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In order to avoid immune recognition in favor of a chronic infection, the malaria parasite Plasmodium falciparum has developed means to express clonally variant antigens at the surface of the infected erythrocyte (IE). Proteins of the var and rif multicopy gene families, encoding PfEMP1 and RIFINs, respectively, have been implicated in these processes. Here, we studied members of the latter family and present data revealing different subcellular localization patterns for RIFIN variants belonging to two distinct subgroups, which have been designated A- and B-type RIFINs. While A-type RIFINs were found to be associated with the parasite and transported to the surface of infected erythrocytes via Maurer's clefts, B-type RIFINs appeared to be mostly retained inside the parasite. However, expression of both subtypes does not seem to be mutually exclusive. Moreover, both A- and B-type variants were also expressed in the merozoite, present either in the apical region (A-type) or in the cytosol (B-type). The presence of RIFINs in merozoites suggests that antigenic variation in P. falciparum is not only restricted to parasite-derived proteins at the IE surface, but the phenomenon also prevails in other life cycle stages. Interestingly, some RIFIN variants were detected only in intracellular stages and not in merozoites, pointing to differential developmental expression patterns for distinct members of this large protein family.

Intraerythrocytic Plasmodium falciparum exports proteins to the cytosol and to the plasma membrane of the host cell. We here present data revealing the existence of a unique common pathway for the surface bound traffic of the clonally variant antigens, repeated-interspersed-antigen (RIFINS) and P. falciparum erythrocyte-membrane-protein-1 (PfEMP1). RIFIN- and PfEMP1-specific antibodies were found to stain single small vesicles (SSV) that bud off from the parasitophorus vacuolar membrane (PMV) at 6-10 h post-invasion. Large multimeric vesicle (LMV) assemblies, composed of subunits each of a similar size to that of a SSV, appeared as the dominant vesicle type carrying the variant antigens in the cytosol as the parasites developed into early trophozoite stages (> or = 16 h post-invasion). Later, more than 24 h post-invasion, large spinle-like vesicles (LSLV) built up as the LMV approached and accumulated underneath the erythrocyte membrane. LMV were found to associate both with the Maurer's cleft antigen Pf332 and with lipids as seen by fluorescent BODIPY-Ceramide staining. Co-traffic of Pf332 with RIFINS and PfEMP1 occurred in sub-compartmentalized LMV, as the variant antigens co-localized at the outer rim while Pf332 occupied the core of the vesicle complex. Formation of LMV for the trafficking of RIFINS and PfEMP1 is a prominent feature of freshly isolated P. falciparum and of in vitro propagated K+ as well as K- parasites, seemingly independent of the knob-associated histidine-rich protein (KAHRP). In vitro cultured 3D7 clones lack LMV formation and traffic the variant antigens in vesicles of a similar size to that of the SSV.