Production of cAMP by HCO₃⁻ sensitive PfACβ and regulation of microneme secretion.

<p>A) HCO⁻₃ sensitive adenylyl cyclase activity in <i>P. falciparum</i> merozoite lysates. <i>P. falciparum</i> merozoite lysates were incubated with increasing concentrations of NaHCO₃ (10 mM, 20 mM and 40 mM) and ATP for 30 min at 30°C and production of cAMP was measured. Levels of cAMP are reported (mean + SD from three independent experiments) as femtomoles per µg of merozoite protein. Merozoite lysates without exogenous ATP were used as negative controls. Production of cAMP increased with increasing concentrations of NaHCO₃. Addition of KH7 in presence of 40 mM NaHCO₃ inhibited cAMP production. B) Intracellular pH (pH_i) of <i>P. falciparum</i> merozoites in different ionic environments with and without treatment with carbonic anhydrase (CA) inhibitor acetazolamide (ACTZ). <i>P. falciparum</i> merozoites loaded with the pH-sensitive fluorescent dye BCECF-AM (inset) were transferred from IC to EC buffer with or without prior treatment with ACTZ. Fluorescence signal from BCECF was measured and used to determine pH_i using a standard curve (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004520#ppat.1004520.s006" target="_blank">S6 Figure</a>) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004520#s4" target="_blank">Materials and Methods</a>. The pH_i of <i>P. falciparum</i> merozoites remains unchanged following transfer from IC to EC buffer. Pre-treatment of <i>P. falciparum</i> merozoites with ACTZ results in a rise in pH_i following transfer from IC to EC buffer (EC+ACTZ). C) ACTZ blocks rise in intracellular cAMP following transfer of <i>P. falciparum</i> merozoites from IC to EC buffer. <i>P. falciparum</i> merozoites were transferred from IC to EC buffer with or without treatment with ACTZ. Fold change (mean ± SD) in cAMP levels is reported from 3 independent experiments. Pre-treatment with ACTZ inhibits rise in cAMP in merozoites following transfer from IC to EC buffer. D) ACTZ blocks microneme secretion following transfer of <i>P. falciparum</i> merozoites from IC to EC buffer. <i>P. falciparum</i> merozoites were transferred form IC to EC buffer with or without prior treatment with CA inhibitor, ACTZ. Secretion of PfAMA1 in merozoite supernatants (AMA1(s)) was detected by Western blotting. Presence of cytoplasmic protein PfNapL was detected in <i>P. falciparum</i> merozoite supernatants (NapL(s)) and lysates of merozoite pellets (NapL(p)) under different conditions by Western blotting to control for merozoite lysis and number of merozoites used in the different conditions, respectively. Treatment of merozoites with ACTZ prior to transfer from IC to EC buffer inhibits secretion of microneme protein PfAMA1. Treatment of merozoites with ACTZ+IBMX, ACTZ+DiB+Epac Agonist and ACTZ+DiB+A23187 restores microneme secretion. However, treatment with ACTZ+DiB does not restore microneme secretion.</p

Model for cAMP and Ca²⁺ mediated signaling pathways that regulate microneme secretion in <i>P falciparum</i> merozoites.

<p>Exposure of <i>P. falciparum</i> merozoites to low K⁺ environment as present in blood plasma leads to production of H⁺ and HCO₃⁻ ions by carbonic anhydrase (CA) to maintain pH. HCO₃⁻ ions activate cytoplasmic adenylyl cyclase (ACβ) leading to rise in cytosolic levels of cAMP. Elevation of cAMP activates its downstream effectors PKA and Epac. Epac activates Rap1 by transferring GTP. Rap1-GTP activates PLC leading to rise in cytosolic Ca²⁺ levels, which leads to activation of calcium dependent protein kinase 1 (CDPK1) and calcium dependent phosphatase, calcineurin (CN), both of which directly play roles in microneme secretion. Mn, micronemes; Rh, rhoptries.</p

Crosstalk between cAMP and Ca²⁺ in <i>P. falciparum</i> merozoites.

<p>A) Ca²⁺ does not regulate cytosolic cAMP levels. <i>P. falciparum</i> merozoites were transferred from IC to EC buffer with or without treatment with BAPTA-AM or U73122. Levels of cytosolic cAMP were measured in merozoite lysates before and after transfer to EC buffer. Fold changes in cAMP levels per µg of merozoite protein (mean ± SD from 3 independent experiments) in different conditions relative to cAMP levels in IC buffer (mean ± SD) from 3 independent experiments are reported. Treatment of merozoites with BAPTA-AM or U73122 does not have any effect on rise in intracellular cAMP levels following transfer from IC to EC buffer. B) Rise in cytosolic Ca²⁺ is inhibited by ACβ inhibitor KH7. <i>P. falciparum</i> merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer with or without treatment with KH7. Cytosolic Ca²⁺ levels in <i>P. falciparum</i> merozoites were measured before and after transfer by flow cytometry. Treatment with KH7 inhibits the rise in cytosolic Ca²⁺ following transfer to EC buffer. C) PKA does not regulate cytosolic Ca²⁺. <i>P. falciparum</i> PHL dhfr-PfPKAr merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer or from IC to IC+DiB or from IC to EC+DiB. Cytosolic Ca²⁺ levels rise normally following transfer of <i>P. falciparum</i> merozoites from IC to EC buffer. Cytosolic Ca²⁺ levels do not rise when <i>P. falciparum</i> PHL dhfr-PfPKAr merozoites in IC buffer are treated with DiB indicating that PKA does not play a role in regulating Ca²⁺ levels in merozoites. D) Regulation of cytosolic Ca²⁺ levels in <i>P. falciparum</i> 3D7 merozoites by Epac. <i>P falciparum</i> merozoites loaded with Fluo-4AM were transferred from IC to EC buffer or from IC to IC buffer containing Epac agonist 8-Pcpt-2’-O-Me-cAMP (IC+Epac agonist), DiB (IC+DiB), or Epac agonist and Epac inhibitors (IC+Epac agonist+ESI-09 or IC+Epac agonist+ESI-05). Cytosolic Ca²⁺ levels rise when merozoites are transferred from IC to EC buffer, or IC buffer to IC+Epac agonist, but not when they are transferred from IC buffer to IC+DiB. EPAC1 antagonist ESI-09 inhibits rise in Ca²⁺ stimulated by Epac agonist. E) Regulation of cytosolic Ca²⁺ levels in <i>P. falciparum</i> 3D7 merozoites by Epac agonist, PLC inhibitor and Rap1 inhibitor. <i>P falciparum</i> merozoites loaded with Fluo-4AM were transferred from IC to EC buffer, or from IC to IC buffer containing Epac agonist (IC+Epac agonist), or IC buffer containing Epac agonist and PLC inhibitor (IC+Epac agonist+U73122), or IC buffer containing Epac agonist and Rap1 inhibitor GGTI298 (IC+Epac agonist+GGTI298). Cytosolic Ca²⁺ levels rise when merozoites are transferred from IC to EC buffer, or from IC to IC+Epac agonist. PLC inhibitor U73122 and Rap1 inhibitor GGTI298 inhibit rise in cytosolic Ca²⁺ stimulated by Epac agonist. F) Cytosolic Ca²⁺ levels in <i>P. falciparum</i> 3D7 merozoites following transfer to EC buffer in presence of Epac and Rap1 inhibitors. <i>P falciparum</i> merozoites loaded with Fluo-4AM were transferred from IC to EC buffer containing Epac inhibitors (EC+ESI-09 or EC+ESI-05), or EC buffer containing Rap1 inhibitor (EC+GGTI298). Cytosolic Ca²⁺ rises when merozoites are transferred from IC to EC buffer. Presence of Epac inhibitor ESI-09 and Rap1 inhibitor GGTI298 inhibits rise in cytosolic Ca²⁺.</p

cAMP responsive kinase activity in <i>P. falciparum</i> merozoites under different ionic conditions and microneme secretion.

<p>A) Kemptide phosphorylation by <i>P. falciparum</i> 3D7 merozoite lysates under different ionic environments. <i>P. falciparum</i> 3D7 merozoites were transferred from IC buffer to EC buffer with or without treatment with ACβ inhibitor, KH7. Merozoite lysates made under different conditions were incubated with kemptide, a protein kinase A (PKA) substrate, and γ-³²P-ATP. Fold change (mean ± SD) in phosphorylation of kemptide under different ionic conditions is reported relative to phosphorylation in IC buffer from 3 independent experiments. B) Kemptide phosphorylation by lysates of transgenic <i>P. falciparum</i> PHL dhfr-<i>Pf</i>PKAr merozoites under different ionic environments. <i>P. falciparum</i> PHL dhfr-<i>Pf</i>PKAr merozoites were isolated in IC buffer and transferred to EC buffer with or without treatment with dibutryl-cAMP (DiB). Phosphorylation of kemptide by merozoite lysates made under different conditions was measured in presence of γ-³²P-ATP. Fold change in kemptide phosphorylation (mean ± SD) by merozoite lysates under different conditions is reported relative to that in IC buffer from 3 independent experiments. C) Microneme secretion in <i>P. falciparum</i> PHL dhfr-PfPKAr merozoites. <i>P. falciparum</i> PHL dhfr-PfPKAr merozoites were transferred from IC to EC buffer, IC buffer+DiB, or EC buffer+DiB and secretion of PfAMA1 into merozoite supernatants (AMA1(s)) was detected by Western blotting. Presence of cytoplasmic protein PfNapL was detected in <i>P. falciparum</i> merozoite supernatants (NapL(s)) and lysates of merozoite pellets (NapL(p)) under different conditions by Western blotting to control for merozoite lysis and number of merozoites used in the different conditions, respectively. PfAMA1 was not secreted when <i>P. falciparum</i> PHL dhfr-PfPKAr merozoites were transferred from IC to IC+DiB, or from IC to EC buffer. PfAMA1 was secreted when <i>P. falciparum</i> PHL dhfr-PfPKAr merozoites were transferred from IC to EC+DiB.</p

Production of cAMP in <i>P. falciparum</i> merozoites by adenylyl cyclase β (PfACβ) and microneme exocytosis.

<p>A) Expression of PfACβ in <i>P. falciparum</i> blood stages. Immunofluorescence assays (IFA) were used to detect PfACβ (green) in <i>P. falciparum</i> rings (i), trophozoites (ii), schizonts (iii) and merozoites (iv) using mouse antisera against a peptide derived from PfACβ. Nuclear DNA was stained with DAPI (blue). Rabbit antiserum against <i>P. falciparum</i> cytoplasmic protein PfNAPL (red) was used for co-localization. Yellow indicates overlap of PfACβ and PfNAPL. B) Exposure of <i>P. falciparum</i> merozoites to low K⁺ triggers production of cAMP. <i>P. falciparum</i> merozoites were isolated in buffer mimicking intracellular ionic environment (IC buffer – 140 mM KCl, 5 mM NaCl, 1 mM MgCl₂, 5.6 mM glucose, 25 mM HEPES, pH 7.2) and transferred to buffer mimicking extracellular ionic environment (EC buffer – 5 mM KCl, 140 mM NaCl, 1 mM MgCl₂, 2 mM EGTA, 5.6 mM glucose, 25 mM HEPES, pH 7.2) or IC-K_low buffer (IC-K_low buffer – 5 mM NaCl, 5 mM KCl, 135 mM choline-Cl, 1 mM EGTA, 5.6 mM glucose, 25 mM HEPES, pH 7.2). Levels of cytosolic cAMP in merozoite lysates were measured using a colorimetric cAMP Direct Immunoassay Kit (Calbiochem) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004520#s4" target="_blank">Materials & Methods</a>. Total protein content in each merozoite sample was determined using Pierce Protein Assay Kit (Pierce). Amount of cAMP per µg of protein was determined for each merozoite sample and used to calculate fold increase compared to cAMP per µg of protein in control merozoites in IC buffer. Graphs represent mean fold change (± SD) in cAMP levels per µg of protein in merozoites under different conditions with respect to cAMP per µg of protein in merozoites in IC buffer from three independent experiments. C) Mammalian soluble AC inhibitor KH7 blocks rise in cytosolic cAMP levels following transfer of <i>P. falciparum</i> merozoites to low K⁺ buffer. <i>P. falciparum</i> merozoites were isolated in IC buffer and transferred to EC buffer with or without treatment with KH7. Levels of cAMP were measured in merozoite lysates as described above. Graphs represent mean fold change (± SD) in cAMP levels per µg of protein in merozoites under different conditions with respect to cAMP per µg of protein in merozoites in IC buffer from three independent experiments. Treatment with KH7 inhibits increase in cytosolic cAMP levels when merozoites are transferred from IC to EC buffer. D) Inhibition of microneme secretion by KH7. <i>P. falciparum</i> merozoites were transferred from IC to EC buffer with or without treatment with KH7. Secretion of PfAMA1 in merozoite supernatants (AMA(s)) was detected by Western blotting. Presence of cytoplasmic protein PfNapL was detected in <i>P. falciparum</i> merozoite supernatants (NapL(s)) and lysates of merozoite pellets (NapL(p)) under different conditions by Western blotting to control for merozoite lysis and number of merozoites used in the different conditions, respectively. Treatment of merozoites with KH7 inhibits secretion of microneme protein PfAMA1 following transfer from IC to EC buffer. Treatment of merozoites with KH7+IBMX, KH7+DiB+Epac Agonist and KH7+DiB+A23187 restores microneme secretion. However, treatment with KH7+DiB does not restore microneme secretion.</p

Expression and purification of surface re-engineered PvDBPII recombinant proteins containing additional N-linked glycan residues.

<p>(A) 2 µg of PvDBPII wild type and DBPII glycosylation variants were run on SDS-PAGE gel and stained with GelCode Blue reagent. A ladder effect is seen consistent with increasing number of N-glycosylation sites present in the STBP glycan, P1 and Max hyperglycosylated variants compared to PvDBPII wild-type. (B) Western blot of 1 µg of PvDBPII and DBPII glycosylation variants probed with anti-His antibody. (C) PvDBPII wild type and DBPII glycosylation variants were either untreated (−) or digested (+) with N-glycosidase PNGaseF, run on SDS-PAGE gel and probed with anti-His antibody, except for Max variant which was probed with anti-PvDBPII serum. The P1 lanes were run separate from the other samples. Molecular mass is shown on the left.</p

PvDBP – DARC interaction.

<p>DARC, a seven transmembrane chemokine receptor on erythrocytes, has been shown to bind to PvDBP <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Horuk1" target="_blank">[16]</a>. Binding has been mapped to the N-terminal 65 amino acids (illustrated as a red tail). The COS-7-RBC cytoadherance assay is based upon a multivalent interaction between PvDBPII present on surface of COS-7 cells and DARC expressed by RBC. In the yeast-PvDBPII display assay, PvDBPII present on the yeast surface interacts with dimeric recombinant DARC-Fc recombinant protein (N-terminal 65 mer region). The two assays offer different platforms to reveal inhibitory effects of antibodies using a potentially higher affinity, multimer-multimer interaction (COS-7 format) or a lower affinity multimer-dimer interaction (yeast display). Bottom of figure, polymorphisms in DARC that generate FyA/FyA^null genotype are associated with half of the number of surface DARC protein and lower <i>P. vivax</i> infection <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Zimmerman1" target="_blank">[4]</a>.</p

Immunogenic characterization of PvDBPII and its hyperglycosylated variants.

<p>(A) Schematic representation of immunization scheme for PvDBPII wild type and DBPII glycosylation variants. Group 1 mice received PvDBPII wild type generated in <i>E. coli</i>, Group 2 mice received PvDBPII wild type generated in HEK-293F cells. Mice in groups 3–6 received a combination of DNA and homologous protein; group 3, PvDBPII wildtype; group 4, STBP glycan; group 5, P1; group 6, Max hyperglycosylated variant. (B) The ELISA end-point antibody titers are shown for each of the immunization groups after the third DNA boost, the first protein immunization, and the second protein boost (final bleed). (C) The inhibitory activity of vaccine plasma in a COS-7-RBC binding assay. COS-7 cells expressing PvDBPII wild type protein were pre-incubated with varying dilutions of PvDBPII wild type and DBPII glycosylation variant immune plasma obtained after the third DNA boost or the second protein immunization (final bleed). The values are an average of duplicate experiments.</p

Glycan Masking of <i>Plasmodium vivax</i> Duffy Binding Protein for Probing Protein Binding Function and Vaccine Development

<div><p>Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of <i>Plasmodium vivax</i> Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to <i>P. vivax</i> vaccine development.</p></div

Effect of DARC phenotype on antibody binding inhibitory activity.

<p>COS-7 cells expressing PvDBPII as a GFP fusion protein were incubated with immune plasma and then RBC expressing the FyA (A) or FyB (B) Duffy blood group antigen were added. The inset shows that FyA RBCs (A) gave smaller rosettes than FyB RBCs (B) in the COS-7 cell–RBC binding assay. Statistical testing and p values as explained in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat-1003420-g006" target="_blank">Figure 6B</a>.</p