The cell cycle-regulated cytoplasmic kinase, TgCRCK1, is required for efficient propagation of human protozoan pathogen,.

Abstract

is an obligate intracellular parasite that relies on a complex network of protein kinases to regulate essential processes such as invasion, replication, and egress. Thegenome encodes approximately 159 kinases, yet only a small subset has been characterized. Our group is interested in defining the role of cell cycle-regulated kinases important forfitness. In this study, we investigated the role of an uncharacterized, cell cycle-regulated cytoplasmic kinase, which we named TgCRCK1, in parasite biology and pathogenesis. Using endogenous tagging and immunofluorescence assays, we demonstrated that TgCRCK1 is a cytosolic kinase exhibiting a cell cycle-dependent expression pattern. Conditional depletion of TgCRCK1 via an auxin-inducible degron system impaired parasite growth, primarily due to defects in cell division and invasion. Transcriptomic analysis of TgCRCK1-deficient parasites revealed 93 differentially expressed genes, many involved in metabolism, gene expression, and intracellular transport. Although TgCRCK1 is essential for parasite growth, its depletion did not reducevirulence in the mouse model. Collectively, these findings identify TgCRCK1 as a key factor contributing topropagation.IMPORTANCEis the primary causative agent of toxoplasmosis, infecting a broad range of warm-blooded animals, including humans. The parasite depends on a complex network of protein kinases for growth and pathogenesis, yet the functions of many remain uncharacterized. In this study, we present the initial characterization of TgCRCK1, a cytoplasmic kinase with temporally regulated expression. Our results demonstrate that loss of TgCRCK1 impairs parasite growthby disrupting parasite division and host-cell invasion. However, studies in an animal model indicate that TgCRCK1 is not essential for acute toxoplasmosis. Together, these findings provide foundational insights into the localization, expression, and function of TgCRCK1 in this important human pathogen.