Elucidation of TgPKG kinase substrates required for Toxoplasma motility

特定的目标

Specific Aims Toxoplasma gondii persists in 25-30% of humans worldwide because there are no vaccines to prevent infections and current therapies are non-curative (1). The first-line therapy for toxoplasmosis consists of pyrimethamine and sulfadiazine, which inhibit intracellular parasite replication but are highly toxic and unable to eliminate the dormant tissue-encysted stage of T. gondii (2). Thus, there is an urgent need for new non-toxic drugs that block T. gondii infectivity in both acute (tachyzoite) and chronic (bradyzoite) life stages. Cyclic guanosine monophosphate (cGMP) has emerged as a master regulator of T. gondii infectivity by coordinating parasite motility (3, 4). Previously, we determined that a T. gondii guanylate cyclase (TgGC) synthesizes cGMP from GTP to stimulate motility for entry and exit of host cells (5). Conditional depletion of TgGC paralyzed T. gondii, rendering parasites incapable of establishing infections and causing disease. Additionally, we determined that a cGMP-dependent protein kinase (TgPKG) at the plasma membrane acts as the central effector of cGMP in T. gondii in both tachyzoites (6) and bradyzoites (7). Chemical inhibition or conditional knockdown of TgPKG phenocopied loss of TgGC, completely blocking parasite motility and infectivity (6, 8). Furthermore, we determined that TgPKG functions by controlling secretion of microneme proteins that are required for parasite motility, host cell invasion, and host cell egress (6). However, the mechanisms by which TgPKG controls microneme secretion and motility remain unclear because its substrates have not been identified or characterized. We hypothesize that TgPKG regulates T. gondii motility and virulence through phosphorylation of proteins that regulate microneme expression and secretion. Here, we will utilize modern genetic, transcriptomic, and proteomic approaches to test our hypothesis with three independent Specific Aims. Specific Aim 1: Identify genes differentially expressed following TgPKG knockdown. TgPKG controls T. gondii infectivity by regulating microneme secretion (6). We speculate that TgPKG directly modifies proteins required for microneme fusion with the plasma membrane. However, TgPKG may also regulate microneme secretion by modulating gene expression since PKGs regulate several transcription factors in other organisms (9). Previously, we generated a T. gondii line that expresses TgPKG fused to an auxin-inducible degron that allows for rapid and robust depletion of TgPKG with auxin treatment (6). Here we will identify genes that are differentially expressed following conditional depletion of TgPKG using RNA-Seq. Specific Aim 2: Identify interactors of TgPKG under basal and activated conditions. As with most kinases, we predict that TgPKG transiently interacts with its protein substrates following activation. We will use liquid chromatography with tandem mass spectrometry (LC-MS/MS) to identify TgPKG interactors captured by TurboID biotin-proximity labeling (10) and co-immunoprecipitation. We will treat extracellular parasites with or without a cell permeable cGMP analog (PET-cGMP) to distinguish proteins that interact with TgPKG basally or following activation. We have already generated an epitope-tagged TgPKG line for co-immunoprecipitation experiments (6) and a new TurboID-tagged TgPKG line for kinetic proximity labeling experiments. These two approaches are complementary since TurboID will provide a snapshot of proteins in close proximity (<15 nm) to TgPKG in live parasites, while co-immunoprecipitation will reveal proteins that directly interact with TgPKG. Furthermore, we will simultaneously assess the phosphorylation status each peptide identified, which could be modulated upon TgPKG activation if it is a direct substrate. Specific Aim 3: Define the TgPKG-dependent phosphoproteome under basal and activated conditions. In addition to knowing what TgPKG directly interacts with, it is critical to determine which interactors are phosphorylated by TgPKG. Using the TgPKG knockdown line, we will use stable isotope labeling using amino acids in cell culture (SILAC) phosphoproteomics to identify and quantify phosphopeptides from extracellular parasites treated with or without PET-cGMP in the presence or absence of TgPKG (+/- auxin treatment). Overall Impact. This study will provide critical insights into how TgPKG controls T. gondii motility and expose additional essential targets for drug development for treating toxoplasmosis. Since PKGs are conserved and essential in other apicomplexan parasites, like Plasmodium (11), this study will likely serve as a model for apicomplexan PKG function. Candidate substrates of TgPKG will be comprehensively investigated further as the subject of NIH R01 proposals going forward.