<i>Mycobacterium tuberculosis</i> protease MarP activates a peptidoglycan hydrolase during acid stress

Botella, Hélène; Vaubourgeix, Julien; Lee, Myung Hee; Song, Naomi; Xu, Weizhen; Makinoshima, Hideki; Glickman, Michael S.; Ehrt, Sabine

doi:10.15252/embj.201695028

Cited by 49 publications

(52 citation statements)

References 51 publications

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“…This opens the possibility to monitor the accessibility of the active site in different RipA constructs using the sulfhydryl‐specific DTNB derivatization spectrophotometrically at 412 nm (Figure ). The two‐domain construct RipApp (residues 39–472), the C‐terminal catalytic domain RipAc including the lid‐module of the inter‐domain linker (residues 260–472), the catalytic domain not including the inter‐domain linker RipActr (residues 321–472), the cysteine to alanine mutant variant of this RipActr(C383A), and RipAcX covering the residue range 236–472 proposed to be released by the periplasmic protease MarP were investigated (Figure ). In the constructs RipApp, RipAc, and RipAcX, the catalytic Cys383 was not solvent exposed (Figure C,D).…”

Section: Resultsmentioning

confidence: 99%

“…19 The N-terminal domain of RipA that shows no PG-cleaving activity was implicated in auto-inhibition, 16,20 and its proteolytic cleavage by the periplasmic protease MarP was suggested to activate the enzyme. 21 In several PG remodeling enzyme classes as penicillin binding proteins (PBPs) and L,D-transpeptidases (Ldts), the catalytic domains are combined with additional domains that mediate the interactions between the catalytic module and the polymeric substrate or other partner proteins. As the PG is a multilayered structure, some of these additional domains may be spacers positioning the catalytic modules to a certain distance from the membrane, as proposed in the case of Ldts producing the peptide cross-links.…”

Section: Characterized Inmentioning

confidence: 99%

“…RipA degrades high molecular weight PG from Bacillus subtilis displaying peptide stem sequence identical to the one found in M. tuberculosis (D‐Ala/D‐γ‐isoGlu/ meso ‐Dap/D‐Ala). 19 The N‐terminal domain of RipA that shows no PG‐cleaving activity was implicated in auto‐inhibition, and its proteolytic cleavage by the periplasmic protease MarP was suggested to activate the enzyme …”

Section: Introductionmentioning

confidence: 99%

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The structure of the N‐terminal module of the cell wall hydrolase RipA and its role in regulating catalytic activity

et al. 2018

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RipA plays a vital role during cell division of Mycobacterium tuberculosis by degrading the cell wall peptidoglycan at the septum, allowing daughter cell separation. The peptidoglycan degrading activity relies on the NlpC/P60 domain, and as it is potentially harmful when deregulated, spatial and temporal control is necessary in this process. The N-terminal domain of RipA has been proposed to play an inhibitory role blocking the C-terminal NlpC/P60 domain. Accessibility of the active site cysteine residue is however not limited by the presence of the N-terminal domain, but by the lid-module of the inter-domain linker, which is situated in the peptide binding groove of the crystal structures of the catalytic domain. The 2.2 Å resolution structure of the N-terminal domain, determined by Se-SAD phasing, reveals an all-α-fold with 2 long α-helices, and shows similarity to bacterial periplasmic protein domains with scaffold-building role. Size exclusion chromatography and SAXS experiments are consistent with dimer formation of this domain in solution. The SAXS data from the periplasmic two-domain RipA construct suggest a rigid baton-like structure of the N-terminal module, with the catalytic domain connected by a 24 residue long flexible linker. This flexible linker allows for a catalytic zone, which is part of the spatiotemporal control of peptidoglycan degradation.

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Section: Resultsmentioning

confidence: 99%

Section: Characterized Inmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The structure of the N‐terminal module of the cell wall hydrolase RipA and its role in regulating catalytic activity

et al. 2018

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“…3A), possibly indicating the presence of partially degraded MptA. There are a number of proteases that have the active site in the periplasmic space, including the serine proteases MarP, HtrA and Rv3671c that are proposed to play roles in cell envelope homeostasis and stress response in mycobacteria (29)(30)(31)(32)(33). It remains to be determined if MptA is proteolytically degraded and which protease fulfills the role.…”

Section: Discussionmentioning

confidence: 99%

The role of LmeA, a mycobacterial periplasmic protein, in stabilizing the mannosyltransferase MptA and its product lipomannan under stress

Rahlwes

Osman

Morita

2020

Preprint

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The mycobacterial cell envelope has a diderm structure, composed of an outer mycomembrane, an arabinogalactan-peptidoglycan cell wall, periplasm and an inner membrane. Lipomannan (LM) and lipoarabinomannan (LAM) are structural and immunomodulatory components of this cell envelope. LM/LAM biosynthesis involves a number of mannosyltransferases and acyltransferases, and MptA is an α1,6-mannosyltransferase involved in the final extension of the mannan backbones. Recently, we reported the periplasmic protein LmeA being involved in the maturation of the mannan backbone in Mycobacterium smegmatis. Here, we examined the role of LmeA under stress conditions. We found that the lmeA transcription was upregulated under two stress conditions: stationary growth phase and nutrient starvation. Under both conditions, LAM was decreased, but LM was relatively stable, suggesting that maintaining the cellular level of LM under stress is important. Surprisingly, the protein levels of MptA were decreased in lmeA deletion mutant (ΔlmeA) in both stress conditions. The transcript levels of mptA in ΔlmeA were similar to or even higher than those in the wildtype, indicating that the decrease of MptA protein was a post-transcriptional event. Consistent with the decrease in MptA, ΔlmeA was unable to maintain the cellular level of LM under stress. Even during active growth, overexpression of LmeA led the cells to produce more LM and become more resistant to several antibiotics. Altogether, our study reveals the roles of LmeA in the homeostasis of the MptA mannosyl-transferase particularly under stress conditions, ensuring the stable expression of LM and the maintenance of cell envelope integrity.

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“…55 In turn, M. tuberculosis and M. smegmatis have evolved the ability to tolerate acidic environments by reprogramming a series of genetic elements 56 including some that are associated with PG biosynthesis. 57,58 To test the potential for PG remodeling in response to acidic environments, M. smegmatis cells were grown in media supplemented with probes at pH 4.0 or pH 7.4 (Figure 5). Remarkably, there was a nearly 8-fold increase in fluorescence levels for cells incubated with TriQmCYT in acidic media but no induction was observed for TriQLLys.…”

mentioning

confidence: 99%

Facile Synthesis and Metabolic Incorporation ofm-DAP Bioisosteres Into Cell Walls of Live Bacteria

Apostolos

Nelson

Pires

2020

Preprint

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Bacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes highly crosslinked by cell wall transpeptidases (TP). Because it is an essential component of the bacterial cell, the PG biosynthetic machinery is often the target of antibiotics. For this reason, cellular probes that advance our understanding of PG biosynthesis and its maintenance can be powerful tools to reveal novel drug targets. While synthetic PG fragments containing L-Lysine in the 3 rd position on the stem peptide are easier to access, those with meso-diaminopimelic acid (m-DAP) pose a severe synthetic challenge. Herein, we describe a solid phase synthetic scheme based on the widely available Fmoc-protected L-Cysteine building block to assemble meso-cystine (m-CYT), which mimics key structural features of m-DAP. To demonstrate proper mimicry of m-DAP, cell wall probes were synthesized with m-CYT in place of m-DAPand evaluated for their metabolic processing in live bacterial cells. We found that m-CYT-based cell wall probes were properly processed by TPs in various bacterial species that endogenously contain m-DAP in their PG. We anticipate that this strategy, which is based on the use of inexpensive and commercially available building blocks, can be widely adopted to provide greater accessibility of PG mimics for m-DAP containing organisms.

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Mycobacterium tuberculosis protease MarP activates a peptidoglycan hydrolase during acid stress

Cited by 49 publications

References 51 publications

The structure of the N‐terminal module of the cell wall hydrolase RipA and its role in regulating catalytic activity

The structure of the N‐terminal module of the cell wall hydrolase RipA and its role in regulating catalytic activity

The role of LmeA, a mycobacterial periplasmic protein, in stabilizing the mannosyltransferase MptA and its product lipomannan under stress

Facile Synthesis and Metabolic Incorporation ofm-DAP Bioisosteres Into Cell Walls of Live Bacteria

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