Structure of the Peptidoglycan Synthase Activator LpoP in Pseudomonas aeruginosa

Caveney, Nathanael; Egan, Alexander J F; Ayala, Isabel; Laguri, Cédric; Robb, C.S.; Breukink, Eefjan; Vollmer, Waldemar; Strynadka, N.C.J.; Simorre, Jean-Pierre

doi:10.1016/j.str.2020.03.012

Cited by 11 publications

(14 citation statements)

References 41 publications

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“…The addition of LpoP Pa (sol) resulted in an increase in the final FRET by ~2.2-fold in the membrane and ~2.1-fold in detergents ( Figure 3G , Figure 3—figure supplement 8A ), and accelerated initial slopes by ~4.2-fold in the membrane and ~11.5-fold in detergents ( Figure 3H , Figure 3—figure supplement 8B ); lipid II consumption was increased under both conditions ( Figure 3G , right, Figure 3—figure supplement 8C ). Overall, these results indicate that LpoP Pa (sol) stimulates both GTase and TPase activities in agreement with a recent report ( Caveney et al, 2020 ).…”

Section: Resultssupporting

confidence: 93%

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

Hernández-Rocamora

Baranova

Peters

et al. 2021

eLife

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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 applied 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 will allow 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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Section: Resultssupporting

confidence: 93%

“…Our assay revealed the effect of Lpo activators on PBP1B analogues from three bacteria. P. aeruginosa uses LpoP to stimulate its PBP1B ( Greene et al, 2018 ; Caveney et al, 2020 ). Here, we identified an LpoP homologue in A. baumannii and showed that it stimulated its cognate PBP1B.…”

Section: Discussionmentioning

confidence: 99%

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

Hernández-Rocamora

Baranova

Peters

et al. 2021

eLife

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“…Our experiments revealed differences in the activities and effect of activators between both PBP1B-homolgoues which we discuss in the following paragraphs. these results indicate that LpoP Pa (sol) stimulates both GTase and TPase activities in agreement with a recent report (Caveney et al, 2020).…”

Section: Activities Of Other Membrane-bound Class a Pbpssupporting

confidence: 94%

“…The following proteins were purified following published protocols: PBP1B Ec (Bertsche et al, 2006), LpoB(sol) (Egan et al, 2014), PBP1B Pa (Caveney et al, 2020), LpoP Pa (sol) (Caveney et al, 2020). All chromatographic steps were performed using an AKTA PrimePlus system (GE Healthcare).…”

Section: Purification and Labelling Of Proteinsmentioning

confidence: 99%

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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“…LpoB conservation is even more limited: LpoB homologs are found only in a subset of gammaproteobacteria (12,112). Interestingly, unique class A PBP cofactors have evolved independently in some Gram-negative and actinobacteria (112,113), some of which also possess intrinsically disordered linkers (114). Mechanisms of cell wall quality control are equally, if not more, mysterious in Gram-positive bacteria.…”

Section: Responses To Acute Cell Wall Damagementioning

confidence: 99%

Bacterial Cell Wall Quality Control during Environmental Stress

Mueller

Levin

2020

mBio

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Single-celled organisms must adapt their physiology to persist and propagate across a wide range of environmental conditions. The growth and division of bacterial cells depend on continuous synthesis of an essential extracellular barrier: the peptidoglycan cell wall, a polysaccharide matrix that counteracts turgor pressure and confers cell shape. Unlike many other essential processes and structures within the bacterial cell, the peptidoglycan cell wall and its synthesis machinery reside at the cell surface and are thus uniquely vulnerable to the physicochemical environment and exogenous threats. In addition to the diversity of stressors endangering cell wall integrity, defects in peptidoglycan metabolism require rapid repair in order to prevent osmotic lysis, which can occur within minutes. Here, we review recent work that illuminates mechanisms that ensure robust peptidoglycan metabolism in response to persistent and acute environmental stress. Advances in our understanding of bacterial cell wall quality control promise to inform the development and use of antimicrobial agents that target the synthesis and remodeling of this essential macromolecule. IMPORTANCE Nearly all bacteria are encased in a peptidoglycan cell wall, an essential polysaccharide structure that protects the cell from osmotic rupture and reinforces cell shape. The integrity of this protective barrier must be maintained across the diversity of environmental conditions wherein bacteria replicate. However, at the cell surface, the cell wall and its synthesis machinery face unique challenges that threaten their integrity. Directly exposed to the extracellular environment, the peptidoglycan synthesis machinery encounters dynamic and extreme physicochemical conditions, which may impair enzymatic activity and critical protein-protein interactions. Biotic and abiotic stressors—including host defenses, cell wall active antibiotics, and predatory bacteria and phage—also jeopardize peptidoglycan integrity by introducing lesions, which must be rapidly repaired to prevent cell lysis. Here, we review recently discovered mechanisms that promote robust peptidoglycan synthesis during environmental and acute stress and highlight the opportunities and challenges for the development of cell wall active therapeutics.

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Structure of the Peptidoglycan Synthase Activator LpoP in Pseudomonas aeruginosa

Abstract: Highlights d Structural organization of LpoP has been resolved d Pa PBP1B is directly stimulated by LpoP but not by Pa CpoB

Cited by 11 publications

References 41 publications

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

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

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

Bacterial Cell Wall Quality Control during Environmental Stress

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