Variable surface molecules of the plasmodium falciparum infected erythrocyte and merozoite

Abstract

As Plasmodium falciparum parasites matures within the infected erythrocyte (IE) it produces a number of various proteins, which are transported out to the cell surface where they are exposed to the host immune system. To avoid recognition and elimination by the liver and spleen these proteins can undergo antigenic variation, a phenomenon where the parasites switch from expressing one variant to' another, both belonging to the same gene-family.

The most well characterised protein family on the surface of P. falciparum IEs is PfEmP1 encoded by the var-gene family comprising approximately 60 gene-copies per haploid genome. These are involved in the binding of IEs to uninfected erythrocytes and to the endothelial cell-lining in the vasculature, which blocks the blood flow in capillaries and cause severe outcome of malaria disease. The largest P. falciparum gene-family comprises the rifgenes encoding small variable RIFIN polypeptides, the function of which is unknown.

Here we show that PfEMP1 and RIFINs are co-transported through the IE cytosol to the surface in a complex trafficking machinery built up by the intracellular developing parasite. It involves the formation of single small vesicles (SSV), large multimeric vesicles (LMV) and large spindle-like vesicles (LSLV). All these structures are stainable with the Giemsa dye, and hence are all Maurer's clefts according to the original definition by Dr. Georg Maurer. The LMVS, and the SSVs to some extent, are also identical to the structures in which Pf332, PfSbp1, PfSarlp, PfSec31, PfEMP3, MAHRP, PP1 and LANCL colocalise, and the LW formation seems to be a default transport pattern in trophozoite IEs of in vitro grown parasites as well as in fresh patient isolates. However, we have observed that some long term in vitro grown parasite strains such as 3D7 have lost their ability to form LMVs and instead transport antigens to the surface in vesicles of a sirnilar size of SSVs. PfEMP2 PfHRP2 EXP1 and Exp2 localise in the cytosol in structures distinct from the SSV/LMV/LSLV pathway, although the PfHRP2 proteinaggregates and the parasitophorous vacuole membrane (PVM)buds carrying Exp1/Exp2 are also stainable with Giemsa and thus are Maurer's clefts by the original definition.

Furthermore, we have detected a new small gene-family of surface exposed molecules called the SURFINs. These are also transported to the IE surface together with PfEMP1 and RIFINs, however the bulk of proteins accumulate within the parasitophorous vacuole (PV) during maturation into late schizont stages. Upon IE rupture SURFIN associates with the individual merozoites in an amorphous cap structure in the apical end, where they possible are involved in the invasion process into new erythrocytes.

RIFINs were also detected in the apex of the merozoite, although using a panel of antisera raised against a number of different RIFIN polypeptides we found that the expression and localisation of various RIFIN variants differed. Detailed bioinformatic analysis of the 3D7 repertoire of RIFINs revealed two major subgroups (A- and B-type RIFINs). The A-type RIFINs are exported to the IE surface via the SSV/LMV/LSLV pathway, where they are exposed to the surrounding environment. They also accumulate in the M membrane and space, as well as in the apical end of the merozoite. In contrast, the B-type RIFINs stay within the PV during maturation and are detected in the cytosol of released merozoites.

Furthermore, we found that there is an intra-clonal switch of RIFIN repertoire between schizonts and merozoites, suggesting an adjustment to these distinct stages by the usage of different variants. The insertion/deletion of a 25 amino acid stretch in the semi-conserved region and different numbers of conserved cysteine residues between the groups has led us to suspect distinct folding properties and possible subfunctionalisation within the RIFIN repertoire. However, further analyses of members of the two groups are needed to be done.