Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, Virginia, USA.
Department of Biomedical Sciences, Edward Via College of Osteopathic Medicine (VCOM), Blacksburg, Virginia, USA.
mSphere. 2024 Nov 21;9(11):e0077924. doi: 10.1128/msphere.00779-24. Epub 2024 Oct 30.
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 biology. The 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 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 propagation and virulence.
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 and essential for virulence . Herein, we show that the TgTKL1 is, indeed, a 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 .
酪氨酸激酶样（TKL）蛋白家族是一组研究较少的激酶，近年来因其在生物学中的作用而受到关注。基因组包含八个 TKL 激酶，其中六个被预测对寄生虫的繁殖很重要。我们之前已经表明，TgTKL1 是一种核激酶，对于寄生虫的裂解周期至关重要，并且对于动物模型中的急性毒力是必需的。然而，激酶结构域对 TgTKL1 功能的贡献尚不清楚。因此，为了确定其催化功能的重要性，我们首先使用纯化的重组蛋白验证了 TgTKL1 是一种真正的激酶。此外，我们通过 CRISPR-Cas9 基因编辑成功地生成了 TgTKL1 激酶突变株。我们的研究表明，TgTKL1 的激酶突变体在寄生虫生长和宿主细胞入侵方面存在缺陷。此外，激酶功能的丧失改变了寄生虫的转录组谱，包括下调与入侵相关的基因 TgSUB1。重要的是，这种 TgSUB1 表达的失调导致微线体蛋白的出芽后加工缺陷，这对于正常的宿主细胞入侵至关重要。此外，TgTKL1 激酶突变体在急性弓形体病的小鼠模型中完全无毒性。由于激酶功能的丧失导致以前与 TgTKL1 敲除寄生虫所见的表型表现，我们得出结论，激酶活性对于 TgTKL1 在寄生虫繁殖和毒力中的功能很重要。
是一种原生动物寄生虫，可导致人类危及生命的疾病。因此，确定寄生虫生长和发病机制所需的关键因素对于开发新型治疗方法很重要。我们之前已经表明，TKL 蛋白激酶家族的一个成员，TgTKL1，是一种植物样激酶，对于有效的弓形虫生长是必需的，并且对于毒力是必不可少的。在这里，我们表明 TgTKL1 确实是一种激酶，并且在弓形虫中失去其激酶功能会导致与完全缺失 TgTKL1 的寄生虫中所见的类似缺陷。更具体地说，TgTKL1 激酶突变体在寄生虫生长，宿主细胞入侵，基因表达谱和动物模型中的毒力方面均存在缺陷。总之，这些发现表明 TgTKL1 是一种真正的激酶，并且其激酶活性的丧失会导致 TgTKL1 在中的功能中断。
