Kinase function of TgTKL1 is essential for its role in Toxoplasma propagation and pathogenesis

Abstract

ABSTRACT The Tyrosine Kinase-Like (TKL) family of proteins are a set of poorly studied kinases that have garnered attention in recent years for their role in Toxoplasma biology. The Toxoplasma genome contains eight TKL kinases, of which six have been predicted to be important for parasite propagation. We have previously shown that TgTKL1 is a nuclear kinase that is critical for the parasite lytic cycle and is essential for acute virulence in the animal model. However, the contribution of the kinase domain to the functioning of TgTKL1 was not known. Hence to determine the significance of its catalytic function, we first validated that TgTKL1 is a true kinase using purified recombinant protein. Furthermore, we successfully generated a TgTKL1 kinase mutant strain of Toxoplasma via CRISPR-Cas9 gene editing. Our studies revealed that the kinase mutant of TgTKL1 displays defects in parasite growth and host-cell invasion. Additionally, loss of kinase function alters the transcriptomic profile of the parasite, including downregulation of the invasion-related gene, TgSUB1. Importantly, this dysregulation of TgSUB1 expression leads to defects in post-exocytosis processing of micronemal proteins, an event critical for normal host-cell invasion. Furthermore, the TgTKL1 kinase mutant is completely avirulent in the mouse model of acute toxoplasmosis. Since the loss of kinase function leads to phenotypic manifestations seen previously with TgTKL1 knockout parasites, we conclude that kinase activity is important for TgTKL1 function in Toxoplasma propagation and virulence.IMPORTANCEToxoplasma gondii is a protozoan parasite that can cause life-threatening disease in humans. Hence, identifying key factors required for parasite growth and pathogenesis is important to develop novel therapeutics. We have previously shown that a member of the TKL protein kinase family, TgTKL1, is a plant-like kinase that is required for effective Toxoplasma growth in vitro and essential for virulence in vivo. Herein, we show that the TgTKL1 is, indeed, a bona fide kinase, and loss of its kinase function in the Toxoplasma leads to similar defects seen in parasites with complete loss of TgTKL1. More specifically, the TgTKL1 kinase mutant exhibits defects in parasite growth, host-cell invasion, gene expression profile, and virulence in the animal model. Together, these findings suggest that TgTKL1 is a true kinase, and loss of its kinase activity leads to disruption of TgTKL1 function in Toxoplasma.

Keywords

• apicomplexan parasites
• Toxoplasma gondii
• kinases