Protein kinases PknA and PknB independently and coordinately regulate essential Mycobacterium tuberculosis physiologies and antimicrobial susceptibility

Author summary Tuberculosis is a major global health threat, with approximately 10 million new cases per year and 1.5 million deaths. Although tuberculosis is treatable, cure requires prolonged antibiotic courses and strains that are resistant to current treatments are increasingly common. Understanding how Mycobacterium tuberculosis adapts to the human host during infection can lead to new approaches to treat tuberculosis. A major adaptive mechanism is reversible protein phosphorylation mediated by protein kinases, which detect signals in the extracellular environment and transmit this information to regulate M. tuberculosis physiology. In this study we analyzed two M. tuberculosis kinases, PknA and PknB, by comparing strains in which these kinases are present to strains in which one or both of these kinases has been depleted. We identified features that differ between the kinase replete and kinase depleted strains including bacterial growth and viability, cell shape, the bacterial cell envelope and gene regulation. We also identified proteins that show decreased phosphorylation in the kinase depleted strains, which can result from direct or indirect effects of kinase depletion. These findings provide insight into how these kinases are important for M. tuberculosis pathogenesis and indicate that these kinases may be valuable targets for development of new TB drugs. The Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB are essential for growth and have been proposed as possible drug targets. We used a titratable conditional depletion system to investigate the functions of these kinases. Depletion of PknA or PknB or both kinases resulted in growth arrest, shortening of cells, and time-dependent loss of acid-fast staining with a concomitant decrease in mycolate synthesis and accumulation of trehalose monomycolate. Depletion of PknA and/or PknB resulted in markedly increased susceptibility to beta-lactam antibiotics, and to the key tuberculosis drug rifampin. Phosphoproteomic analysis showed extensive changes in protein phosphorylation in response to PknA depletion and comparatively fewer changes with PknB depletion. These results identify candidate substrates of each kinase and suggest specific and coordinate roles for PknA and PknB in regulating multiple essential physiologies. These findings support these kinases as targets for new antituberculosis drugs and provide a valuable resource for targeted investigation of mechanisms by which protein phosphorylation regulates pathways required for growth and virulence in M. tuberculosis.