Multisystem Analysis of Mycobacterium tuberculosis Reveals Kinase-Dependent Remodeling of the Pathogen-Environment Interface.

Xavier Carette,Robert N Husson,Jumei Zeng,David C Young,Cameron Stuver Moody,Kyu Y Rhee,Sladjana Prisic,Ashoka V R Madduri,John Platig,Zhe Wang,Jan Muntel,Michaela Helmel,Lakshmi-Prasad Potluri,John Quackenbush,Jacob A Mayfield,Tiansheng Wang,Hanno Steen,D Branch Moody,Joseph N Paulson,Jorge J Velarde ,A E Young ,Christopher P Locher

doi:10.1128/mbio.02333-17

Abstract

ABSTRACTTuberculosis 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.

Highlights

Tuberculosis is the leading killer among infectious diseases worldwide
PknA and PknB are potentially valuable targets for antituberculosis drug development based on their essentiality for growth in vitro and in mice [5,6,7] and on the successful development of several kinase inhibitors as drugs to treat human disease [4]
From a series of small molecules developed by Vertex Pharmaceuticals Incorporated to inhibit PknA and PknB, we selected a 5-substituted pyrimidine (Fig. 1A) based on its potent in vitro inhibition of PknB and PknA (Ki ϭ 0.018 ␮M), its ability to inhibit growth of M. tuberculosis H37Ra [minimal inhibitory concentration (MIC) ϭ 4.5 ␮M], and its selectivity for PknA and PknB relative to human kinases [19]

Summary

Introduction

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. 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. 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 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. PknA and PknB are potentially valuable targets for antituberculosis drug development based on their essentiality for growth in vitro and in mice [5,6,7] and on the successful development of several kinase inhibitors as drugs to treat human disease [4]

Methods

Results

Conclusion