Abstract
ESCRT protein complexes play essential roles in membrane remodeling for multivesicular body formation,
intercellular communication, formation of the nuclear envelope, and cell division. We recently showed that the
obligate intracellular parasite Toxoplasma gondii repurposes host ESCRT machinery to acquire macromolecular
resources from infected cells. Host-derived resources critically fuel rapid growth of T. gondii acute stage
parasites, which are responsible for debilitating disease in immunodeficient or congenitally infected individuals.
Although we identified one parasite effector protein (GRA14) that directly binds the ESCRT-I complex protein
TSG101 and recruits it to the parasite’s replicative compartment, this protein only accounts for ~50% of TSG101
recruitment. Using a novel combination of CRISPR/Cas9 screening, high content imaging, and machine learning-
driven image analysis, in addition to GRA14, we identified 7 new parasite secretory proteins that putatively
contribute to TSG101 recruitment. Among the newly identified parasite proteins was ROP18, which is a major
virulence factor that is best known for effector function in evading innate immunity. Importantly, ROP18 deficient
parasites showed significantly less recruitment of TSG101, thus validating it as a hit and exposing a novel
function for this parasite kinase. However, additional studies are necessary to determine how ROP18 contributes
to TSG101 recruitment and which of the other 6 hits function in such recruitment. To meet this need, in Aim 1
we will determine if the kinase activity of ROP18 is required for TSG101 recruitment to the PV and identify
ROP18-linked parasite and host proteins that regulate TSG101 recruitment. In Aim 2, we will validate other hits
from the screen and determine if they are working in conjunction with GRA14 or ROP18. Together, these studies
will identify a novel protein network responsible for engaging host ESCRT machinery to ensure the successful
replication of an important human pathogen.