Hypervariability within the Rifin, Stevor and Pfmc-2TM superfamilies in Plasmodium falciparum

Abstract

The human malaria parasite, Plasmodium falciparum, possesses a broad repertoire of proteins that are proposed to be trafficked to the erythrocyte cytoplasm or surface, based upon the presence within these proteins of a Pexel/VTS erythrocyte-trafficking motif. This catalog includes large families of predicted 2 transmembrane (2TM) proteins, including the Rifin, Stevor and Pfmc-2TM superfamilies, of which each possesses a region of extensive sequence diversity across paralogs and between isolates that is confined to a proposed surface-exposed loop on the infected erythrocyte. Here we express epitope-tagged versions of the 2TM proteins in transgenic NF54 parasites and present evidence that the Stevor and Pfmc-2TM families are exported to the erythrocyte membrane, thus supporting the hypothesis that host immune pressure drives antigenic diversity within the loop. An examination of multiple P.falciparum isolates demonstrates that the hypervariable loop within Stevor and Pfmc-2TM proteins possesses sequence diversity across isolate boundaries. The Pfmc-2TM genes are encoded within large amplified loci that share profound nucleotide identity, which in turn highlight the divergences observed within the hypervariable loop. The majority of Pexel/VTS proteins are organized together within sub-telomeric genome neighborhoods, and a mechanism must therefore exist to differentially generate sequence diversity within select genes, as well as within highly defined regions within these genes.

Figure 1

Schematic representation of the catalog of P.falciparum Pexel/VTS-containing proteins. Signal peptide sequences are represented by gray boxes; transmembrane domains are represented by black boxes. All proteins are encoded by two exon genes; the location of the intron is represented by a gray triangle. The Pexel/VTS motif is encoded near the 5′ end of the second exon and is represented by a narrow open box. The locations of signal peptide cleavage sites have not been determined, and the N-termini of mature proteins are not known. Predicted globular domains are indicated by open boxes.

Figure 2

Immunolocalization of epitope-tagged proteins. (A) Schematic representation of the SFM (Stevor-FLAG-myc) and 2TMFM (Pfmc-2TM-FLAG-myc) recombinant proteins. For the respective genes, two FLAG epitopes (F) were placed between the 2TM domains and three myc epitopes (M) were inserted at the carboxy terminus. The lengths, in amino acids, of the chimeric proteins are indicated at the bottom. The restriction sites specific for the insertion of tags are indicated. (B) Immunofluorescence assays (IFA) showing the localization of SFM and 2TMFM in the transformed lines, pHL-SFM and pHL-2TMFM. IFA studies were performed on air-dried P.falciparum transformed parasites. Infected erythrocytes were stained with FITC-conjugated anti-c-myc mAb and with anti-FLAG polyclonal antibodies followed by goat anti-rabbit Alexa 594-conjugated IgG. As a negative control, air-dried pHL-dhfr-luc P.falciparum parasites gave no signal (data not shown).

Figure 3

Immunogold localization of SFM and 2TMFM in the transformed lines pHL-SFM and pHL-2TMFM. Thin sections of late trophozoites and schizonts from pHL-SFM line (a–f) and pHL-2TMFM line (g–l) were probed with an anti-c-myc mAb and revealed by 6 nm gold-labeled anti-mouse IgG. Gold particles were associated with Maurer's clefts (a, g and h) or the erythrocyte surface (b–f, h, i, k and l). Some particles were associated with knobs (c, f, i and l). Bar, 0.25 μm.

Figure 4

Alignments of the divergent loop and flanking sequences for Pfmc-2TM family members from isolates 3D7, GB4, Liberia, Dd2, 7G8, HB3 and Ghana. 3D7 isolate genes are denoted by their protein names; genes from other isolates are numbered in the order of their discovery. The TM domains are indicated by blue boxes and a green box indicates the hypervariable loop. Translated sequences corresponding to the sense and antisense PCR primers are indicated in lowercase font. The bottom line reflects 85% consensus. Cysteine residues are highlighted in yellow.

Figure 5

Alignments of the divergent loop and flanking sequences for Stevor family members from isolates 3D7 Dd2, HB3 and Ghana. 3D7 isolate genes are denoted by their protein names; genes from other isolates are numbered in the order of their discovery. The TM domains are indicated by blue boxes and a green box indicates the hypervariable loop. Translated sequences corresponding to the sense and antisense PCR primers are indicated in lowercase font. The bottom line reflects 85% consensus. Cysteine residues are highlighted in yellow.

Figure 6

A locus containing a central Pfmc-2TM gene that is duplicated 13 times in the P.falciparum 3D7 isolate genome sequence. (A) The predicted length (Kb) of locus duplication, determined as nucleotide identities shared with the extended PFF1525c locus (upper row, indicated by an open rectangle). The left-hand columns indicate for each locus the predicted chromosome and telomere location (left, L; right, R); orientation (sense, S; antisense, AS); and Pfmc-2TM gene identifier. Adjacent loci are indicated by a gray star (Chr2, R) and black star (Chr11, L). A vertical dashed line, at approximately the 12.4 Kb mark, indicates the location of the Pfmc-2TM gene within each locus. Gray horizontal bars indicate >85% nt identity with the PFF1525c locus, and black horizontal bars indicate >95% identity. The curly brackets unify additional sequence similarities (dashed gray bars) that are specific to PF11_0025 and PF10_0390; PF11_0014 and MAL8P1.213; and PFA0065w, PF11_0014 and MAL8P1.213. (B) Predicted ORFs within the 13 loci extending ∼12 Kb upstream and 20 Kb downstream of each centralized Pfmc-2TM gene. ORFs on the sense strand are shown in gray, and ORFs on the antisense strand are in black. All ORFs encode Pexel/VTS-containing genes, var genes or predicted pseudogenes.

Figure 7

Two models for the function of the 2TM proteins. In the upper panel (A) the proteins participate in formation of a multi-subunit, multi-TM solute pore. An alternative model is proposed in which the 2TM proteins modulate endogenous erythrocyte solute channels. In the lower panel (B) the proteins form the knob structure containing PfEMP1 proteins and mediating cytoadherence. A third model might integrate the knob and solute pore and knobs into a single erythrocyte surface structure.