Functional exploration of a deep Mycobacterium tuberculosis phosphoproteome

摘要 磷酸化信号提供了细菌适应的主要渠道。双组分系统（TCS） 长期以来一直被视为细菌中典型的磷酸化信号系统，但越来越多地，O- 由Ser/Thr激酶介导的磷酸化被认为是相关的细菌磷酸化信号传导 机制也是如此。我们现在表明，事实上，结核分枝杆菌（Mtb）具有广泛的， 分布式的，合作的O-磷酸化系统的大小和复杂性，通常只与 真核生物通过使用全面的STPK功能丧失和获得突变面板和定量分析， 通过质谱分析，我们发现>70%的Mtb蛋白在Ser/Thr/Tyr上磷酸化，鉴定出数千种Mtb蛋白。 的单个Ser/Thr激酶底物，并显示Ser/Thr激酶共同控制的表达， 约30%的Mtb基因。在这里，我们将测试一个新的和广泛的调节连接之间的丝氨酸/苏氨酸激酶 以及TCS的His激酶，并测试O-磷酸化在转录调节中的作用 因素作为我们对结核分枝杆菌磷酸化的详尽分析的结果，我们还获得了结核分枝杆菌磷酸化的第一个证据。 Mtb中的Arg磷酸化-革兰氏阳性细菌外的第一个细菌Arg磷酸化蛋白质组。我们将 鉴定相关的磷酸酶并测试pArg作为ClpP-降解标签的功能 介导的蛋白水解。

ABSTRACT Phosphosignaling provides the major conduit for bacterial adaptation. The two component systems (TCSs) have long been viewed as the canonical phosphosignaling systems in bacteria, but increasingly, O- phosphorylation mediated by Ser/Thr kinases is recognized as a relevant bacterial phosphosignaling mechanism as well. We now show that in fact, Mycobacterium tuberculosis (Mtb) has an expansive, distributed, and cooperative O-phosphorylation system of a size and complexity that is typically only associated with eukaryotes. By using a comprehensive STPK loss- and gain-of-function mutant panel and quantitative mass spectrometry, we show that >70% of Mtb proteins are phosphorylated on Ser/Thr/Tyr, identify thousands of individual Ser/Thr kinase substrates, and show that the Ser/Thr kinases collectively control the expression of ~30% of Mtb genes. Here, we will test a new and extensive regulatory connection between the Ser/Thr kinases and the His kinases of the TCSs and test the role of O-phosphorylation on the regulation of transcription factors. As a result of our exhaustive analysis of Mtb phosphorylation, we also obtained the first evidence of Arg phosphorylation in Mtb- the first bacterial Arg phosphoproteome outside of gram-positive bacteria. We will identify the relevant phosphoenzymes and test the idea that pArg functions as a degradation tag for ClpP- mediated proteolysis.