ABSTRACT: Tuberculosis is the leading killer among infectious diseases worldwide. Increasing multidrug 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 multisystem 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 levels of phosphorylation of many proteins and extensive changes in levels of 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 that these kinases are potentially valuable targets for new antituberculosis drugs. In addition, the data from these linked multisystems provide a valuable resource for future targeted investigations into the pathways regulated by these kinases in the M. tuberculosis cell.IMPORTANCE Tuberculosis is the leading killer among infectious diseases worldwide. Increasing drug resistance threatens efforts to control this epidemic; thus, new antitubercular drugs are urgently needed. We performed an integrated, multisystem analysis of Mycobacterium tuberculosis responses to inhibition of its two essential serine/threonine protein kinases. These kinases allow the bacterium to adapt to its environment by phosphorylating cellular proteins in response to extracellular signals. We identified differentially phosphorylated proteins, downstream changes in levels of specific mRNA and protein abundance, and alterations in the metabolite and lipid content of the cell. These results include changes previously linked to growth arrest and also reveal new roles for these kinases in regulating essential processes, including growth, stress responses, transport of proteins and other molecules, and the structure of the mycobacterial cell envelope. Our multisystem data identify PknA and PknB as promising targets for drug development and provide a valuable resource for future investigation of their functions.
