Multisystems analysis of Mycobacterium tuberculosis reveals kinase-dependent remodeling of the pathogen-environment interface

Tuberculosis is the leading killer among infectious diseases worldwide. Increasing multi-drug resistance has prompted new approaches for tuberculosis drug development, including targeted inhibition of virulence determinants and of signaling cascades that control many downstream pathways. We used a multisystems approach to determine the effects of a potent small molecule inhibitor of the essential Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB. We observed differential phosphorylation of many proteins and extensive changes in gene expression, protein abundance, cell wall lipids and intracellular metabolites. The patterns of these changes indicate regulation by PknA and PknB of several pathways required for cell growth, including ATP synthesis, DNA synthesis and translation. These data also highlight effects on pathways for remodeling of the mycobacterial cell envelope via control of peptidoglycan turnover, lipid content, a SigE-mediated envelope stress response, transmembrane transport systems, and protein secretion systems. Integrated analysis of phosphoproteins, transcripts, proteins, and lipids identified an unexpected pathway whereby threonine phosphorylation of the essential response regulator MtrA decreases its DNA binding activity. Inhibition of this phosphorylation is linked to decreased expression of genes for peptidoglycan turnover, and of genes for mycolyl transferases, with concomitant changes in mycolates and glycolipids in the cell envelope. These findings reveal novel roles for PknA and PknB in regulating multiple essential cell functions and confirm these kinases as potentially valuable targets for new anti-tuberculosis drugs. In addition, these linked multisystems data provide a valuable resource for future targeted investigations into the pathways regulated by these kinases in the M. tuberculosis cell. RNA-seq for comparison of transcriptomic effects of a chemical inhibitor of Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB, to an inactive control compound. Two experiments: 1) a time course comparing active inhibitor (T4) vs. control compound (T10) in wild type M. tuberculosis H37Rv, and 2) comparison of treatment with T4 vs. T10 at 48 hours in pknD and pknL mutant strains derived from M. tuberculosis H37Rv.结核病（Tuberculosis）是全球范围内感染性疾病的首要致死病因。日益严峻的多药耐药性推动了结核病药物研发的新策略，包括靶向抑制毒力决定簇以及调控众多下游通路的信号级联反应。我们采用多系统研究策略，探究了强效小分子抑制剂对结核分枝杆菌（Mycobacterium tuberculosis）必需丝氨酸/苏氨酸蛋白激酶（Ser/Thr protein kinases）PknA和PknB的作用效果。 我们观察到大量蛋白质发生差异磷酸化，同时基因表达、蛋白质丰度、细胞壁脂质以及细胞内代谢物均出现广泛改变。这些变化模式表明，PknA与PknB可调控细胞生长所需的多条通路，包括ATP合成、DNA合成与蛋白质翻译过程。本研究数据还揭示了其通过调控肽聚糖周转、脂质含量、SigE介导的包膜应激反应、跨膜转运系统以及蛋白质分泌系统，进而重塑分枝杆菌细胞包膜的相关通路。 对磷酸化蛋白质、转录本、蛋白质与脂质的整合分析发现了一条意外通路：必需应答调节蛋白MtrA的苏氨酸磷酸化会降低其DNA结合活性。该磷酸化过程的抑制与肽聚糖周转相关基因以及分枝菌酸转移酶编码基因的表达下调相关，同时伴随细胞包膜中分枝菌酸与糖脂的含量变化。 这些研究结果揭示了PknA和PknB在调控多项必需细胞功能中的全新作用，并证实这两种激酶可作为新型抗结核病药物的潜在优质靶点。此外，这套关联式多系统数据集为后续针对结核分枝杆菌细胞内这些激酶调控通路的靶向研究提供了宝贵的资源。 本研究包含两组RNA测序（RNA-seq）实验，用于对比结核分枝杆菌Ser/Thr蛋白激酶PknA和PknB的化学抑制剂与无活性对照化合物的转录组效应： 1. 野生型结核分枝杆菌H37Rv中，活性抑制剂（T4）与对照化合物（T10）的时间进程对比实验； 2. 以结核分枝杆菌H37Rv为亲本构建的pknD与pknL基因突变株中，T4与T10处理48小时后的对比实验。