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 ϳ3.3 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 ϳ54-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.

Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

“…Parasites with a disrupted TgAMA1 gene (designated "⌬ama1/AMA1-myc") were identified by PCR and Western blotting with mAb B3.90, and disruption of the wild-type locus was confirmed by Southern blot analysis. Western blotting with antisera against residues 21-36, 69 -410, and 166 -379 of TgAMA1 (Hehl et al, 2000; generously provided by Dr. John Boothroyd, Stanford University) confirmed the absence of partial TgAMA1 translation products in the ⌬ama1/AMA1-myc parasites. …”

“…Apical membrane antigen-1 (AMA1) is a microneme protein that is highly conserved among apicomplexan parasites (Waters et al, 1990;Donahue et al, 2000;Hehl et al, 2000;Gaffar et al, 2004). Since its initial discovery Ͼ15 years ago, AMA1 has received considerable attention as a malaria vaccine candidate (Deans et al, 1988;Collins et al, 1994;Anders et al, 1998;Kennedy et al, 2002;Stowers et al, 2002).…”

“…After the apical end of a parasite comes in contact with the host cell membrane, apical secretory organelles (micronemes and rhoptries) sequentially discharge their contents (Dubremetz et al, 1993;Carruthers and Sibley, 1997) and a zone of tight interaction forms between the two cells (Nichols and O'Connor, 1981;Dubremetz et al, 1985;Grimwood and Smith, 1995). An invagination in the host cell plasma membrane develops at the point of entry and progressively deepens, ultimately surrounding the fully internalized parasite (Suss-Toby et al, 1996).Apical membrane antigen-1 (AMA1) is a microneme protein that is highly conserved among apicomplexan parasites (Waters et al, 1990;Donahue et al, 2000;Hehl et al, 2000;Gaffar et al, 2004). Since its initial discovery Ͼ15 years ago, AMA1 has received considerable attention as a malaria vaccine candidate (Deans et al, 1988;Collins et al, 1994;Anders et al, 1998;Kennedy et al, 2002;Stowers et al, 2002).…”

“…AMA1 proteins are type I transmembrane proteins, with a short C-terminal cytoplasmic tail and a large N-terminal extracellular domain (ectodomain) containing 12-16 conserved cysteine residues (Waters et al, 1990;Hodder et al, 1996;Donahue et al, 2000;Hehl et al, 2000;Gaffar et al, 2004). Like other microneme proteins, AMA1 is secreted onto the parasite surface, where its ectodomain is proteolytically cleaved and shed (Narum and Thomas, 1994;Donahue et al, 2000;Hehl et al, 2000;Howell et al, 2003).The recently determined crystal structure of Plasmodium vivax AMA1 demonstrates that the conserved cysteines divide the ectodomain into three distinct subdomains (Pizarro et al, 2005). Domains I and II belong to the PAN module superfamily, suggesting that they may function in adhesion to protein or carbohydrate receptors (Pizarro et al, 2005).…”

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

“…(Hehl et al, 2000;Triglia et al, 2000). Analysis of a conditional knock-out of TgAMA1 established its important role in invasion and was found to be dispensable for gliding motility (Mital et al, 2005).…”