Figure 1.
Selection of P. falciparum-IEs on different endothelial cell types.
(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 [19]. (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.
Figure 2.
Interaction of P. falciparum-IEs with different endothelial cell types.
(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 var gene [19]. 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 P. falciparum-IE (black dots in image). (C) P. falciparum-infected erythrocytes expressing the DC8 variant (IT4var19) were compared for binding to untreated or TNF-α activated endothelial cells.
Figure 3.
DC8 and DC13 var transcripts are consistently upregulated on different microvascular endothelial cell types.
Transcription of var 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 var genes transcribed relative to the total of 56 var genes analyzed. Genes are organized by Ups category; UpsA (red), UpsB (dark blue), UpsC (yellow), UpsE (grey), undetermined (white). Group B/A var6 and var19 are indicated with an asterisk.
Figure 4.
Production of PfEMP1 adhesion domains.
(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 [14]. 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.
Figure 5.
Multiple domains in the DC8-IT4var19 variant exhibit widespread endothelial binding activity.
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.
Figure 6.
Dose-dependent binding of IT4var19 adhesion domains to endothelial cells.
Binding of recombinant proteins to THBMEC, HPMEC, and CDC-BMEC was assessed by flow cytometry using anti-StrepII tag antibodies.
Figure 7.
IT4var19 domains display distinct protease-sensitive binding profiles for brain endothelial cells.
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).
Figure 8.
Binding of CIDR1 domains present in the semi-conserved head structures of DC8/DC13 or other UpsA PfEMP1 variants.
(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.