Erythrocyte-binding preferences of 6 PvRBPs.

<p>A: Bar charts showing the percentage of binding of CD71-PECy5 (control), PvRBP1a, PvRBP1b, PvRBP2a, PvRBP2b, PvRBP2cNB, PvRBP2-P2 to mature erythrocytes (not stained with thiazole orange, TO-) vs reticulocytes (stained with thiazole orange, TO+) populations. Error bars represent SEM of 7 or 9 independent repeats. B: Dot plots show the binding of six PvRBP proteins to an enriched reticulocyte population. Binding was detected using an anti-PvRBP rabbit IgG antibody followed by a secondary anti-rabbit Alexa 647 antibody.</p

Total and IgG subclass reactivity to PvRBPs in young Papua New Guinean children.

<p>Total and IgG subclass reactivity to PvRBPs in young Papua New Guinean children.</p

Detection of PvRBPs by rabbit anti-PvRBP polyclonal antibodies by ELISA.

<p>Microtiter wells were coated with each PvRBP per well as shown by symbols on the left. Solid lines show specific anti-PvRBPs polyclonal antibodies (top label) added to each plate in a dilution series. The optical density (OD) was measured at 405 nm. Mean OD values from duplicated wells and standard deviation are shown.</p

Association between total IgG to 6 PvRBPs age and exposure in 224 young Papua New Guinea children.

<p>A & B: Data are plotted as Lowess smoothed curves of total IgG levels (arbitrary units) by age and life-time exposure respectively. Life-time exposure was calculated as age multiplied by molFOB. Correlation coefficients (rho) and P-values from Spearman’s rank test. <b>C:</b> Boxplots show median total IgG levels (arbitrary units) and range (whiskers) by infection status detected by PCR. Clear boxes show low antibody levels, dotted boxes show medium antibody levels and grey boxes show high antibody levels. P-values from Kruskal-Wallis test. For all analysis, P < 0.05 were deemed significant.</p

Association between total and IgG subclasses to 6 PvRBPs and protection against clinical malaria (density > 500/μL) in 224 young Papua New Guinean children.

<p>Data are plotted as exposure (molFOB), age, season and village of residency adjusted incidence rate ratios and 95% confidence intervals. Incidence rate ratios, 95% confidence intervals and P-values from GEE models. P < 0.05 were deemed significant. ****P < 0.001; ***P = 0.001; ** P > 0.001 to 0.01; *P > 0.01 to 0.05.</p

Specific amino acid residues in the cytoplasmic domain are essential for PfRh4 function.

<p>(A) Localization of PfRh4 and PfRh2 in Rh4-WT tail, RH4-AMA1tail, Rh4-mut4tail and RH4-mut5tail lines as detected using anti-PfRh4 monoclonal and anti-PfRh2 polyclonal antibodies. Parasite nuclei were stained with DAPI. (B) Expression of PfRh4 and PfRh2 in W2mefΔ175 and all transgenic lines as detected by anti-PfRh4 and anti-PfRh2 antibody. All transgenic lines migrated as a slightly larger doublet compared to PfRh4 in W2mefΔ175, consistent with the addition of the hexa-histidine tag at the C-terminus of the protein. (C) Growth assays of transgenic lines with four and five mutations at serine and tyrosine amino acid residues in the PfRh4 cytoplasmic tail. (D) Growth assays of single mutations in the PfRh4 cytoplasmic tail. (E) Growth assays of double mutations in the PfRh4 cytoplasmic tail. In all three panels, parasitaemia was measured in neuraminidase-treated, and untreated erythrocytes after every 48 hours incubation (labelled as cycles). The parasite lines used in this experiment are displayed on the X-axis. The y-axis represents parasitaemia of neuraminidase-treated erythrocytes as a percentage of parasitaemia of the same line grown on untreated erythrocytes. Error bars represent +1 standard error of the mean. Assay performed three times on separate days, each in triplicate.</p

Identification of putative phosphosites and parasite kinase involved in modification of PfRh4 cytoplasmic tail.

<p>(A) <i>In vitro</i> kinase assays of wildtype and putative phosphosite mutations in PfRh4 cytoplasmic domains. The amino acid sequence of the PfRh4 cytoplasmic tail is shown with serines and tyrosines highlighted with the residue number. Each potential phosphosite on the PfRh4 tail was individually mutated to alanine. The all 4 lane has mutations in S1667A, S1674A, Y1680A and Y1684A and the all 5 lane has S1652A, S1667A, S1674A, Y1680A and Y1684A putative kinase sites mutated. The phosphorylation signal was quantitated and adjusted for protein loading. The loading-adjusted mutant phosphorylation signals were divided by the wildtype and plotted as a percentage of the wildtype signal (Y-axis). Autoradiograph of proteins after incubation in the <i>in vitro</i> phosphorylation assay and Coomassie gel from which protein loading was quantitated are shown in lower panels. Lane labels (X-axis) denote residues mutated to alanine. Mean percentage of wildtype phosphorylation +1 standard error of the mean are displayed. Data was averaged from four experiments performed on separate days. (B) Dosage-response curve for PfRh4 tail phosphorylation by merozoite lysate in the presence of increasing concentrations of the CK2 inhibitor TBB. PfRh4 tail phosphorylation was quantitated after incubation in <i>in vitro</i> phosphorylation assay with TBB. The phosphorylation signal for each condition was adjusted to reflect the average amount of protein loaded across each condition, determined by densitometry of the Coomassie brilliant blue stained gel. Y-axis represents loading-adjusted phosphorylation signal as a percentage of phosphorylation in the presence of DMSO (control). Autoradiograph of wildtype GST-fused PfRh4 proteins after incubation in the <i>in vitro</i> phosphorylation assay. X-axis indicates the TBB concentration with which the phosphorylation assay was incubated or DMSO. (C) <i>In vitro</i> kinase assays of PfRh and EBL cytoplasmic tails. The phosphorylation signal was quantitated and adjusted for protein loading. Autoradiograph of proteins after incubation in the <i>in vitro</i> phosphorylation assay and Coomassie brilliant blue stained gel from which protein loading was quantitated are shown. Data was averaged from four experiments performed on separate days (right panel) and standard error of the mean is shown. The following sites were mutated: EBA140 (S1159A, S1168A, T1173A), EBA175 (T1466A, mut A) and (S1489A, mut B) and in combination (mut A and B), EBA181 (S1528A, S1553A, S1557A, T1564A), PfRh2a (S3128A) and PfRh2b (S3233).</p