A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells

Abstract

Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, but specific interactions involved in cerebral homing of infected erythrocytes (IEs) are poorly understood. In this study, P. falciparum-IEs were characterized for binding to primary human brain microvascular endothelial cells (HBMECs). Before selection, CD36 or ICAM-1-binding parasites exhibited punctate binding to a subpopulation of HBMECs and binding was CD36 dependent. Panning of IEs on HBMECs led to a more dispersed binding phenotype and the selection of three var genes, including two that encode the tandem domain cassette 8 (DC8) and were non-CD36 binders. Multiple domains in the DC8 cassette bound to brain endothelium and the cysteine-rich interdomain region 1 inhibited binding of P. falciparum-IEs by 50%, highlighting a key role for the DC8 cassette in cerebral binding. It is mysterious how deadly binding variants are maintained in the parasite population. Clonal parasite lines expressing the two brain-adherent DC8-var genes did not bind to any of the known microvascular receptors, indicating unique receptors are involved in cerebral binding. They could also adhere to brain, lung, dermis, and heart endothelial cells, suggesting cerebral binding variants may have alternative sequestration sites. Furthermore, young African children with CM or nonsevere control cases had antibodies to HBMEC-selected parasites, indicating they had been exposed to related variants during childhood infections. This analysis shows that specific P. falciparum erythrocyte membrane protein 1 types are linked to cerebral binding and suggests a potential mechanism by which individuals may build up immunity to severe disease, in the absence of CM.

Fig. 1.

Binding of P. falciparum-IEs to primary HBMECs. (A) Parasites exhibiting different binding phenotypes were compared for binding to resting or TNF-α–activated HBMECs from patient donor 13. The A4long^HBMEC and ItG-ICAM-1^HBMEC lines were panned three times on HBMECs from donor 13. Results are expressed as mean number of IEs per square millimeter ± SDs from two to three independent experiments (***P < 0.001). (B) The starting A4long and ItG-ICAM-1 parasite lines exhibited a concentrated binding to a subpopulation of HBMECs that became more dispersed after panning of IEs three times on HBMECs.

Fig. 2.

The binding specificity of P. falciparum-IEs changed after selection on HBMECs. (A) The initial and HBMEC-panned parasite lines were compared for binding to CHO cells that differed in CSA, CD36, ICAM-1, VCAM-1, or ELAM-1 surface expression. (B) The initial and HBMEC-panned parasite lines were compared for binding to recombinant proteins. (C) The ability of anti-CD36 or anti-ICAM-1 antibodies to block IE binding to primary HBMECs (donor 13) was compared between the initial and HBMEC-panned parasite lines. Percentage of inhibition (%) is indicated between bars. Binding results in A–C are expressed as means ± SDs from two to three independent experiments.

Fig. 3.

HBMEC-panned IEs adhere to brain microvascular endothelial cells from different patient sources. (A) Binding was compared between the starting (ItG-ICAM-1) and selected (ItG-ICAM-1^HBMEC) parasite lines on HBMECs harvested from donors 13, 56, and 123 and a transformed HBMEC line (THBMEC) from donor 13. The anatomic origin of HBMEC cultures is illustrated in the brain schematic. (B) A DC8-var-expressing clonal parasite line derived from ItG-ICAM-1^HBMEC bound to primary microvascular cells from heart, lung, and dermis.

Fig. 4.

New var genes are up-regulated in parasite lines panned on primary HBMECs. (A and B) Transcription of var genes was compared from ring-stage parasites before and after panning on primary HBMECs (donor 13). Results were normalized to the housekeeping control gene adenylosuccinate lyase (asl). Genes are organized by Ups category, UpsA (red), UpsB (blue), UpsC (yellow), and UpsE (gray), as well as three genes for which the Ups type has not been determined (white). The names of parasite lines are indicated at the left, and var genes that expressed onefold or more of asl are indicated. (C) The extracellular domain architecture and predicted binding features of the three main genes up-regulated on HBMECs are shown. The DC8 cassette in IT4var6 and IT4var19 and the DC17 cassette in IT4var13 are underlined. The percentage of amino acid domain identity is shown for the two DC8-var products. Binding predictions are from repertoire-wide analysis of PfEMP1 recombinant proteins between CIDR-CD36 (14) and DBLβ-ICAM-1 (38).

Fig. 5.

Cloned parasite lines expressing IT4var6 or IT4var19 var products were weak CD36 binders and did not bind ICAM-1. (A) A panel of eight clonal parasite lines was generated from A4long^HBMEC and ItG-ICAM-1^HBMEC, using limited dilution cloning. Five parasite lines from the ItG-ICAM1^HBMEC line express a unique predominant var transcript IT4var19, IT4var6, IT4var31, or IT4var25, whereas three parasite lines from A4long^HBMEC express a mixture of var transcripts including the frequent-switch event IT4var31. (B) Profiling of var transcription, performed by Q-RT-PCR, for the clonal parasite lines expressing IT4var6, IT4var19, or IT4var31. (C) Parasite lines expressing IT4var19 and IT4var6 exhibit the dispersed HBMEC binding of the parental line, whereas the parasite line expressing IT4var31 binds to only a subpopulation of HBMECs. (D) Parasite lines were compared for binding to recombinant proteins. Results are expressed as means ± SDs from two independent experiments.

Fig. 6.

DC8-var19–encoded recombinant proteins exhibit binding capacity for brain endothelium. (A) Schematic of the protein construct. Protein boundaries are the following: DBLα2, M1-V484; CIDRα1.1, C485-C732; DBLα2-CIDRα1.1, M1-C732; and DBLβ12, P733-C1220. Proteins were analyzed on SDS/PAGE gel and visualized with GelBlue code. (B) Recombinant proteins binding to unfixed THBMEC cells were determined by flow cytometry. Recognition via the anti-strepII tag antibody tag is shown. (C) Protein-coupled Dynal Bead binding assays to THBMEC cells or CHO-745 cells as a negative control. Results are expressed as mean of beads binding per 100 cells ± SDs. (D) Binding of the IT4var19-expressing parasite clone (1E2) to transformed HBMECs in the presence of each of the E. coli fusion recombinant proteins, from 0.4 mg/mL to 0.05 mg/mL final concentration. Mix A corresponds to 0.4 mg/mL of three single domains DBLα2, CIDRα1.1, and DBLβ12 combined and mix B to 0.4 mg/mL of the tandem domain plus the DBLβ12 domain combined. The percentage of binding is expressed relative to binding in the presence of control protein MBP. Results are expressed as mean ± SDs from two independent experiments.

Fig. 7.

Endemic sera recognition of HBMEC-panned parasite lines. (A) Acute and convalescent sera pairs were collected from 10 children with cerebral malaria and 10 age-matched children with nonsevere malaria. (B) Surface recognition of infected erythrocytes was analyzed by flow cytometry. The mean fluorescence intensity (MFI) of uninfected erythrocytes was subtracted from the MFI of the infected erythrocyte population to give a specific MFI of the infected erythrocytes. The specific MFI reported in the graph was further corrected by subtracting antibody reactivity of a nonimmune European control plasma, which had an MFI of 1 on ItG-ICAM-1^HBMEC and no reactivity on ItG-ICAM-1. Antibody reactivity between starting and selected parasite lines was significantly different at the time of acute disease and at convalescence (***P < 0.001, Mann–Whitney test).

Fig. P1.

Selection of P. falciparum-infected erythrocytes on human brain endothelial cells. (Upper) CD36 or CD36 plus ICAM-1–binding parasites exhibited a concentrated binding pattern to a subpopulation of brain endothelial cells, and binding was CD36-dependent. Selection of infected erythrocytes for adherence to primary HBMECs led to a more dispersed binding phenotype and the enrichment of two related var genes associated with CD36^low/ICAM-1⁻–binding parasite variants (both encode a DC8 cassette) and a distinct CD36⁺/ICAM-1⁺–binding variant (encodes a DC17 cassette; not shown). (Lower) The extracellular region of the DC8-var (IT4var19) encodes multiple receptor-like domains termed DBL and CIDRs. The first three domains in the DC8 cassette (highlighted by blue boxes) were shown to encode adhesive activity for HBMEC.