The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth

Turapov, Obolbek; Loraine, Jessica; Jenkins, Christopher; Barthe, Philippe; McFeely, Daniel; Forti, Francesca; Ghisotti, Daniela; Hesek, Dušan; Lee, Mijoon; Bottrill, Andrew R.; Vollmer, Waldemar; Mobashery, Shahriar; Cohen‐Gonsaud, Martin; Mukamolova, Galina V.

doi:10.1098/rsob.150025

Cited by 22 publications

(24 citation statements)

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“…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).…”

Section: Discussionmentioning

confidence: 99%

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

Alqaseer

Turapov

Barthe

et al. 2019

Molecular Microbiology

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Summary Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi‐drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H‐NS‐like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H‐NS, Lsr2 binds DNA in sequence‐dependent and non‐specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA‐binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP‐sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

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Section: Discussionmentioning

confidence: 99%

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

Alqaseer

Turapov

Barthe

et al. 2019

Molecular Microbiology

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“…This regulation is essential for adjusting bacterial growth and synthesis of the cell wall. The external PASTA domain of PknB 40, 41 is essential for PknB-mediated signalling and its disruption results in bacterial death and alteration of antimicrobial susceptibility 42,5,31 . Here we present data demonstrating that PknB also controls global gene expression via another substrate, the DNA-binding protein Lsr2.…”

Section: Discussionmentioning

confidence: 99%

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the global transcriptional regulator Lsr2

Alqaseer

Turapov

Jagatia

et al. 2019

Preprint

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Mycobacterium tuberculosis is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the essential protein kinase B (PknB), that control transitions between growth states. Here, we establish that depletion of PknB in replicating M. tuberculosis results in transcriptional adaptations that implicate the DNA-binding protein Lsr2 in coordinating these changes. We show that Lsr2 is phosphorylated by PknB, and that phosphorylation of Lsr2 at threonine 112 is important for M. tuberculosis growth and survival under hypoxic conditions. Fluorescence anisotropy and electrophoretic mobility shift assays demonstrate that phosphorylation reduces Lsr2 binding to DNA, and ChIP-sequencing confirms increased DNA binding of a phosphoablative (T112A) Lsr2 mutant in M. tuberculosis. Altered expression of target genes in T112A Lsr2 compared to wild type Lsr2 M. tuberculosis offers further evidence that phosphorylation mediates expression of the Lsr2 regulon. Structural studies reveal increased dynamics of the Lsr2 DNA binding domain from a T112D phosphomimetic Lsr2 mutant, providing a molecular basis for decreased DNA binding by phosphorylated Lsr2. Our findings suggest that, the essential protein kinase, PknB controls M. tuberculosis growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2. expression (WhiB6) 24 . The transcriptional signature of PknB-depletion resembled features of intracellular growth, which significantly overlapped with M. tuberculosis macrophage-derivedRNA profiles from several studies as reflected by hypergeometric probability values: 6.7x10 -23 25 ; 7.34x10 -18 26 ; 3.57x10 -17 27 (Fig. 1). This profile was exemplified by induction of pathways involved in mycobactin synthesis (mbtB/C/D), complex lipid phthiocerol dimycocerosate (PDIM) biosynthesis (fadD26, ppsA/B/C/D), metabolism of alternative lipid carbon sources, the glyoxylate shunt (icl), the methylcitrate cycle (prpD/C, prpR) and triacylglycerol synthase (tgs1). The isoniazid inducible genes (iniB/A/C), responsive to cell wall stress and cell wall targeting drugs were also upregulated 28 . Four of the nine genes coding for alternative ribosomal proteins, rpmB1, rpmB2, rpmG1, rpsN2 29 were induced by PknB depletion.The 34 genes that were significantly repressed in PknB-depleted bacteria included pknB itself (6 fold down-regulated, as expected); nuoA/B/C, encoding subunits of NADH dehydrogenase I, which is part of the aerobic respiratory chain, and several genes involved in intermediary metabolism (Table S1). Overall these changes in replicating bacteria were comparable, in number of differentially expressed genes, to depletion of other regulators, for example DosR 30 . This is in contrast to the large-scale changes in gene expression after treatment with an inhibitor of PknB and PknA that would likely impact M. tuberculosis viability 31 . In summary, PknB depletion in replicating bacteria resulted in co-ordinated change...

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“…For example, PknB, the only STPK containing PASTA in M. tuberculosis , is essential for cell survival . This STPK kinase possesses a modular organization characterized by an extracellular (sensor) domain composed by four PASTA motives, which were shown to be required for PknB localization to the division septum and also involved in the regulation of PGN biosynthesis and maintenance of cell‐wall architecture in mycobacteria . A likely explanation for the muropeptide‐driven resuscitation mediated by STPKs is that the binding of muropeptides to the extracellular PASTA domains brings the kinases close enough to facilitate intracellular phosphorylation of the kinase catalytic domains …”

Section: Pgn Degradation and Bacterial Return To Lifementioning

confidence: 99%

Chemistry of Peptidoglycan in Mycobacterium tuberculosis Life Cycle: An off‐the‐wall Balance of Synthesis and Degradation

Squeglia

Ruggiero

Berisio

2017

Chemistry A European J

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The cell wall envelope of mycobacteria is structurally distinct from that of both Gram-positive and Gram-negative bacteria. In Mycobacterium tuberculosis, this cell wall has unique structural features and plays a crucial role in drug resistance and macrophage survival under stress conditions. Peptidoglycan is the major constituent of this cell wall, with an important structural role, giving structural strength, and counteracting the osmotic pressure of the cytoplasm. Synthesis of this complex polymer takes place in three stages that occur at three different locations in the cell, from the cytoplasm to the external side of the cell membrane, where polymerization occurs. A fine balance of peptidoglycan synthesis and degradation is responsible for a plethora of molecular mechanisms which are key to the pathogenicity of M. tuberculosis. Enlargement of mycobacterial cells can occur through the synthesis of new peptidoglycan, autolysis of old peptidoglycan, or a combination of both processes. Here, we discuss the chemical aspects of peptidoglycan synthesis and degradation, in relation to metabolic stages of M. tuberculosis. Going from inside the mycobacterial cytoplasm to outside its membrane, we describe the assembly line of peptidoglycan synthesis and polymerization, and continue with its depolymerization events and their consequences on mycobacterial life and resuscitation from dormancy.

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The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth

Cited by 22 publications

References 68 publications

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

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

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the global transcriptional regulator Lsr2

Chemistry of Peptidoglycan in Mycobacterium tuberculosis Life Cycle: An off‐the‐wall Balance of Synthesis and Degradation

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