Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells

Abstract

Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of approximately 3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A approximately 54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.

FIG. 1

(A) To-scale graphic depiction of the TgAMA1 gene locus, depicting exon (black boxes) and intron segments. Black line, noncoding genomic sequence. (B) Graphic representation of the predicted full-length TgAMA1 protein (GenBank accession number AF010264). Black boxes, hydrophobic sequences. Cysteines are represented by vertical lines. Domains I, II, and III are defined by analogy to those described by Hodder et al. (20) for the P. knowlesi AMA1 ectodomain. (C) Southern blot analysis. Genomic DNA from RH tachyzoites was isolated and digested overnight with NheI (N), KpnI (K), HindIII (H), EcoRI (R), or BglII (B). The DNA was resolved by agarose gel electrophoresis, and blots were probed sequentially with a 600-bp AMA1 5′ probe and a 1,200-bp AMA1 3′ probe. The migration of size markers is indicated (in kilobases).

FIG. 2

Multiple alignment of AMA1 amino acid sequences from P. berghei, P. yoelii, P. chabaudi, P. knowlesi, P. vivax, and P. falciparum, and TgAMA1. Identical residues in four or more of the seven sequences are boxed. Gaps inserted to optimize alignments are represented by dashes.

FIG. 3

Western blot strips of parasite-associated TgAMA1 separated on SDS-PAGE. (A) Triton X-100 extracts of RH strain tachyzoites incubated with mouse polyclonal antibodies raised against MBP-LF (LF) or preimmune serum (PI). (B) Reactivity of C-pep (C) and N-pep (N) antibodies with tachyzoite extracts separated on SDS-PAGE gels. A putative precursor band, detected with anti-N-pep, is indicated by an arrow. The migration of size markers is indicated (in kilodaltons).

FIG. 4

Immunofluorescence localization of TgAMA1 in T. gondii tachyzoites. (A, C, E, and G) Phase-contrast images; (B, D, F, and H) corresponding immunofluorescence images. (A and B) Formaldehyde-fixed and detergent-permeabilized intracellular parasites treated with MBP-SF antiserum. (C and D) Fixed and nonpermeabilized extracellular tachyzoites labeled with MBP-SF antiserum, showing apical localization of TgAMA1 on the surface of intact cells, with distinct circumferential staining. (E and F) Permeabilized intracellular tachyzoites incubated with N-pep antiserum, showing distinct perinuclear staining in addition to the subpellicular apical signal. (G and H) N-pep antiserum labeled mostly a small area at the conoid of extracellular parasites not treated with detergent.

FIG. 5

Immunofluorescence localization of TgAMA1 in intracellular tachyzoites detected with the mouse MAb CL22 (A) and MIC2 detected with a rabbit polyclonal antiserum using intracellular tachyzoites that had been fixed and permeabilized (B). The merged image shows colocalization in large areas of overlap of the two signals (C). The corresponding phase-contrast image is depicted in panel D. (E) Localization of TgAMA1 by immunoelectron microscopy; intracellular tachyzoite section was labeled with MBP-SF antiserum. M, micronemes; R, rhoptries; D, dense granules. Immunogold staining of TgAMA1 in micronemes is marked with arrows. Bar, 0.2 μm.

FIG. 6

Western blot of intracellular organelles from T. gondii fractionated on a Percoll gradient. Lane 1, lysed cells; lane 2, top fraction containing tachyzoite ghosts; lane 3, middle fraction enriched in micronemes; lane 4, bottom fraction containing rhoptries and dense granules. Blots were probed with MAbs to organellar proteins AMA1 (CL22), MIC2 (6D10), ROP2, ROP3, ROP4 (T34A7), and GRA3 (2H11). The migration of size markers is indicated (in kilodaltons).

FIG. 7

Secretion assay. Washed parasites were incubated in serum-free RPMI medium at 37°C. Excreted-secreted fractions (S) collected at 0, 5, 10, 20, 30, and 60 min of incubation of extracellular tachyzoites in serum-free medium and corresponding cell pellets (P) were separated on SDS–10% PAGE. Detection of membrane-bound TgAMA1 and secreted TgAMA1 was done with MBP-LF antibodies and subsequent incubation with peroxidase-conjugated goat anti-mouse Ig secondary antibodies.

FIG. 8

Antibodies against recombinant TgAMA1 inhibit infection of HFF. Invasion in the presence of different concentrations of MBP-LF antiserum from mouse 1 (black column) and mouse 2 (stippled column) and MBP-SF antiserum from mouse 3 (white column) is shown as a percentage of invasion in the presence of pooled normal mouse serum as measured by uracil incorporation. Error bars represent the standard deviation for duplicate wells.