Toxoplasma gondii: cyst wall glycobiology

Toxoplasma gondii is a prevalent human parasite that can cause severe disease in immune compromised individuals and fetuses. It is recognized as an important opportunistic pathogen in patients with Acquired Immunodeficiency Syndrome (AIDS) where it causes necrotizing encephalitis. It is believed that these episodes of central nervous system (CNS) toxoplasmosis are due to transition of latent encysted bradyzoites (found in tissue cysts) into tachyzoites leading to increased inflammation and necrosis. The persistence of bradyzoites within tissue cysts is critical for latency and the major risk factor for reactivation during immune suppression. We have established that cysts are enclosed in highly glycosylated cyst wall, but the role(s) of glycosylation in persistence and the pathogenesis of parasites are not fully understood. Several glycoproteins are known to be expressed and localized to the cyst wall. We have demonstrated that one of these cyst wall glycoproteins, CST1, is critical in the differentiation of the parasites and formation of the cyst wall structure. Furthermore, we have demonstrated that CST1 is primarily modified by o-linked glycosylation and that this glycosylation of CST1 is critical for cyst wall stability. A limitation of studies on glycosylation of proteins in intracellular parasites has been the ability to get selective labeling of parasite proteins without those of the host. This R21 will focus on a glycomic analysis of T. gondii using novel reagents we have developed that employ click chemistry and genetically modified T. gondii strains. This technique is applicable to many compounds and, once validated, will provide a tool for selective delivery of small molecules to obligate intracellular pathogens, permitting selective labeling of these organisms as well as selective targeting of pathways. The proposed studies utilize a proteomic approach employing click chemistry and mass spectrometry to validate this new strategy. This will enable us to characterize the T. gondii glycoproteome during cyst formation. The interdisciplinary team that has been assembled for this project includes internationally recognized expertise in T. gondii bradyzoite biology (Dr. Louis Weiss), orthogonal click chemistry (Dr. Peng Wu) and glycobiology (Dr. Pamela Stanley).