SURFIN is a polymorphic antigen expressed on Plasmodium falciparum merozoites and infected erythrocytes

Abstract

The surfaces of the infected erythrocyte (IE) and the merozoite, two developmental stages of malaria parasites, expose antigenic determinants to the host immune system. We report on surface-associated interspersed genes (surf genes), which encode a novel polymorphic protein family, SURFINs, present on both IEs and merozoites. A SURFIN expressed in 3D7 parasites, SURFIN4.2, was identified by mass spectrometric analysis of peptides cleaved off the surface of live IEs with trypsin. SURFINs are encoded by a family of 10 surf genes, including three predicted pseudogenes, located within or close to the subtelomeres of five of the chromosomes. SURFINs show structural and sequence similarities with exported surface-exposed proteins (PvSTP1, PkSICAvar, PvVIR, Pf332, and PfEMP1) of several Plasmodium species. SURFIN4.2 of a parasite other than 3D7 (FCR3S1.2) showed polymorphisms in the extracellular domain, suggesting sequence variability between genotypes. SURFIN4.2 not only was found cotransported with PfEMP1 and RIFIN to the IE surface, but also accumulated in the parasitophorous vacuole. In released merozoites, SURFIN4.2 was present in an amorphous cap at the parasite apex, where it may be involved in the invasion of erythrocytes. By exposing shared polymorphic antigens on IEs and merozoites, the parasite may coordinate the antigenic composition of these attachment surfaces during growth in the bloodstream.

Figure 1.

SURFINs share structural features with exported proteins of several Plasmodium species. (A) Common structure of SURFIN proteins and their relation to other Plasmodiae surface proteins. SURFINs and a putative transmembrane protein of P. vivax, PvSTP1, form a clade with a common structure (gray box). A predicted TM separates a variable NH₂-terminal region from a semiconserved COOH-terminal region. The NH₂-terminal domain features a moderately conserved CRD with six highly conserved cysteine residues and a modified Pexel, which precedes a region of higher sequence variability. The COOH-terminal domain is characterized by one (PvSTP1) or three to four (SURFIN) relatively conserved WRDs. SURFIN–PvSTP1 is related to the surface/exported proteins Pf332, PfEMP1, PvVIR, and PkSICAvar. PSI-BLAST and ClustalW alignment analysis revealed conserved cysteine residues shared between the CRD of SURFIN–PvSTP1 and the variable extracellular NH₂-terminal domain of VIR. In contrast, the COOH-terminal WRDs feature similarities with the exported protein Pf332 and the surface-exposed proteins PfEMP1 and PvSICAvar. Two sequence segments can be distinguished in the SURFIN WRDs: S1 (30 aa) and S2 (60 aa) which both occur with high homology in a WRD found at the COOH-terminal end of Pf332, here intersected by a stretch of ∼130 aa containing a partial duplication of S2 (S2*; Fig. S3). A modified S1 segment, characterized by a highly conserved Pexel-like motif (Fig. S3), is also found in the COOH-terminal domain of PkSICAvar, which lacks an S2 segment. S2 is present in the ATS-domain of PfEMP1, which lacks a S1 segment, although a strictly conserved Pexel-like motif related to S1 is ∼150 aa upstream of S2. (B) General structural features shared by related surface proteins in P. vivax and P. falciparum include a variable external domain containing conserved cysteine residues and a conserved internal domain.

Figure 2.

surf _4.2 gene expression profile in 3D7. (A) RT-PCR with RNA from synchronized 3D7S8 cultures. Samples were taken at the trophozoite stage (<6 h), the midtrophozoite stage (20–24 h), and the matured schizont stage (>40 h). Forward primer P1 and reverse primer P2 were used to amplify a segment of 440 bp of the PFD1160w transcript across the intron boundary. To further control for a genomic DNA-derived transcript, the RT step was performed in the presence (+) or absence (−) of reverse transcriptase. (B) Northern blot analysis with total RNA prepared from an asynchronous parasite culture. Total RNA separated by agarose gel electrophoresis and stained with ethidium bromide before transfer to a membrane (lane 1) is shown. Northern blot analysis with a probe specific for the 3D7surf _4.2 identified a full-length transcript of ∼9 kb, which was in the expected size range for SURFIN-encoding mRNA (lane 2).

Figure 3.

Anti-3D7surf_4.2 (PFD1160w) antibodies identify SURFIN_4.2 as a Triton X-100 insoluble protein strongly labeled in surface biotinylation experiments. (A) Relative position of S1.3 and S1.4 peptides, and rSURFIN_4.2 used to raise anti-SURFIN_4.2 serum. (B) Western blot analysis with anti-S1.3 serum on protein fractions of 3D7S8 sequentially extracted with 1% Triton X-100 (Triton soluble), 2% SDS (Triton insoluble), and boiling sample buffer (SDS insoluble). Also, a transient total protein stain with Ponceau S is shown. Anti-S1.3 serum reacted specifically with a 280–300-kD band, as is predicted for SURFIN_4.2 (286 kD), which separated predominantly into the Triton-insoluble and SDS-insoluble fraction (*, SURFIN_4.2). A copartitioning band of the same size was also visualized with Ponceau S. (C) Anti-rSURFIN_4.2 and anti-S1.3 react with the same antigen in Western blots with Triton-insoluble fractions. Lanes were stained with Ponceau S, cut in half, and then probed with either S1.3 or rSURFIN_4.2 antibodies. (D) Immunoprecipitation of surface-biotinylated 3D7S8 parasites with anti-SURFIN_4.2 serum S1.3. Total lysate and antibody-precipitated materials were analyzed in Western blots with streptavidin reagent. A major biotinylated band in total lysates was specifically precipitated (lanes shown were from the same gel). A control with preimmune serum did not precipitate any biotinylated protein (not depicted). (E) Western blot analysis of lysates of surface-biotinylated 3D7S8 parasites probed with streptavidin reagent or anti-PfEMP1 revealed SURFIN_4.2 (*) as of considerably higher molecular mass to an expressed PfEMP1 (^O). Lanes shown were run in the same gel and the position of spectrin was deduced in a transient stain with Ponceau S. (F) 3D7S8 parasites were surface iodinated. Triton-insoluble fractions were analyzed by 5% SDS-PAGE and exposed by phosphoimaging. The position of SURFIN_4.2 and PfEMP1 was deduced by Western blot with 3D7S8 lysate incubated with anti-SURFIN_4.2 or anti-PfEMP1 antibodies (not depicted). Note that a band comigrating with SURFIN_4.2 is barely visible in the exposed surface-iodinated lysate, whereas a strongly labeled protein comigrated with anti-PfEMP1 reactive antibodies.

Figure 4.

SURFIN_4.2 in developmental stages of 3D7S8. Samples of IEs of a synchronized 3D7S8 parasite culture were taken at various time points. IEs were then either lysed and analyzed in Western blots with anti-SURFIN_4.2 serum S1.3 (A) or surface biotinylated and developed in Western blots with streptavidin reagent (B). Also, the total protein stain with Ponceau S is shown. SURFIN_4.2 detected by either antibody anti-S1.3 or surface biotinylation/streptavidin was increasingly visible from 20 h on. A very faint band of SURFIN_4.2 (*) was also found in early stages (4–8 h). Note that a comparably stronger band comigrating with SURFIN_4.2 was visible in early stages by Ponceau S.

Figure 5.

SURFINs expressed in 3D7S8 and FCR3S1.2 are cotransported with PfEMP1 to the host PM. (A) Indirect immunofluorescence assay (IFA) on air-dried monolayers of 3D7S8 IEs at various stages of maturation. (top) Conventional IFA with SURFIN_4.2-directed anti-S1.3 antibodies at 4, 24, 36, and 44 h a.i.; (middle and bottom) merged double-stained confocal IFA of PI-stained parasite nuclei (red) with Alexa 488–labeled SURFIN_4.2 (green). SURFIN was detected with either anti-S1.3 (middle) or anti-rSURFIN_4.2 antibodies (bottom); yellow, label overlay. Both antibodies detected SURFIN_4.2 in ∼25% of examined parasites at ∼24 h a.i. in dot-like transport vesicles outside the parasitophorous vacuole. (B) Dual localization of TRITC-labeled SURFIN_4.2 (red) and Alexa 488–labeled PfEMP1 (green) showed that SURFIN_4.2 and PfEMP1 are colocalized in cytosolic structures of ∼25% 3D7S8 IEs (compare with the merged fluorescence pattern). An expressed SURFIN was detected in >90% of trophozoite-stage FCR3S1.2 IEs (∼24 h a.i.) and colocalized with PfEMP1 in LMVs. Dashed circles indicate the area of the IE. (C) Immunoelectron microscopy studies on 3D7S8 and FCR3S1.2 IEs. SURFIN detected with either anti-S1.3 serum or affinity-purified anti-S1.4 immunoglobulins is present in the PM, as well as in the erythrocyte cytosol associated with Maurer's clefts (MC) as indicated by arrows. Bars: (A and B), 2 μm; (C), 0.5 μm.

Figure 6.

SURFIN accumulates within the parasitophorous vacuole in late schizonts and is associated with released merozoites. (A) Alexa 488–labeled SURFIN_4.2 (green) detected with anti-S1.3 antibodies in parasites developing from late trophozoite stage to schizonts. PI staining (red) was used to visualize nuclei. (B–D) SURFIN_4.2 was found localized in MAM and the PM of late and bursting schizonts (>48 h), and associated with the released merozoite surface. Dashed circles indicate the area of the IE. (B) IFA with anti-S1.3 + PI on bursting schizonts. IFA (anti-rSURFIN_4.2) and immunoelectron microscopy (anti-S1.4 Ig) on late schizonts (C) and released merozoites (D). SURFIN associated with PM or MAM is indicated by arrows. (E) SURFIN_4.2 did not colocalize with the microneme AMA-1. A detailed analysis of SURFIN_4.2 (anti-S1.3 antibodies) and AMA-1 in relation to the nuclei and to each other in merozoites is shown. (B and E) A schematic representation of the antigen localization is shown on the right. Bars: IFA, 2 μm; EM, 0.5.