, n = 68). In 69 of these children, we measured these same factors once when they experienced a malaria episode and twice when they were healthy (i.e., before and after the malaria transmission season). We found that levels of UA, sICAM-1, sVCAM-1, sE-Selectin and sTM increase during a malaria episode and return to basal levels at the end of the transmission season (p<0.0001). Plasma levels of UA and these four endothelial biomarkers correlate with parasite density and disease severity. In children with UM, UA levels correlate with parasite density (r = 0.092, p = 0.043), sICAM-1 (r = 0.255, p<0.0001) and sTM (r = 0.175, p = 0.0001) levels. After adjusting for parasite density, UA levels predict sTM levels. CONCLUSIONS/SIGNIFICANCE: Elevated UA levels may contribute to malaria pathogenesis by damaging endothelium and promoting a procoagulant state. The correlation between UA levels and parasite densities suggests that parasitized erythrocytes are one possible source of excess UA. UA-induced shedding of endothelial TM may represent a novel mechanism of malaria pathogenesis, in which activated thrombin induces fibrin deposition and platelet aggregation in microvessels. This protocol is registered at clinicaltrials.gov (NCT00669084)

Effects of Age, Hemoglobin Type and Parasite Strain on IgG Recognition of <i>Plasmodium falciparum</i>–Infected Erythrocytes in Malian Children

<div><p>Background</p><p>Naturally-acquired antibody responses to antigens on the surface of <i>Plasmodium falciparum</i>-infected red blood cells (iRBCs) have been implicated in antimalarial immunity. To profile the development of this immunity, we have been studying a cohort of Malian children living in an area with intense seasonal malaria transmission.</p> <p>Methodology/Principal Findings</p><p>We collected plasma from a sub-cohort of 176 Malian children aged 3-11 years, before (May) and after (December) the 2009 transmission season. To measure the effect of hemoglobin (Hb) type on antibody responses, we enrolled age-matched HbAA, HbAS and HbAC children. To quantify antibody recognition of iRBCs, we designed a high-throughput flow cytometry assay to rapidly test numerous plasma samples against multiple parasite strains. We evaluated antibody reactivity of each plasma sample to 3 laboratory-adapted parasite lines (FCR3, D10, PC26) and 4 short-term-cultured parasite isolates (2 Malian and 2 Cambodian). 97% of children recognized ≥1 parasite strain and the proportion of IgG responders increased significantly during the transmission season for most parasite strains. Both strain-specific and strain-transcending IgG responses were detected, and varied by age, Hb type and parasite strain. In addition, the breadth of IgG responses to parasite strains increased with age in HbAA, but not in HbAS or HbAC, children.</p> <p>Conclusions/Significance</p><p>Our assay detects both strain-specific and strain-transcending IgG responses to iRBCs. The magnitude and breadth of these responses varied not only by age, but also by Hb type and parasite strain used. These findings indicate that studies of acquired humoral immunity should account for Hb type and test large numbers of diverse parasite strains.</p> </div

Plasma uric acid levels correlate with inflammation and disease severity in Malian children with Plasmodium falciparum malaria.

Plasmodium falciparum elicits host inflammatory responses that cause the symptoms and severe manifestations of malaria. One proposed mechanism involves formation of immunostimulatory uric acid (UA) precipitates, which are released from sequestered schizonts into microvessels. Another involves hypoxanthine and xanthine, which accumulate in parasitized red blood cells (RBCs) and may be converted by plasma xanthine oxidase to UA at schizont rupture. These two forms of 'parasite-derived' UA stimulate immune cells to produce inflammatory cytokines in vitro.We measured plasma levels of soluble UA and inflammatory cytokines and chemokines (IL-6, IL-10, sTNFRII, MCP-1, IL-8, TNFα, IP-10, IFNγ, GM-CSF, IL-1β) in 470 Malian children presenting with uncomplicated malaria (UM), non-cerebral severe malaria (NCSM) or cerebral malaria (CM). UA levels were elevated in children with NCSM (median 5.74 mg/dl, 1.21-fold increase, 95% CI 1.09-1.35, n = 23, p = 0.0007) and CM (median 5.69 mg/dl, 1.19-fold increase, 95% CI 0.97-1.41, n = 9, p = 0.0890) compared to those with UM (median 4.60 mg/dl, n = 438). In children with UM, parasite density and plasma creatinine levels correlated with UA levels. These UA levels correlated with the levels of seven cytokines [IL-6 (r = 0.259, p<0.00001), IL-10 (r = 0.242, p<0.00001), sTNFRII (r = 0.221, p<0.00001), MCP-1 (r = 0.220, p<0.00001), IL-8 (r = 0.147, p = 0.002), TNFα (r = 0.132, p = 0.006) and IP-10 (r = 0.120, p = 0.012)]. In 39 children, UA levels were 1.49-fold (95% CI 1.34-1.65; p<0.0001) higher during their malaria episode [geometric mean titer (GMT) 4.67 mg/dl] than when they were previously healthy and aparasitemic (GMT 3.14 mg/dl).Elevated UA levels may contribute to the pathogenesis of P. falciparum malaria by activating immune cells to produce inflammatory cytokines. While this study cannot identify the cause of elevated UA levels, their association with parasite density and creatinine levels suggest that parasite-derived UA and renal function may be involved. Defining pathogenic roles for parasite-derived UA precipitates, which we have not directly studied here, requires further investigation.ClinicalTrials.gov NCT00669084

Breadth of IgG responses to parasite strains stratified by season, Hb type and age.

<p>The number of parasite strains (range, 0-7) recognized by IgG were counted and stratified by season, Hb type and age. <b>a</b>, Tukey whisker plots showing the total number of parasite strains recognized by IgG in paired plasma samples taken before and after the 2009 transmission season. p-values were calculated using Wilcoxon matched-pairs signed rank test. **p<0.01. <b>b</b>, <b>c</b>, Tukey whisker plots showing the total number of parasite strains recognized by IgG before (<b>b</b>) and after (<b>c</b>) the 2009 transmission season, stratified by age and Hb type. p-values were calculated using Kruskal-Wallis test followed by Dunn’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001.</p

The breadth of IgG responses to parasite strains correlates with increased IgG titers to merozoite antigens.

<p>IgG titers to 4 merozoite antigens (AMA-1, EBA-175, MSP-1, MSP-2) were measured in plasma obtained before the transmission season, and stratified by the total number of parasite strains recognized by IgG. Tukey whisker plots are shown. IgG titers were compared using Kruskal-Wallis test followed by Dunn’s multiple comparison test. *p<0.05. The numbers of children recognizing 0-2, 3, 4, 5 and 6-7 parasite strains were 45, 34, 32, 36 and 30.</p

Effect of Hb type on IgG recognition of different parasite strains.

<p>The proportions of children responding to each parasite strain were compared between Hb types, before and after the transmission season. The age effect at each time point was evaluated using Chi-square likelihood ratio test; significant findings were then confirmed by Odds ratio test. The seasonal effect of each Hb type was evaluated using Fisher’s exact test. *p<0.05, **p<0.01, ***p<0.001. Error bars shown are the upper 95% Confidence Interval for the % responder estimates.</p

Effect of age on IgG recognition of different parasite strains.

<p>The proportions of children responding to each parasite strain were compared between age groups, before and after the transmission season. The age effect at each time point was evaluated using Chi-square likelihood ratio test; significant findings were then confirmed by Odds ratio test. The seasonal effect in each age group was evaluated using Fisher’s exact test. *p<0.05, **p<0.01, ***p<0.001. Error bars shown are the upper 95% Confidence Interval for the % responder estimates.</p

Correlations between plasma levels of UA and those of sICAM-1 or sTM during an acute episode of <i>P. falciparum</i> malaria.

<p>The levels of UA, sICAM-1 and sTM were quantified in plasma samples from 487 Malian children at their first episode of uncomplicated <i>P. falciparum</i> malaria and their relationships analyzed. A. The graph shows a positive linear correlation between UA and sICAM-1 levels, but not at the lowest and highest UA levels measured. B. The graph shows a positive linear correlation between UA and sTM levels, but not at the lowest and highest UA levels measured. The blue line in each graph is the loess smooth using the default values <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054481#pone.0054481-WS1" target="_blank">[47]</a>, showing a moving average line with 95% pointwise confidence intervals.</p