Abstract:In humans clinically relevant disease with Toxoplasma gondii a common intracellular parasite results fromT.gondiis tropism for and life-long persistence in the CNS. To persist T.gondii switches from a fast replicatingform (tachyzoite) to a slow replicating encysted form (bradyzoite). While T.gondii infection is asymptomatic inmost T. gondii can cause severe neurologic complications in individuals with incomplete immune responses(e.g. AIDS patients developing fetuses) including cognitive visual and motor deficits and death. The treatmentsfor symptomatic toxoplasmosis most commonly pyrimethamine and sulfadiazine are poorly tolerated have lowtherapeutic indices and are ineffective against the persistent encysted form. The development of therapiestargeting T. gondiis encysted form requires a deep mechanistic understanding of how parasites encyst. Priorstudies have focused on identifying parasite genes that drive the tachyzoite/bradyzoite transition and parasiteproteins that form the cyst wall. Less work has been done on identifying host cell genes that influenceencystment and almost no work has been done on how parasite manipulations of the host cell affect encystment.The goal of this proposal is to address this gap by building upon my preliminary work showing that a well-knownkinase (ROP16) which parasites injects into the cytoplasm of host cells and which shows allelic variationbetween the canonical T. gondii strains (type I II and III) facilitates encystment in a strain-specific manner.Type I and III alleles (rop16I/III/ROP16I/III) which are 99% identical cause prolonged activation of STAT3 6 andpossibly 5 signaling pathways during acute tachyzoite infection while the type II allele does not. To determineif ROP16 play a role in encystment I generated type II and type III strains that lack ROP16 (IIrop16 andIIIrop16) and tested them in an in vitro encystment assay. Remarkably in contrast to IIrop16 which showedlittle change in encystment IIIrop16 has a greater than 2-fold decrease in forming cysts in fibroblasts andneurons. In addition I found that this defect can be complemented in trans by co-infecting a host cell withIIIrop16 and parental type III (WTIII) parasites but not with type II parasites suggesting a role for strain-specifichost cell manipulations. My overall hypothesis therefore is that ROP16III facilitates cyst development throughstrain-specific host cell manipulations. I will address this hypothesis by: i) determining which ROP16III functionsare required for encystment by complementing the IIIrop16 strain with a panel of ROP16 mutants that lackkinase activity nuclear localization or STAT-binding and assessing encystment (Aim 1) and ii) identifying theROP16III-dependent host genes that drive encystment by transcriptionally profiling cells in encysted conditionsand infected with WTIII IIIrop16 or complemented parasites (Aim 2). The completion of this work willsignificantly advance our understanding of how T.gondii encystment varies by strain type a necessary first steptoward developing strain-specific drugs against the encysted form of T. gondii.