No abstract

Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Apicomplexan parasites to invade host cells. We report the 2.0 Å resolution x-ray crystal structure of the full ectodomain (domains I, II, and III) of AMA1 from the pervasive protozoan parasite Toxoplasma gondii. The structure of T. gondii AMA1 (TgAMA1) is the most complete of any AMA1 structure to date, with more than 97.5% of the ectodomain unambiguously modeled. Comparative sequence analysis reveals discrete segments of divergence in TgAMA1 that map to areas of established functional importance in AMA1 from Plasmodium vivax (PvAMA1) and Plasmodium falciparum (PfAMA1). Inspection of the TgAMA1 structure reveals a network of apical surface loops, reorganized in both size and chemistry relative to PvAMA1/ PfAMA1, that appear to serve as structural filters restricting access to a central hydrophobic groove. The terminal portion of this groove is formed by an extended loop from DII that is 14 residues shorter in TgAMA1. A pair of tryptophan residues (Trp 353 and Trp 354 ) anchor the DII loop in the hydrophobic groove and frame a conserved tyrosine (Tyr 230 ), forming a contiguous surface that may be critical for moving junction assembly. The minimalist DIII structure folds into a cystine knot that probably stabilizes and orients the bulk of the ectodmain without providing excess surface area to which invasion-inhibitory antibodies can be generated. The detailed structural characterization of TgAMA1 provides valuable insight into the mechanism of host cell invasion by T. gondii.Toxoplasma gondii, the etiological agent of toxoplasmosis, is a prevalent global pathogen capable of establishing acute and chronic infections in nearly all warm blooded animals (1, 2). Although largely asymptomatic in healthy individuals, T. gondii infections can be lethal to a developing fetus and immunocompromised cancer and AIDS patients (3-6). Toxoplasmosis can also result in severe ocular infections in both children and adults, and encysted forms of the parasite have recently been implicated in neuropsychiatric disorders, such as schizophrenia (7-9).The success of T. gondii stems from its ability to persist in the environment, utilize several modes of transmission (10), and, importantly, to infect a broad range of host cells (1). A dominant feature that endows T. gondii and, in fact, all Apicomplexan parasites, including Plasmodium, Babesia, Cryptosporidium, and Neospora, with the ability to efficiently invade host cells is a multiprotein complex assembled at the moving junction (MJ) 4 (2, 11). The MJ is an electron-dense, ringlike structure formed between the plasma membranes of the apical tip of the motile parasite and the target host cell (12). During invasion, T. gondii is rapidly engulfed within a parasitophorous vacuole (PV) as the MJ traverses in a posterior direction along the length of the parasite (13, 14). As it migrates, the MJ serves as a molecular sieve, selectively filtering host proteins from the PV (12, 15), thereby protec...

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects nearly one-third of the human population. The success of T. gondii is based on its complex life cycle; a lytic tachyzoite form disseminates infection, whereas an encysted bradyzoite form establishes a latent, chronic infection. Persistence and transmissibility is central to the survival of the parasite and is, in part, mediated by a family of antigenically distinct surface antigen glycoprotein (SAG)-related sequences (SRS) adhesins that play a dual role in host cell attachment and host immune evasion. More than 160 members of the SRS family have been identified with only the tachyzoite-expressed SAG1 structurally characterized. Here we report the first structural description of the bradyzoite adhesin BSR4 using x-ray crystallography and small angle x-ray scattering. The 1.90-Å crystal structure of BSR4 reveals an architecture comprised of tandem ␤ sandwich domains organized in a head to tail fashion with the N-terminal domain responsible for dimer formation. A restructured topology in BSR4 results in a ligand-binding site that is significantly reorganized in both structure and chemistry relative to SAG1, consistent with BSR4 binding a distinct physiological ligand. The small angle x-ray scattering solution structure of BSR4 highlights a potentially important structural role for the interdomain polymorphic linker that imparts significant flexibility that may promote structural adaptation during ligand binding. This study reveals an unexpected level of structural diversity within the SRS superfamily and provides important insight into the role of these virulence factors.The protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, is a serious global pathogen that infects nearly one-third of the human population (1-3). T. gondii infections can be lethal to a developing fetus and immunocompromised, cancer, AIDS, and organ transplant patients. Clinical features range from asymptomatic infection to lymphadenopathy, ileitis, encephalitis, and/or blinding ocular infections in both children and adults with the severity of symptoms tending to increase with age (1, 4 -9). During infection, T. gondii cycles between the rapidly growing, lytic tachyzoite stage and the slow growing, cyst-forming bradyzoite stage. Upon challenge by the immune system, the tachyzoites, which are responsible for rapid dissemination of the parasite, differentiate into encysted bradyzoites that promote chronic infection. The molecular switches that regulate interconversion of the two parasitic stages in the host remain largely undefined. Despite the complex life cycle of T. gondii, it exists as a single species and has been referred to as one of the most, if not the most, successful protozoan parasite of animals on this planet (3).The success of T. gondii is largely due to its ability to infect a broad range of host cells (10), which is, in part, mediated by a family of developmentally expressed, antigenically distinct surface antigen glycoprotein (SAG)...

Toxoplasma gondii is a widespread zoonotic pathogen capable of causing serious disease in humans and animals. As an obligate intracellular parasite, T. gondii relies on the orchestrated secretion of proteins from its apical complex organelles including the multimodular, transmembrane micronemal protein 2 (MIC2) that couples recognition of the host cell with cytoskeletal reorganization of the parasite to drive invasion. To probe the basis by which the von Willebrand Factor A (vWA)-Integrin like module of TgMIC2 engages the host cell, we solved the crystal structure of a truncated form of TgMIC2A/I (TgMIC2A/Ic) phased by iodide SIRAS and refined to a resolution of 2.05 Å . The TgMIC2A/Ic core is organized into a central twisted beta sheet flanked by a-helices consistent with a canonical vWA fold. A restricted basic patch serves as the putative heparin binding site, but no heparin binding was detected in native gel shift assays. Furthermore, no metal was observed in the metal ion dependent adhesion site (MIDAS). Structural overlays with homologous A/I domains reveal a divergent organization of the MIDAS b4-a4 loop in TgMIC2A/Ic, which is stabilized through the burial of Phe195 into a deep pocket formed by Gly185. Intriguingly, Gly185 appears to be unique among A/I domains to TgMIC2A/I suggesting that the divergent loop conformation may also be unique to TgMIC2A/I. Although lacking the C-terminal extension, the TgMIC2A/Ic structure reported here is the first of an A/I domain from an apicomplexan parasite and provides valuable insight into defining the molecular recognition of host cells by these widespread pathogens.

Toxoplasma gondii, the etiological agent of toxoplasmosis, utilizes stage-specific expression of antigenically distinct glycosylphosphatidylinositol-tethered surface coat proteins to promote and establish chronic infection. Of the three infective stages of T. gondii, sporozoites are encapsulated in highly infectious oocysts that have been linked to large scale outbreaks of toxoplasmosis. SporoSAG (surface antigen glycoprotein) is the dominant surface coat protein expressed on the surface of sporozoites. Using a bioinformatic approach, we show that SporoSAG clusters with the SAG2 subfamily of the SAG1-related superfamily (SRS) and is non-polymorphic among the 11 haplogroups of T. gondii strains. In contrast to the immunodominant SAG1 protein expressed on tachyzoites, SporoSAG is non-immunogenic during natural infection. We report the 1.60 Å resolution crystal structure of SporoSAG solved using cadmium single anomalous dispersion. SporoSAG crystallized as a monomer and displays unique features of the SRS ␤-sandwich fold relative to SAG1 and BSR4. Intriguingly, the structural diversity is localized to the upper sheets of the ␤-sandwich fold and may have important implications for multimerization and host cell ligand recognition. The structure of SporoSAG also reveals an unexpectedly acidic surface that contrasts with the previously determined SAG1 and BSR4 structures where a basic surface is predicted to play a role in binding negatively charged glycosaminoglycans. Our structural and functional characterization of SporoSAG provides a rationale for the evolutionary divergence of this key SRS family member.