Structural Characterization of the Bradyzoite Surface Antigen (BSR4) from Toxoplasma gondii, a Unique Addition to the Surface Antigen Glycoprotein 1-related Superfamily

Abstract

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)...

Structure of thePlasmodium6-cysteine s48/45 domain

“…This family of surface antigens from T. gondii is defined by the presence of an N-terminal secretion signal, a GPI anchor, and a set of conserved amino acids, among which are six cysteines involved in the formation of three disulfide bonds important in maintaining the structure of the SRS fold (40). To date, more than 160 DNA sequences from T. gondii have been identified as belonging to the SRS superfamily (45), but only the structures for SAG1, SporoSAG, and BSR4 have been solved (39,40,46,47). Although there is some structural divergence among these three proteins, and within each protein the N-terminal (D1) and C-terminal (D2) domains are also structurally distinct, the SRS fold is observed as a common feature in the domains of all three (40) (Fig.…”

Structural and Functional Characterization of SporoSAG

“…2, B and C). The absence of an observed dimer for SporoSAG contrasts with the structures of SAG1, which crystallized as a dimer (25), and BSR4 that showed a clear dimer along the crystallographic 2-fold axis (28), suggesting that SporoSAG may exist as a monomer on the cell surface of the parasite. However, the physiological GPI tether that is absent in our recombinant construct may be required to promote dimerization of SporoSAG on the parasite cell surface.…”

“…Initial crystallization trials produced a P6 crystal form that diffracted to 1.60 Å. Attempts to solve the SporoSAG structure using molecular replacement with the individual core domains of SAG1 (25) and BSR4 (28,46) as search models were unsuccessful. Additional rounds of crystallization produced a cadmium-derivatized P2 1 2 1 2 crystal form from which we were able to generate high quality phases using cadmium single-wavelength anomalous dispersion.…”

“…To date, only two structural descriptions have been reported for the 160ϩ SRS superfamily: one for the tachyzoite-expressed SAG1 (25) and the second for the bradyzoite-expressed BSR4 (28). To determine the structural and functional implications of a sporozoite-expressed SRS protein, we report the 1.60 Å resolution crystal structure and immunoreactivity profile of the major GPI-tethered cell surface antigen expressed in sporozoites.…”

“…Sequencing of the T. gondii genome has revealed more than 160 SRS family members (27). Recent structural studies of the predominantly tachyzoite-expressed SAG1 and bradyzoiteexpressed BSR4 identified a topologically defined groove that is postulated to coordinate host cell surface molecules such as heparin (25,28).…”