<i>Plasmodium falciparum</i> Expressing Domain Cassette 5 Type PfEMP1 (DC5-PfEMP1) Bind PECAM1

<div><p>Members of the <i>Plasmodium falciparum</i> Erythrocyte Membrane protein 1 (PfEMP1) family expressed on the surface of malaria-infected erythrocytes mediate binding of the parasite to different receptors on the vascular lining. This process drives pathologies, and severe childhood malaria has been associated with the expression of particular subsets of PfEMP1 molecules. PfEMP1 are grouped into subtypes based on upstream sequences and the presence of semi-conserved PfEMP1 domain compositions named domain cassettes (DCs). Earlier studies have indicated that DC5-containing PfEMP1 (DC5-PfEMP1) are more likely to be expressed in children with severe malaria disease than in children with uncomplicated malaria, but these PfEMP1 subtypes only dominate in a relatively small proportion of the children with severe disease. In this study, we have characterised the genomic sequence characteristic for DC5, and show that two genetically different parasite lines expressing DC5-PfEMP1 bind PECAM1, and that anti-DC5-specific antibodies inhibit binding of DC5-PfEMP1-expressing parasites to transformed human bone marrow endothelial cells (TrHBMEC). We also show that antibodies against each of the four domains characteristic for DC5 react with native PfEMP1 expressed on the surface of infected erythrocytes, and that some of these antibodies are cross-reactive between the two DC5-containing PfEMP1 molecules tested. Finally, we confirm that anti-DC5 antibodies are acquired early in life by individuals living in malaria endemic areas, that individuals having high levels of these antibodies are less likely to develop febrile malaria episodes and that the antibody levels correlate positively with hemoglobin levels.</p> </div

Antibody cross-reactivity to DC5-PfEMP1-expressing 3D7 and FCR3, shown using flow cytometry and confocal microscopy.

<p>(A) Flow cytometry histograms showing the reactivity of a <i>P. falciparum</i> line expressing IT4var02 containing DC5 and a parasite line expressing VAR2CSA (FCR3 BeWo) to antibodies elicited to the FCR3-DC5 cassette (DBLγ12-DBLδ5-CIDRβ4-DBLβ9) or antibodies against VAR2CSA. (B) Flow cytometry histograms showing the reactivity of parasite lines expressing 3D7 PF11_0008 (3D7-DC5), FCR3 IT4var02 (FCR3-DC5) and 3D7 PFD1235w (3D7-DC4) with antibodies against domains DBLδ5-CIDRβ4 from 3D7-DC5 or FCR3-DC5. Below the flow cytometry histograms are confocal microscopy pictures of the same infected erythrocytes. Surface reactivity with FITC-labeled rat antibodies is seen as green dots and the DNA in the nuclei is stained blue by DAPI.</p

Schematic presentation of known PfEMP1 domain cassette 5.

<p>The four-domain DC5 is only found in PfEMP1 molecules of group A. The association of the specific domain type to the DC5 is listed as the count of said domain types found in the DC5 context compared to the total count of the domain type in 399 PfEMP1 sequences from seven near-complete assembled <i>P. falciparum</i> genomes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069117#B29" target="_blank">29</a>]. Percentage shared amino acids between domains of the cassette is shown in comparison to percentage shared amino acids of all domains of the major domain class. Lower panel shows domain architecture within DC5 and DC5-like sequences. Dotted lines indicate that only a partial sequence was available for the domain.</p

Distance trees of DBLγ, δ, β subdomains S1-3 and CIDR M and M1-2 (p-distance/Neighbour Joining method).

<p>Black dots indicate sub-domains found in DC5 sequences. Pink triangles indicate DBLγ S3 domains of DC8 sequences. Numbers at the nodes represent bootstrap proportions on 1000 replicates. DBLγ S3 subdomains of DC5 and 8 as well as all DC5 DBLδ S1 sub-domains form bootstrap supported clusters indicating that these regions are the most characteristic of DC5.</p

<i>Var</i> transcript profiles of 3D7 and FCR3 parasites selected for DC5-PfEMP1 expression.

<p>Bars show <i>var</i> transcript abundances in 3D7 and FCR3 lines before (blue bars) and after (red bars) selection with DC5 antibodies specific for PF11_0008 (3D7) and IT4var02 (FCR3), respectively. Copy numbers (y axis) are calculated from qPCRs using primers and standard curves specific for each gene, and given relative to 1000 copies of internal <i>seryl-tRNA synthetase</i> and <i>aldolase</i> controls. Pie charts show the <i>var</i> transcript distribution in the selected lines.</p

Adhesion of FCR3 parasites expressing IT4var02 (FCR3-DC5) to endothelial cells (TrHBMEC).

<p>Parasites were labeled with radioactivity and the y-axis shows the amount of radioactivity bound to the endothelial cells in percentage of the total amount of radioactivity added to the well. Parasites were tested after incubation with anti-PECAM1, anti-ICAM1, antibodies raised against the four-domain cassette 5 of IT4var02 (Rat 1-3), antibodies raised against a double-domain (DBLδ5-CIDRβ4) in IT4var02 DC5 (Rat 54) and control antibodies against a recombinant protein corresponding to full-length IT4var13. Error-bars are standard deviations, and the results are one representative experiment of three binding assays, except for inhibition with Rat 1-3, which was tested once.</p

Correlation between <i>var</i> transcript levels and cytoadhesive phenotypes of <i>P</i>. <i>falciparum</i> isolates from Mozambican children.

<p>The relationship between adhesion and transcript levels of <i>var</i>/DCs was assessed by Spearman correlation analysis, with * indicating <i>P</i><0.05 and ** if statistically significant after Benjamini-Hochberg correction for the six adhesive phenotypes tested. PM-agg: platelet-mediated agglutination.</p

Breadth of IgG recognition of <i>P</i>. <i>falciparum</i> isolates according to <i>var</i> transcript levels and origin.

<p>Geometric Mean Fluorescence Intensity (GMFI) values from each parasite/plasma combination were scored in relation to the threshold of positivity (GMFI of negative controls plus two standard deviations), with a score of 0 assigned if GMFI values were below the cut-off; 1 if the value was between one- and two-fold the cut-off; 2 if the value was between two- and three-fold the cut-off; and so on until a maximum score of 5. Breadth of IgG recognition (BoR) was calculated as the sum of scores obtained for each parasite and expressed as percentage of the maximum score possible. BoR was compared between A) isolates transcribing <i>var</i>/DCs at low- or high- levels by negative binomial regression models adjusted by age and B) between isolates collected from travelers (n = 3), severe malaria (SM, n = 23), uncomplicated malaria (UM, n = 15) children and adults (Moz adults, n = 4) by test for trend across ordered groups. Bars represent the mean of BoR and standard deviation. * indicates <i>P</i><0.05 and ** <i>P</i>≤0.001.</p

Phenotypical and molecular characterization of <i>Pf</i>3D7^gC1qR and <i>Pf</i>3D7^CD36.

<p><b>A)</b> Binding assays of <i>Pf</i>3D7^<i>gC1qR</i> and <i>Pf</i>3D7^<i>CD36</i> over gC1qR, CD36, ICAM1 and CSA receptors. <b>B)</b> Transcriptional analysis of the <i>var</i> gene repertoire of <i>Pf</i>3D7. Transcript levels of <i>var</i> genes were determined by qPCR using primers specific for each of the <i>P</i>. <i>falciparum</i> 3D7 <i>var</i> genes and were expressed as copy number relative to the seryl-tRNA synthetase gene. <b>C)</b> Levels of IgG recognition by plasma from Mozambican children were compared by Wilcoxon matched pair test, with * indicating P≤0.001. Bars represent mean and standard deviation of geometric mean fluorescence intensity (GMFI). D) Domain structure of PFD002c.</p

Transcript levels of <i>var</i>/DCs in <i>P</i>. <i>falciparum</i> isolates from travelers, children with uncomplicated malaria and adults.

<p>Transcript levels (y axis) correspond to relative copy number relative to seryl-tRNA synthetase gene copies (X100). Bars represent the median and interquartile range. Transcript levels were compared between groups by Mann-Whitney test, with * indicating <i>P</i><0.05 and ** if statistically significant after Benjamini-Hockberg correction. RCN: Relative copy number; DC: Domain Cassette; Tv: travelers, Ch: Mozambican children, Ad: Mozambican adults.</p