The essential kinase TgGSK regulates centrosome division and endodyogeny in Toxoplasma gondii

Abstract

Intracellular replication is crucial for the success of apicomplexan parasites, including Toxoplasma gondii. Therefore, essential players in parasite replication present potential targets for drug development. In this study, we have characterized TgGSK, a glycogen synthase kinase homolog that plays an important role in Toxoplasma endodyogeny. We have shown that TgGSK has a dynamic localization that is concurrent with the cell cycle. In non-dividing parasites, this kinase is highly concentrated in the nucleus. However, during division, TgGSK displays a cytosolic localization, with concentration foci at the centrosomes, a key organelle involved in parasite division, and the basal end. Conditional knockdown of TgGSK determined that it is essential for the completion of the lytic cycle and proper parasite division. Parasites lacking endogenous protein levels of TgGSK exhibited defects in division synchronicity and the segregation of the nucleus and apicoplast into forming daughter cells. These phenotypes are associated with defects in centrosome duplication and fission. Global phosphoproteomic analysis determined TgGSK-dependent phosphorylation of RNA-processing, basal end, and centrosome proteins. Consistent with the putative regulation of RNA-processing proteins, global transcriptomic analysis suggests that TgGSK is needed for proper splicing. Finally, we show that TgGSK interacts with GCN5b, a well-characterized acetyltransferase with roles in transcriptional control. Conversely, GCN5b chemical inhibition results in specific degradation of TgGSK. Thus, these studies reveal the involvement of TgGSK in various crucial processes, including endodyogeny and splicing, and identify acetylation as a possible mechanism by which this essential kinase is regulated. AUTHOR SUMMARYToxoplasma gondii infects nearly a third of the worlds human population. While infection is largely asymptomatic in healthy adults, in immunocompromised or immunosuppressed individuals it can lead to brain lesions and even death. Similarly, toxoplasmosis can result in stillbirth, birth defects, and blindness of a developing fetus in the case of a congenital infection. With minimal treatments for Toxoplasmosis available, it is crucial to study parasite-specific processes that could be potential drug targets for the treatment of Toxoplasmosis. In this study, we investigated the protein TgGSK that is essential for parasite survival and proper division. We showed that TgGSK may perform its essential functions through interaction with the centrosome, an organelle that plays a major role in cell division in many organisms. We also show in this study a role for TgGSK in proper processing of messenger RNAs. Taken together, we have performed an in-depth study of the functional role of the essential protein TgGSK in Toxoplasma gondii. Importantly, TgGSK was shown to have more similarity to plant proteins than mammalian proteins which may allow for the possibility of targeting of this protein for therapeutic treatment of toxoplasmosis.