TgTKL4 Is a Novel Kinase That Plays an Important Role in Toxoplasma Morphology and Fitness.
TgTKL4 Is a Novel Kinase That Plays an Important Role in Toxoplasma Morphology and Fitness.
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弓形虫的蛋白激酶已被证明在调节寄生虫在宿主体内的运动、入侵、复制、出口和生存中起关键作用。酪氨酸激酶样（TKL）激酶家族是一组研究较少的激酶，我们最近的研究表明在弓形虫生物学中起关键作用。在这项研究中，我们关注的是TgTKL4，这是TKL家族的另一个成员，预计会赋予寄生虫适应性。TgTKL4的内源性标记表明它是一种在寄生虫中具有动态定位的暂时振荡激酶。基因破坏实验表明，TgTKL4对弓形虫的体外繁殖很重要，该激酶的缺失会导致弓形虫的复制和侵袭缺陷。在寄生虫分裂过程中，TgTKL4的表达仅限于合成和有丝分裂-细胞分裂阶段，有趣的是，TgTKL4的缺失会导致弓形虫形态的缺陷。进一步的细胞骨架分析表明，缺乏TgTKL4的寄生虫的膜下微管更短，间距更宽。虽然TgTKL4的缺失只引起基因表达谱的适度变化，但TgTKL4无突变体在其全局磷酸化蛋白质组中表现出显著变化，包括构成寄生虫细胞骨架的蛋白质。此外，腹腔接种TgTKL4敲除寄生虫的小鼠存活率增加，表明TgTKL4在急性弓形虫病中起重要作用。综上所述，这些发现表明TgTKL4介导了一条调节寄生虫形态的信号通路，是寄生虫在体内和体外适应度所需的重要因素。刚地弓形虫是一种原生动物寄生虫，可在哺乳动物中引起危及生命的疾病；因此，确定寄生虫生长和发病所需的关键因素对于开发新的治疗方法非常重要。在这项研究中，我们鉴定并表征了新描述的TKL家族的另一个成员，TgTKL4，一种细胞周期调节激酶。通过破坏TgTKL4，我们确定该激酶是体外正常寄生虫生长所必需的，并且该激酶的缺失导致寄生虫在复制和入侵过程中的能力降低。具体而言，缺乏TgTKL4的弓形虫在细胞骨架排列上存在缺陷，导致弓形虫形态异常。磷酸化蛋白质组学研究提供了进一步的线索，表明构成弓形虫细胞骨架的蛋白质磷酸化降低可能是导致tgtkl4缺陷寄生虫形态改变的原因。此外，TgTKL4的缺失导致动物模型的毒力衰减，表明TgTKL4是一个重要的毒力因子。因此，这项研究为TgTKL4作为弓形虫体外繁殖和体内发病的健康决定因子的重要性提供了新的见解。
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.
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作者：
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通讯作者：
White MW
DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
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