Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects

Vigouroux, A.; Cordier, Baptiste; Aristov, Andrey; Álvarez, Laura; Özbaykal, Gizem; Chaze, Thibault; Oldewurtel, Enno R.; Matondo, Mariette; Cava, Felipe; Bikard, David; Teeffelen, Sven van

doi:10.7554/elife.51998

Cited by 105 publications

(149 citation statements)

References 65 publications

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“…EPs (see a more detailed discussion below) for productive PG incorporation. These interpretations are in line with several recent observations in E. coli, i.e., aPBPs have more of a damage repair function than a functional role in promoting cell elongation (14) and that upregulated EP activity promotes aPBP function (28), likely indirectly through the creation of PG incisions that allow for an interaction between aPBPs and their OM- Taken together, our data suggest that two main cell wall synthases, the aPBPs and the Rod system have differential relationships with autolysins, and especially endopeptidases. While the Rod system likely relies on a "make-before-break" strategy, the aPBPs seem capable of the reverse, "break-before-make", i.e.…”

Section: Mreb Movement Continues In Ep-insufficient Cellssupporting

confidence: 92%

“…the ability to efficiently recognize and patch holes in the cell wall sacculus. As such, our data also provide additional support for a functional independence of the aPBPs and the Rod system (14,54), at least during cell elongation.…”

Section: Mreb Movement Continues In Ep-insufficient Cellssupporting

confidence: 72%

“…In many rod-shaped Gram-negative bacteria, cell wall synthesis during cell elongation is mediated by two separate types of cell wall synthase complexes: the Rod complex (which includes the glycosyltransferase RodA in conjunction with a class B Penicillin-Binding Protein [bPBP] and accessory proteins) and the class A PBPs in conjunction with their lipoprotein activators (7)(8)(9)(10)(11)(12). The differential physiological roles of these seemingly redundant systems have only recently been begun to be dissected (13,14), but remain incompletely understood.…”

Section: Introductionmentioning

confidence: 99%

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Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

Murphy

Murtha

Zhao

et al. 2020

Preprint

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AbstractThe bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth and cellular structural integrity. PG is synthesized by two different types of PG synthase complexes (class A Penicillin-binding Proteins [PBP]s/Lpos and Shape, Elongation, Division, Sporulation [SEDS]/class B PBP pairs) and degraded by ‘autolytic’ enzymes (e.g., endopeptidases, EPs) to accommodate growth processes. It is thought that autolsyin activity (and particulary the activity of EPs) is required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and the separate synthesis machineries remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae. We found that EP depletion resulted in severe morphological defects, increased cell mass, and a decline in viability, but continuing (yet aberrant) incorporation of cell wall material. Mass increase and cell wall incorporation proceeded in the presence of Rod system inhibitors, but was abolished upon inhibition of aPBPs. However, the Rod system remained functional (i.e., exhibited sustained directed motion) even after prolonged EP depletion, apparently without effectively inserting significant PG material. Lastly, heterologous expression of an EP from Neisseria gonorrhoeae could fully complement growth and morphology of an EP-insufficient V. cholerae. Overall, our findings suggest that in V. cholerae, only the Rod system absolutely requires endopeptidase activity (but not necessarily direct interaction with EPs) for productive PG incorporation, whereas aPBPs are able to engage in sacculus construction even during severe EP insufficiency.ImportanceSynthesis and turnover of the bacterial cell wall must be tightly co-ordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes (“autolysins”, e.g., endopeptidases, EPs) are required for activity of the different cell wall synthesis machines, the Rod system and the class A penicillin-binding proteins (aPBPs). Our results suggest that in Vibrio cholerae, endopeptidases are required only for cell expansion mediated by the Rod system, while the aPBPs maintain structural integrity during EP insufficiency. Overall, our results imply a complex relationship between cell wall synthesis and cleavage and suggest that aPBPs are more versatile than the Rod system in their ability to repair cell wall gaps formed by autolysins other than the major EPs, adding to our understanding of the co-ordination between autolysins and cell wall synthases.

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Section: Mreb Movement Continues In Ep-insufficient Cellssupporting

confidence: 92%

Section: Mreb Movement Continues In Ep-insufficient Cellssupporting

confidence: 72%

Section: Introductionmentioning

confidence: 99%

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Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

Murphy

Murtha

Zhao

et al. 2020

Preprint

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“…Remarkably, we detected a reduction of PBP1B Ec diffusivity in the presence of lipid II ( Figure 3). Previous in vivo single-molecule tracking of fluorescentprotein tagged class A PBPs reported the presence of two populations of molecules, a fast diffusing one and an almost immobile one with a near-zero diffusing rate which was assumed to be the active population (Cho et al, 2016, Lee et al, 2016, Vigouroux et al, 2020. Our result supports this interpretation, although more experiments are required to further explore this point.…”

Section: Towards Single-molecule Pg Synthesissupporting

confidence: 87%

Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

Hernández-Rocamora

Baranova

Peters

et al. 2020

Preprint

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Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here we developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we demonstrate this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay allows unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high throughput screening for new antimicrobials.

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“…In contrast, the role of class A PBPs is less clear, and notably, their numbers vary from one species to the other. It was recently shown, however, that class A PBPs can work independently of the SEDS–class B PBP complexes and contribute to the remodeling of the PG mesh required during expansion and/or after cell wall damages ( 21 , 22 ). The class A PBPs PBP1a, PBP1b, and PBP2a encoded by the pneumococcus are not essential, and individual deletion leads to weak morphological and growth defects.…”

Section: Introductionmentioning

confidence: 99%

A CozE Homolog Contributes to Cell Size Homeostasis of Streptococcus pneumoniae

Stamsås

Restelli

Ducret

et al. 2020

mBio

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Control of peptidoglycan assembly is critical to maintain bacterial cell size and morphology. Penicillin-binding proteins (PBPs) are crucial enzymes for the polymerization of the glycan strand and/or their cross-linking via peptide branches. Over the last few years, it has become clear that PBP activity and localization can be regulated by specific cognate regulators. The first regulator of PBP activity in Gram-positive bacteria was discovered in the human pathogen Streptococcus pneumoniae. This regulator, named CozE, controls the activity of the bifunctional PBP1a to promote cell elongation and achieve a proper cell morphology. In this work, we studied a previously undescribed CozE homolog in the pneumococcus, which we named CozEb. This protein displays the same membrane organization as CozE but is much more widely conserved among Streptococcaceae genomes. Interestingly, cozEb deletion results in cells that are smaller than their wild-type counterparts, which is the opposite effect of cozE deletion. Furthermore, double deletion of cozE and cozEb results in poor viability and exacerbated cell shape defects. Coimmunoprecipitation further showed that CozEb is part of the same complex as CozE and PBP1a. However, although we confirmed that CozE is required for septal localization of PBP1a, the absence of CozEb has no effect on PBP1a localization. Nevertheless, we found that the overexpression of CozEb can compensate for the absence of CozE in all our assays. Altogether, our results show that the interplay between PBP1a and the cell size regulators CozE and CozEb is required for the maintenance of pneumococcal cell size and shape. IMPORTANCE Penicillin-binding proteins (PBPs), the proteins catalyzing the last steps of peptidoglycan assembly, are critical for bacteria to maintain cell size, shape, and integrity. PBPs are consequently attractive targets for antibiotics. Resistance to antibiotics in Streptococcus pneumoniae (the pneumococcus) are often associated with mutations in the PBPs. In this work, we describe a new protein, CozEb, controlling the cell size of pneumococcus. CozEb is a highly conserved integral membrane protein that works together with other proteins to regulate PBPs and peptidoglycan synthesis. Deciphering the intricate mechanisms by which the pneumococcus controls peptidoglycan assembly might allow the design of innovative anti-infective strategies, for example, by resensitizing resistant strains to PBP-targeting antibiotics.

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Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects

Cited by 105 publications

References 65 publications

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

A CozE Homolog Contributes to Cell Size Homeostasis of Streptococcus pneumoniae

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