The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1

Abstract

Toxoplasma gondii is an Apicomplexan parasite with a complex life cycle that includes a rapidly dividing asexual stage known as the tachyzoite. The tachyzoite surface has been reported to comprise five major antigens, the most abundant of which is designated SAG1 (for surface antigen 1). At least one of the other four (SAG3) and another recently described minor antigen (SRS1 [for SAG1-related sequence 1]) have previously been shown to be structurally related to SAG1. To determine if further SAG1 homologs exist, we searched a Toxoplasma expressed sequence tag (EST) database and found numerous ESTs corresponding to at least three new genes related to SAG1. Like SAG1, these new SRS genes encode apparently glycosylphosphatidylinositol-anchored proteins that share several motifs and a set of conserved cysteine residues. This family appears to have arisen by divergence from a common ancestor under selection for the conservation of overall topology. The products of two of these new genes (SRS2 and SRS3) are shown to be expressed on the surface of Toxoplasma tachyzoites by immunofluorescence. We also identified strain-specific differences in relative expression levels. A total of 10 members of the SAG1 gene family have now been identified, which apparently include three of the five major surface antigens previously described and one antigen expressed only in bradyzoites. The function of this family may be to provide a redundant system of receptors for interaction with host cells and/or to direct the immune responses that limit acute T. gondii infections.

FIG. 1

EST contig assembly for SRS2 (A) and SRS3 (B). Sequence data from overlapping EST clones (y axis) from RH (shaded bars) and ME49 (open bars) tachyzoites were assembled into contigs with Gelassemble (GCGV8.0).

FIG. 2

Amino acid sequence alignment of eight members of the SAG1 family produced by ClustalW. Dashes represent gaps introduced to produce optimal alignment. Amino acids conserved in four or more sequences are boxed, and homologies are shaded when present in two or more sequences. Each allele shown is from the RH strain, except for BSR4, which is from the ME49 strain.

FIG. 3

Western blot strips labeled with antisera to SAG1 (lane 1), SRS1 (lane 2), SRS2 (lane 3), SRS3 (lane 4), and CRD (lane 5). Triton X-100 lysates were produced from equal numbers of tachyzoites from RH (A), the ME49 wild type (B), and the ME49 Sag1⁻ mutant (C). Each lysate was treated with trypanosomal GPI-PLC prior to reducing SDS-PAGE. Variant surface glycoprotein (VSG) is a contaminant present in the GPI-PLC preparation. Sizes (kilodaltons) are shown on the left.

FIG. 4

Western blot strips of ME49 lysates (without GPI-PLC treatment) labelled with SAG1, SAG3, and SRS2 antisera (from nonreducing SDS-PAGE). Sizes (kilodaltons) are shown on the right.

FIG. 5

Surface immunofluorescence (left) and phase-contrast microscopy (right) of RH tachyzoites following live staining with antisera to SRS2 (top) and SRS3 (bottom) and fixation with paraformaldehyde. Preimmune sera failed to stain T. gondii tachyzoites (not shown).