Structural and Functional Characterization of SporoSAG

Abstract

Toxoplasma gondii, the etiological agent of toxoplasmosis, utilizes stage-specific expression of antigenically distinct gly-cosylphosphatidylinositol-tethered surface coat proteins to promote and establish chronic infection. Of the three infec-tive stages of T. gondii, sporozoites are encapsulated in highly infectious oocysts that have been linked to large scale out-breaks of toxoplasmosis. SporoSAG (surface antigen glyco-protein) 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. Intrigu-ingly, 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 pro-vides a rationale for the evolutionary divergence of this key SRS family member. Toxoplasma gondii is a highly prevalent, obligate, intracellu-lar protozoan parasite that infects nearly one-third of the human population (1, 2). Since its recognition as the causative agent of toxoplasmosis in the late 1930s, many clinical manifes-tations have been attributed to T. gondii infections including lymphadenopathy, ileitis, encephalitis, and/or blinding ocular infections in both children and adults (1, 3– 8). T. gondii infec-tions can also be lethal to a developing fetus and immunocom-promised, cancer, AIDS, and organ transplant patients. A key feature of the ability of T. gondii to infect and multiply in virtu-ally any warm blooded animal is a complex life cycle that encompasses both sexual (sporozoite) and asexual (tachyzoite and bradyzoite) stages of development. Sexual replication oc-curs exclusively in felines, whereas asexual division occurs in all warm blooded hosts. Three main routes of infection exist for T. gondii. Congenital infection occurs with maternal transmission of tachyzoites via the placenta to a fetus, whereas ingestion of bradyzoites occurs when tissue cysts in undercooked meat are consumed, and finally, infection with sporozoites occurs when food or water contaminated with oocysts is consumed. A remarkable trait of T. gondii is its ability to differentiate from the ingested bradyzoite or sporo-zoite into the fast replicating tachyzoite, facilitating rapid dissemination throughout the host. Upon challenge by the immune system, the tachyzoite converts to the slow growing, encysted bradyzoite responsible for establishing a chronic, transmissible infection. Historically, meat containing T. gondii tissue cysts was con-sidered the major route of transmission to humans; however, improved farming practices have considerably reduced the like-lihood of infected livestock (9). The prevalence of T. gondii in felines, however, remains high (10 –14). Shedding of T. gondii oocysts containing infectious sporozoites by both feral and domestic cats continues to result in widespread environmental contamination, due both to large numbers of parasites shed (as many as 1 million/cat) and to the resistance of oocysts to envi-ronmental degradation (15). Localized incidences of water-borne transmission as well as substantial outbreaks due to contaminated water supplies or soil have been extensively documented (16 –21). Due to the potentially broad scale of an outbreak, environmental transmission of T. gondii through sporozoite ingestion arguably poses the most significant global risk. Stage conversion in T. gondii coincides with a major change in expression of surface antigens belonging to the surface