Serum Lipoproteins Promote Efficient Presentation of the Malaria Virulence Protein PfEMP1 at the Erythrocyte Surface

Abstract

The virulence of the malaria parasite Plasmodium falciparum is related to its ability to express a family of adhesive proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) at the infected red blood cell surface. The mechanism for the transport and delivery of these adhesins to the erythrocyte membrane is only poorly understood. In this work, we have used specific immune reagents in a flow cytometric assay to monitor the effects of serum components on the surface presentation of PfEMP1. We show that efficient presentation of the A4 and VAR2CSA variants of PfEMP1 is dependent on the presence of serum in the bathing medium during parasite maturation. Lipid-loaded albumin supports parasite growth but allows much less efficient presentation of PfEMP1 at the red blood cell surface. Analysis of the serum components reveals that lipoproteins, especially those of the low-density lipoprotein fraction, promote PfEMP1 presentation. Cytoadhesion of infected erythrocytes to the host cell receptors CD36 and ICAM-1 is also decreased in infected erythrocytes cultured in the absence of serum. The defect appears to be in the transfer of PfEMP1 from parasite-derived structures known as the Maurer's clefts to the erythrocyte membrane or in surface conformation rather than a down-regulation or switching of particular PfEMP1 variants.

Improved In Vitro Culture of Plasmodium falciparum Permits Establishment of Clinical Isolates with Preserved Multiplication, Invasion and Rosetting Phenotypes

“…Studies investigating the rosetting phenomena need to take into consideration that the display of the rosetting phenotype and expression of PfEMP1, and possibly other parasite ligands, can be dependent on the presence of human serum. One possible mechanism is the role of lipid components during translocation of the PfEMP1 molecule from the Maurer’s clefts to the pRBC membrane [31], [32].…”

“…P. falciparum propagated in vitro under static conditions employing the classical candle jar method looses its capacity to form knobs at the surface of the pRBC, adhere to host cells and also to switch PfEMP1 expression [27], [28], [29], [30]. In previous work it has been suggested that serum-lipid components are required for the final relocation of the PfEMP1 molecule from the Maurer’s clefts to the pRBC surface [31], [32]. Other factors such as complement factor D, albumin and naturally occurring antibodies to the anion transport protein band 3 have been attributed a role in rosetting [33].…”

Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum‐infected erythrocytes reveals novel structural features

“…Briefly, infected RBCs (10 8 cells) were fixed in RPMI containing 2% paraformaldehyde, treated with purified recombinant His‐tagged EqtII (10 μg) (Anderluh et al ., 1996), refixed in PBS containing 2% paraformaldehyde and blocked with 3% bovine serum albumin in PBS. Permeabilized cells were incubated with primary antibody (rabbit anti‐GFP; Roche), rabbit anti‐SBP1 (Cooke et al ., 2006) or rabbit anti‐PfEMP1 (Kriek et al ., 2003; Frankland et al ., 2007) (all at 1:20 in 3% bovine serum albumin in PBS). Cells were washed and incubated with 6 nm gold‐conjugated protein A (Aurion), then fixed with glutaraldehyde, post‐fixed in osmium tetroxide and ‘en‐bloc’ stained with uranyl acetate prior to embedding in LR White resin.…”

A Method for Positive and Negative Selection of Plasmodium falciparum Platelet-Mediated Clumping Parasites and Investigation of the Role of CD36

“…The IT/A4 clone is derived from the IT/FCR3 strain [10]. Parasites were cultured in RPMI 1640 medium (Lonza, catalogue number 12-167F) supplemented with 2 mM glutamine, 25 mM Hepes, 20 mM glucose, 25 µg/ml gentamicin, and either 10% pooled human serum or 5% serum +0.25% Albumax II (Invitrogen) [11], with the pH adjusted to 7.2–7.4 with 1 M NaOH. Cultures were set up at 1% haematocrit with blood group O erythrocytes (donors from the Scottish National Blood Transfusion Service) and incubated at 37°C with 3% CO 2 , 1% O 2 , and 96% N 2 .…”