DC8 and DC13 <i>var</i> Genes Associated with Severe Malaria Bind Avidly to Diverse Endothelial Cells

<div><p>During blood stage infection, <i>Plasmodium falciparum</i> infected erythrocytes (IE) bind to host blood vessels. This virulence determinant enables parasites to evade spleen-dependent killing mechanisms, but paradoxically in some cases may reduce parasite fitness by killing the host. Adhesion of infected erythrocytes is mediated by <i>P. falciparum</i> erythrocyte membrane protein 1 (PfEMP1), a family of polymorphic adhesion proteins encoded by <i>var</i> genes. Whereas cerebral binding and severe malaria are associated with parasites expressing DC8 and DC13 <i>var</i> genes, relatively little is known about the non-brain endothelial selection on severe malaria adhesive types. In this study, we selected <i>P. falciparum</i>-IEs on diverse endothelial cell types and demonstrate that DC8 and DC13 <i>var</i> genes were consistently among the major <i>var</i> transcripts selected on non-brain endothelial cells (lung, heart, bone marrow). To investigate the molecular basis for this avid endothelial binding activity, recombinant proteins were expressed from the predominant upregulated DC8 transcript, IT4var19. In-depth binding comparisons revealed that multiple extracellular domains from this protein bound brain and non-brain endothelial cells, and individual domains largely did not discriminate between different endothelial cell types. Additionally, we found that recombinant DC8 and DC13 CIDR1 domains exhibited a widespread endothelial binding activity and could compete for DC8-IE binding to brain endothelial cells, suggesting they may bind the same host receptor. Our findings provide new insights into the interaction of severe malaria adhesive types and host blood vessels and support the hypothesis that parasites causing severe malaria express PfEMP1 variants with a superior ability to adhere to diverse endothelial cell types, and may therefore endow these parasites with a growth and transmission advantage.</p></div

DC8 and DC13 <i>var</i> transcripts are consistently upregulated on different microvascular endothelial cell types.

<p>Transcription of <i>var</i> genes was analyzed by Q-RT-PCR from ring-stage IEs isolated before selection (blue) or after selection on HPMEC (green), HCMEC (pink), or CDC-BMEC (orange). Results were normalized to the housekeeping gene adenylosuccinate lyase (asl) and expressed as percentage of <i>var</i> genes transcribed relative to the total of 56 <i>var</i> genes analyzed. Genes are organized by Ups category; UpsA (red), UpsB (dark blue), UpsC (yellow), UpsE (grey), undetermined (white). Group B/A <i>var6</i> and <i>var19</i> are indicated with an asterisk.</p

IT4var19 domains display distinct protease-sensitive binding profiles for brain endothelial cells.

<p>Binding of DC8-var19 recombinant protein to THBMEC was assessed by flow cytometry via anti-StrepII tag antibodies on THBMEC cells pretreated with trypsin, chymotrypsin, neuraminidase or V8 protease. Inset shows example histograms. Results are expressed as relative surface detection ( = proportion of cells antibody reactive × MFI of reactive cells).</p

Selection of <i>P. falciparum</i>-IEs on different endothelial cell types.

<p>(A) A panel of six parasites lines was generated from the A4long and ItG-ICAM1 parasite lines by three rounds of selection on HPMEC (pulmonary), HCMEC (cardiac), and CDC-BMEC (bone marrow) cells. Selection on HBMEC (brain) was reported previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003430#ppat.1003430-Avril1" target="_blank">[19]</a>. (B) IEs exhibited a concentrated binding pattern to a subpopulation of endothelial cells before selection and a more diffuse binding pattern on the entire population of cells after three rounds of selection.</p

Binding of CIDR1 domains present in the semi-conserved head structures of DC8/DC13 or other UpsA PfEMP1 variants.

<p>(A) Representative images for bead coupled CIDR domain binding to endothelial cells (THBMEC, HPMEC, HCMEC, CDC-BMEC), CHO-CD36, or CHO-745 cells. (B) Surface staining of each recombinant CIDR domain to live THBMEC determined by FACS analysis. Normalized MFI indicated inside parenthesis.</p

Production of PfEMP1 adhesion domains.

<p>(A) Schematic of the MBP-fusion protein constructs and a general PfEMP1 protein domain architecture. The complete extracellular domain architecture is illustrated for IT4var19 and the head structure for the other proteins. Tandem domain arrangements associated with DC8, DC13, or DC16 cassettes are underlined <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003430#ppat.1003430-Rask1" target="_blank">[14]</a>. PfEMP1 protein domains with bold outlines were expressed as recombinant proteins. (B) Proteins were visualized using SDS/PAGE gel and GelBlue code staining. Due to the number of samples, separate gels were used to visualize the entire panel of proteins used in this study.</p

Multiple domains in the DC8-IT4var19 variant exhibit widespread endothelial binding activity.

<p>Top panel: The coupling of recombinant protein to Dynal beads was quantified by flow cytometry with anti-StrepII antibodies; MBP control (grey histogram), PfEMP1 domains (black histogram). The MBP protein used as a negative control lacks the StrepII tag added to the C-terminus of the PfEMP1 domains. Bottom panel: Representative images for each bead coupled PfEMP1 domain to THBMEC, HPMEC, HCMEC, CDC-BMEC, or non-endothelial control CHO-745 cell. A control PfEMP1 domain (var14DBLα0.23) is also represented.</p

Interaction of <i>P. falciparum</i>-IEs with different endothelial cell types.

<p>(A) The ability of anti-CD36 (FA6-152) and anti-ICAM1 (15.2) monoclonal antibodies to block IE binding to different endothelial cell types was compared between starting and selected parasite lines. IT4var19 (DC8) and IT4var31 (CD36 binder) are cloned parasite lines that predominately transcribe a single <i>var</i> gene <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003430#ppat.1003430-Avril1" target="_blank">[19]</a>. Binding results are expressed as percentage of inhibition relative to antibody-free controls. (B) Binding of starting and selected A4long parasite lines to HCMEC in the presence or absence of CD36 or ICAM1 monoclonal antibodies. Arrow points to a <i>P. falciparum</i>-IE (black dots in image). (C) <i>P. falciparum</i>-infected erythrocytes expressing the DC8 variant (IT4var19) were compared for binding to untreated or TNF-α activated endothelial cells.</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

High Avidity Antibodies to VAR2CSA were Associated with Absence of Placental Malaria in Ngali II.

<p>(<b>a</b>) The proportion (%) of Ab that remains bound to FV2 after incubation with 3M NH₄SCN (i.e., % high avidity Ab) in the plasma of primigravidae and multigravidae living in Ngali II is shown. Results represent the mean + SEM for 11 to 51 data points per bar. Throughout the course of pregnancy, the proportion of high avidity Ab to FV2 was higher in multigravid than primigravid women (p<0.0001; multilevel polynomial regression analysis). (<b>b</b>) The scattergram shows that the proportion of high avidity Ab to FV2 was highly variable. However, the proportion (%) of high avidity Ab in plasma of women who were PM⁻ was significantly higher than in plasma of PM⁺ women during pregnancy (PM⁻, n = 13; PM⁺, n = 14) (p = 0.0009), based on multilevel polynomial regression analysis. (<b>c</b>) A significantly higher proportion of high avidity Ab was present in plasma of PM⁻ women than PM⁺ women at 5–6 months (p<0.0001), but not at 3–4 and 7–8 months.</p