Toxoplasma gondii is a widespread human pathogen that is an important opportunistic infection in HIV-AIDS patients. Disease in AIDS patients is typically the result of reactivation of the bradyzoite cyst form of the parasite that maintains the chronic infection throughout the life of the host. In spite of the central role of bradyzoites in establishing this chronic infection, remarkably little is known regarding the secreted parasite factors that mediate cyst formation and resistance to host immune clearance. Elucidating these processes has been hampered by limitations in the biochemical approaches for purifying the parasite-containing vacuole to isolate and study the secreted proteins that comprise the cyst. To overcome this barrier, we have recently adapted an in vivo biotinylation approach (called BioID) that employs a promiscuous biotin ligase that can label proteins in subcellular compartments in T. gondii. We have optimized this technique for dense granule (GRA) proteins that are constitutively secreted into the parasitophorous vacuole and shown that we can label a wide array of vacuolar components from the fast-growing tachyzoite stage of the parasite that mediates the acute infection. In this application, we build on this strong foundation by utilizing BioID for robust labeling of the vacuole in bradyzoites, enabling us to fill the gap in our knowledge of the parasite constituents secreted into the bradyzoite cyst. We will also couple this advance with an improved CRISPR/Cas9 approach that facilitates rapid gene deletions in ?ku80 strains of Toxoplasma for ascertaining the function of the novel bradyzoite GRAs identified by BioID. Together, these methods will allow us to identify and functionally evaluate cyst components that promise to reveal how Toxoplasma parasites persist within their host and cause disease during this unique life-cycle stage.
Toxoplasma gondii is an opportunistic pathogen that causes serious disease in immunocompromised patients by reactivation of an encysted form of the parasite called the bradyzoite upon loss of immune surveillance. The bradyzoite maintains its lifelong persistence in the host by secreting dense granule (GRA) proteins that ensure parasite survival and cyst maintenance. This proposal uses an approach named BioID to identify these secreted cyst factors and study their function by gene knockout.
| Nadipuram, Santhosh M; Kim, Elliot W; Vashisht, Ajay A et al. (2016) In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis. MBio 7: |
