Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria-infected red blood cells

Abstract

Adhesion of parasite‐infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein‐3 (PfEMP3), is deposited under the membrane of the parasite‐infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.falciparum‐infected cell. Truncation of the C‐terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane‐associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite‐infected red blood cell.

PfEMP1 expression is reduced on the surface of knoblessPlasmodium falciparuminfected erythrocytes

“…4). The efficiency of PfEMP1 export in the K + clones used here compared with K + 3D7, where only approximately 50% is similarly exposed on the IE surface (Waterkeyn et al, 2000), probably reflects differences in the parasites clones employed in these respective studies. The 3D7 clone appears to have an inherent deficiency in PfEMP1 export (C.I.N., unpublished).…”

“…These electron dense structures act as sites for anchoring PfEMP1 on the outer face of the plasma membrane and are thus implicated in mediating IE cytoadhesion (Baruch et al, 1995;Cooke et al, 2000). Knobs consist of a number of parasite-encoded proteins, including knob-associated histidine-rich protein (KAHRP), PfEMP3 and…”

“…MESA interacts with components of the IE cytoskeleton, although no evidence suggests a similar bridging role between the cytoskeleton and PfEMP1 (Cooke et al, 2002a). An interaction between PfEMP3 and the cytoskeleton has not yet been demonstrated, but this protein is thought to play a role in the efficiency of export of PfEMP1 to the knobs (Waterkeyn et al, 2000).…”

“…Although several studies have investigated the contribution of these non-PfEMP1 knobassociated proteins (Crabb et al, 1997;Cooke et al, 2002a;Waterkeyn et al, 2000) it has not hitherto been possible to compare adhesion of K + and K -IEs expressing the same PfEMP1 variant because, in the absence of selection, rapid transcriptional switching between members of the gene family (var) that encodes PfEMP1 quickly renders an in vitro population heterogeneous with respect to the PfEMP1 variant expressed (Horrocks et al, 2004a;Roberts et al, 1992). Additionally, previous analysis of adhesion by K + and K -IEs assumed that the amount and distribution of PfEMP1 is equivalent in both K + and K -IEs, something that has never been definitively established and which may obviously impact on the nature of the cytoadhesive process.…”

Maurer's clefts, the enigma of Plasmodium falciparum

“…In that case, PfEMP1 was retained at Maurer's clefts (124). When proteins were deleted that are part of the knob complex in the erythrocyte membrane, such as PfEMP3, a reduced cytoadherence phenotype was observed with a reduction of PfEMP1 (125). In the case of KAHRP knockout, PfEMP1 was correctly trafficked to the surface but showed a significantly reduced cytoadherence under flow conditions, probably because of the lack of cytoskeleton anchoring (126).…”