Investigating the Host Binding Signature on the Plasmodium falciparum PfEMP1 Protein Family

Abstract

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family plays a central role in antigenic variation and cytoadhesion of P. falciparum infected erythrocytes. PfEMP1 proteins/var genes are classified into three main subfamilies (UpsA, UpsB, and UpsC) that are hypothesized to have different roles in binding and disease. To investigate whether these subfamilies have diverged in binding specificity and test if binding could be predicted by adhesion domain classification, we generated a panel of 19 parasite lines that primarily expressed a single dominant var transcript and assayed binding against 12 known host receptors. By limited dilution cloning, only UpsB and UpsC var genes were isolated, indicating that UpsA var gene expression is rare under in vitro culture conditions. Consequently, three UpsA variants were obtained by rosette purification and selection with specific monoclonal antibodies to create a more representative panel. Binding assays showed that CD36 was the most common adhesion partner of the parasite panel, followed by ICAM-1 and TSP-1, and that CD36 and ICAM-1 binding variants were highly predicted by adhesion domain sequence classification. Binding to other host receptors, including CSA, VCAM-1, HABP1, CD31/PECAM, E-selectin, Endoglin, CHO receptor “X”, and Fractalkine, was rare or absent. Our findings identify a category of larger PfEMP1 proteins that are under dual selection for ICAM-1 and CD36 binding. They also support that the UpsA group, in contrast to UpsB and UpsC var genes, has diverged from binding to the major microvasculature receptor CD36 and likely uses other mechanisms to sequester in the microvasculature. These results demonstrate that CD36 and ICAM-1 have left strong signatures of selection on the PfEMP1 family that can be detected by adhesion domain sequence classification and have implications for how this family of proteins is specializing to exploit hosts with varying levels of anti-malaria immunity.

A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

“…Although at least two previously described DBLb3 domains contain an ICAM-1-binding site, many others do not (23,24). By testing hybrids of the ICAM-1-binding PFD1235w-DBLb3_D4 domain and immediate downstream DBLb3_D5 domain that does not bind ICAM-1, we could show that only aa 1029-1209 of DBLb3_D4 were required for binding to ICAM-1.…”

“…Furthermore, it has been proposed that group A PfEMP1 proteins are not under selection for ICAM-1-binding DBLb domains (22), and large group B and group C PfEMP1 proteins appear to be under selection for concomitant adhesion to ICAM-1 and CD36 (24,44). Because IE adhesion to CD36 is associated with uncomplicated malaria (21), these observations can be seen as arguments against an important role for ICAM-1-specific IE adhesion in the pathogenesis of severe P. falciparum malaria, not least CM.…”

“…Until now, the majority of described ICAM-1-binding sites in PfEMP1 are located in DBLb5 domains of group B PfEMP1 proteins (22)(23)(24) (Fig. 5).…”

“…However, IEs selected for expression of DC8 or DC13 by panning on cerebral microvascular endothelial cells were found not to bind to ICAM-1 (16,17). Furthermore, all but one of the ICAM-1-binding DBL domains identified so far belong to a fairly diverse set of DBLb domains from group B or C PfEMP1 proteins (22)(23)(24), and it has been proposed that group A PfEMP1 proteins are not under selection for ICAM-1 binding (24). These and other findings have cast doubt on the importance of ICAM-1 binding and the involvement of group A PfEMP1 proteins in the pathogenesis of cerebral malaria.…”

A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells

“…The var gene groups have functional and clinical significance. Group B and C var genes encode PfEMP1 variants that bind CD36 (14,15) and are linked to nonvirulent clinical disease, whereas group A var genes encode non-CD36-binding variants linked to severe clinical disease including CM (16)(17)(18)(19). Some group A var genes encode PfEMP1 variants that bind to uninfected erythrocytes to form rosettes (20)(21)(22)(23)(24)(25); however, the adhesion phenotype of the majority of group A variants is unknown currently.…”