TgTKL4 Is a Novel Kinase That Plays an Important Role in Toxoplasma Morphology and Fitness

Protein kinases of the protozoan parasite Toxoplasma gondii have been shown to play key roles in regulating parasite motility, invasion, replication, egress and survival within the host. The tyrosine kinase-like (TKL) kinase family of proteins are a set of poorly studied kinases that our recent studies have indicated play a critical role in Toxoplasma biology. In this study, we focused on TgTKL4, another member of the TKL family that is predicted to confer parasite fitness. Endogenous tagging of TgTKL4 identified it as a temporally oscillating kinase with dynamic localization in the parasite. Gene disruption experiments suggested that TgTKL4 is important for Toxoplasma propagation in vitro, and loss of this kinase resulted in replication and invasion defects. During parasite division, TgTKL4 expression was limited to the synthesis and mitosis-cytokinesis phases and, interestingly, loss of TgTKL4 led to defects in Toxoplasma morphology. Further analysis of the parasite cytoskeleton indicated that the subpellicular microtubules are shorter and more widely spaced in parasites lacking TgTKL4. Although loss of TgTKL4 caused only moderate changes in the gene expression profile, TgTKL4 null mutants exhibited significant changes in their global phospho-proteome, including in proteins that constitute the parasite cytoskeleton. Additionally, mice inoculated intraperitoneally with TgTKL4 knockout parasites showed increased survival rates, suggesting that TgTKL4 plays an important role in acute toxoplasmosis. Together, these findings suggest that TgTKL4 mediates a signaling pathway that regulates parasite morphology and is an important factor required for parasite fitness in vitro and in vivo. IMPORTANCE Toxoplasma gondii is a protozoan parasite that can cause life-threatening disease in mammals; hence, identifying key factors required for parasite growth and pathogenesis is important to develop novel therapeutics. In this study, we identified and characterized another member of the newly described TKL family, TgTKL4, a cell cycle-regulated kinase. By disrupting TgTKL4, we determined that this kinase is required for normal parasite growth in vitro and that loss of this kinase results in parasites with reduced competence in replication and invasion processes. Specifically, Toxoplasma parasites lacking TgTKL4 had defects in cytoskeletal arrangement, resulting in parasites with abnormal morphology. Phospho-proteome studies provided further clues that decreased phosphorylation of proteins that constitute the Toxoplasma cytoskeleton could be responsible for altered morphology in TgTKL4-deficient parasites. Additionally, loss of TgTKL4 resulted in attenuation of virulence in the animal model, suggesting that TgTKL4 is an important virulence factor. Hence, this study provides a novel insight into the importance of a TgTKL4 as a fitness-determining factor for Toxoplasma propagation in vitro and pathogenesis in vivo.