Two Faces of CwlM, an Essential PknB Substrate, in Mycobacterium tuberculosis

Abstract

SummaryTuberculosis claims >1 million lives annually, and its causative agent Mycobacterium tuberculosis is a highly successful pathogen. Protein kinase B (PknB) is reported to be critical for mycobacterial growth. Here, we demonstrate that PknB-depleted M. tuberculosis can replicate normally and can synthesize peptidoglycan in an osmoprotective medium. Comparative phosphoproteomics of PknB-producing and PknB-depleted mycobacteria identify CwlM, an essential regulator of peptidoglycan synthesis, as a major PknB substrate. Our complementation studies of a cwlM mutant of M. tuberculosis support CwlM phosphorylation as a likely molecular basis for PknB being essential for mycobacterial growth. We demonstrate that growing mycobacteria produce two forms of CwlM: a non-phosphorylated membrane-associated form and a PknB-phosphorylated cytoplasmic form. Furthermore, we show that the partner proteins for the phosphorylated and non-phosphorylated forms of CwlM are FhaA, a fork head-associated domain protein, and MurJ, a proposed lipid II flippase, respectively. From our results, we propose a model in which CwlM potentially regulates both the biosynthesis of peptidoglycan precursors and their transport across the cytoplasmic membrane.

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112

“…B) and generated several bands, indicative of multiple phosphorylated forms. Mass spectrometry confirmed the previously observed phosphosite on threonine 112 (Turapov et al , ) and detected additional phosphorylations at threonine 8, threonine 22 and threonine 31 (Fig. C and D).…”

“…B. SDS‐PAGE revealed a shift in Lsr2 mobility upon phosphorylation (lanes identical to panel A). C. Schematic presentation of phosphosites identified in phopshoproteomics studies (top) (Turapov et al, ), and in vitro (bottom). D. Phosphopeptides detected by mass spectrometry; phosphorylated residues shown in bold font.…”

“…Transcriptome profiling of PknB-depleted Mtb revealed an Lsr2-regulated gene expression signature PknB is essential for growth in standard conditions; however, we have recently developed an osmoprotective medium (SMM) that supported growth of PknB-depleted Mtb and allowed us to identify PknB substrates (Turapov et al, 2018). Using the same system, we compared the transcriptional profile of PknB-depleted versus PknBproducing Mtb (Fig.…”

“…One of these regulatory systems is protein phosphorylation involving 11 serine/threonine kinases, including protein kinase B. PknB is expressed during active replication (Kang et al, 2005) and is essential for growth in standard media (Fernandez et al, 2006;Forti et al, 2009). PknB has been implicated in the regulation of peptidoglycan biosynthesis (Bellinzoni et al, 2019); its main substrate CwlM stimulates biosynthesis of peptidoglycan precursors (Boutte et al, 2016) and may facilitate their transport to the cell surface (Turapov et al, 2018). This regulation is crucial for adjusting bacterial growth and synthesis of the cell wall.…”

“…This regulation is crucial for adjusting bacterial growth and synthesis of the cell wall. The external PASTA domain of PknB (Barthe et al, 2010;Prigozhin et al, 2016) is essential for PknB-mediated signalling and its disruption results in bacterial death and alteration of antimicrobial susceptibility (Chawla et al, 2014;Turapov et al, 2015;Turapov et al, 2018). Furthermore, PknB can phosphorylate sigma factor SigH and its cognate anti-sigma factor RshA; phosphorylation disrupts interaction of these two proteins and result in increased expression of the SigH regulon (Park et al, 2008).…”