ABSTRACT: Toxoplasma gondii is a ubiquitous Apicomplexan protozoan parasite of mammals andbirds. It is unusual in that propagation does not require passage through its definitive host enabling T.gondii to propagate clonally through its intermediate hosts. T. gondii causes congenital infections inimmune competent hosts and opportunistic infections in immune compromised hosts. The predilectionof this parasite for the central nervous system causing necrotizing encephalitis and for the eye causingchorioretinitis constitutes its major threat to patients. The development of these diseases is aconsequence of the transition of bradyzoites, found within tissue cysts into actively replicatingtachyzoites. It is believed that tissue cysts are not static structures, but regularly rupture reinvading newhost cells. It is likely that in chronic toxoplasmosis, i.e. latent infection, tissue cysts within host cells,regularly transform to tachyzoites which are removed or sequestered by the immune system.Degenerating cysts are often seen in the brains of mice with chronic toxoplasmosis. Such a dynamicequilibrium between encysted and replicating forms leads to recurrent antigenic stimulation and thepersistent antibody titers found in chronically infected hosts. The widespread distribution of T. gondii inhumans and other animals is due to the ability of tissue cysts to permit oral transmission of this infection.The cyst wall is the critical structure for survival, reactivation and transmission of T. gondii.Understanding T. gondii developmental biology and formation of the cyst wall will inform strategies suchas vaccine development and therapeutic agents to eliminate latency and prevent reactivationtoxoplasmosis. Several lines of evidence suggest that bradyzoite differentiation is stress mediatedand that the cyst wall (a modified parasitophorous vacuole membrane) contains many stage specificproteins and glycoproteins. Our laboratory group has identified several cyst wall specific proteinsseveral of which have mucin type domains that are o-glycosylated and demonstrated thatglycosylation is important for cyst wall stability. CST1, a cyst wall glycoprotein, appears to be ascaffolding protein for formation of the cyst wall and we hypothesize that other cyst wall proteinsinteract with CST1 in establishing the cyst wall. Our laboratory group has developed techniques topurify the cyst wall enabling proteomic characterization of this structure as well as adapted BirAtagging techniques to enable definition of the cyst wall interactome. Furthermore, we haveestablished ppGalNAcTs knockout T. gondii strains that enable studies on the role of o-glycosylationin cyst wall formation. An integrated approach employing proteomic, immunologic and genetictechniques will be used to fully characterize the T. gondii cyst wall proteome and the importance andinteractions of the identified cyst wall components. The improved understanding of the formation ofthe cyst wall provide by these studies will provide the basic underpinnings of new strategies toeliminate latent infection thereby preventing reactivation toxoplasmosis.