SECRETED EFFECTORS OF TOXOPLASMA GONDII BRADYZOITES

Project Summary/AbstractToxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa, a diverse group ofparasites that cause various diseases in humans, such as malaria and cryptosporidiosis. T. gondii isubiquitous worldwide, estimated to infect one-third of the human population, and is a public healthconcern in immunocompromised and pregnant individuals. The acute phase of infection ischaracterized by the tachyzoite, a fast-replicating life stage that disseminates throughout the body ofa warm-blooded host. The chronic phase of infection is characterized by the bradyzoite, a slowly-replicating life stage which encysts in muscle and neural tissue via the formation of a cyst wall withina host cell. Bradyzoite biology is poorly understood; the exact composition of the cyst wall, howbradyzoites persist within host cells indefinitely, or how bradyzoites manipulate host cell function isunknown. Previous studies have demonstrated the secretion of several protein effectors bytachyzoites into their host cells across the parasitophorous vacuole, the site where parasites replicatewithin host cells. These include the proteins GRA16, GRA24, GRA28, and TgIST, most of which havebeen shown to localize to the host cell nucleus and directly interact with host cell proteins, alteringhost cell signaling cascades and transcription. Whether these proteins, or other unidentified secretedproteins, cross the cyst wall and enter bradyzoite infected host cells has not been previouslyexplored. In this proposal, the first aim will be to determine the localization of GRA16, GRA24,GRA28, and TgIST after bradyzoite differentiation in vitro by epitope tagging secreted effectors attheir endogenous loci and in vivo in the mouse brain using an optical clearing approach. In thesecond aim, the role of TgIST in bradyzoite infected cells will be investigated by knocking out andcomplementing the TgIST gene in the parasite and subsequently determining the inhibition of STAT1mediated transcription in bradyzoite infected host cells. In the third aim, proximity-based biotinlabeling enzymes will be fused to MYR1, a protein implicated in parasite protein export into the hostcell. This will allow the identification of secreted effectors during the bradyzoite stage by massspectrometry of biotinylated proteins following validation of positive hits. The discovery of secretedeffectors by bradyzoites into host cells would challenge a paradigm in the field of Toxoplasmaresearch, where the bradyzoite has classically been viewed as an inert but transmissible stage of theparasite. This will lead to further studies on understanding how bradyzoites alter host cell function andmaintain persistency in their hosts.