Defining the Role of the Kinase TgGSK in Toxoplasma gondii

TgGSK is a serine/threonine kinase in T. gondii that is most closely related to the glycogen synthase kinase BIN2 in plants. In plants, BIN2 is a negative regulator of brassinosteroid signaling, which plays a role in plant growth and development and stress responses. Previously, we have shown that another T. gondii brassinosteroid signaling homolog, PPKL, is essential for the regulation of proper parasite division. Therefore, we wanted to investigate if TgGSK plays a similar role. We have established a conditional knockdown strain and determined that TgGSK is essential for parasite propagation. Using immunofluorescence, we have determined that TgGSK has dynamic localization, which is concurrent with the parasite"s division cycle. Moreover, based on expansion microscopy we observe TgGSK in the centrosomes, corroborating a possible role in parasite division. Partial knockdown of TgGSK causes abnormal division phenotypes such as asynchronous division, uneven segregation of nuclear material, and abnormal amounts of centrosomes in each parasite as shown by both immunofluorescence and expansion microscopy. Immunoprecipitation has shown that TgGSK interacts with the GCN5b lysine acetyltransferase complex. This complex acetylates histones to open up chromatin for gene transcription and has been shown to interact with ~40% of T. gondii genes. While global transcriptomics did not show a clear pattern of differential gene transcription after TgGSK knockdown, we are currently performing global phosphoproteome analysis to determine differences in phosphorylation. These experiments will begin to elucidate a unique signaling pathway that is essential for T. gondii survival and replication.