Project SummaryA distinguishing characteristic of tissue cyst forming Apicomplexa, like Toxoplasma gondii, is the accumulationof amylopectin (starch) granules (AG) within bradyzoites and their absence within the actively growingtachyzoite forms. As polymers of glucose, AG functions as a battery serving as a reserve for energy productionand biosynthetic functions. Our recent work established that bradyzoites retain significant replicative potentialwithin tissue cysts in vivo. Notably, most bradyzoite replication within tissue cysts occurs asynchronously withclustered bursts of growth interceded with non-replicative periods. While the specific signals triggering thesebursts remain unknown, what is clear is that they would require substantial energy and metabolite inputs toexecute. Historical evidence and our findings reveal that AG levels within encysted bradyzoites are highlyvariable with clusters of parasites lacking AGs adjacent to others that are loaded with starch. This suggeststhat AG metabolism involving both synthesis and turnover are active within tissue cysts and may play a centralrole in the progression of chronic toxoplasmosis. We will directly address the dynamics and biologicalcontributions of AGs in the progression of the chronic infection at the level of individual bradyzoites using noveltools and concepts developed in our recently published work. The variable levels of AG, within encystedbradyzoites, suggest that both AG accumulation and depletion are under regulatory control. Indeed theenzymatic machinery required for both the synthesis and regulated turnover of starch are encoded in theToxoplasma genome. We will directly test the importance of AG in the acute infection, the regulation of stageconversion and the establishment/ progression of the chronic infection by targeting the commitment enzyme forstarch synthesis, the UDP-glucose pyrophosphorylase (TgUDP-GPP, TgME49_218200), and the starchsynthase (TgSS, TgME49_222800). Recent evidence suggests that despite being morphologically (though notbiochemically) detectable, amylopectin may play a role in tachyzoite intermediary metabolism as well. Inaddition, the contribution of AG's to reactivation, the primary trigger of symptomatic disease in HIV-AIDS will beaddressed in the context of induced immune suppression. With the proposed studies we aim to dissect thepreviously unexplored role of AG in tachyzoites, as factors in tachyzoite to bradyzoite conversion, theprogression of the chronic infection and clinically critical reactivation in vivo. These studies will establish thegroundwork for targeting AG metabolism for therapeutic intervention in the chronic infection where the paucityof effective drugs remains a significant issue in the clinical context of HIV-AIDS.