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Allosteric activation of cell wall synthesis during bacterial growth.

ABSTRACT: The peptidoglycan (PG) cell wall protects bacteria against osmotic lysis and determines cell shape, making this structure a key antibiotic target. Peptidoglycan is a polymer of glycan chains connected by peptide crosslinks, and its synthesis requires precise spatiotemporal coordination between glycan polymerization and crosslinking. However, the molecular mechanism by which these reactions are initiated and coupled is unclear. Here we use single-molecule FRET and cryo-EM to show that an essential PG synthase (RodA-PBP2) responsible for bacterial elongation undergoes dynamic exchange between closed and open states. Structural opening couples the activation of polymerization and crosslinking and is essential in vivo. Given the high conservation of this family of synthases, the opening motion that we uncovered likely represents a conserved regulatory mechanism that controls the activation of PG synthesis during other cellular processes, including cell division.

SUBMITTER: Shlosman I

PROVIDER: S-EPMC10257715 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Allosteric activation of cell wall synthesis during bacterial growth.

Shlosman Irina I Fivenson Elayne M EM Gilman Morgan S A MSA Sisley Tyler A TA Walker Suzanne S Bernhardt Thomas G TG Kruse Andrew C AC Loparo Joseph J JJ

Nature communications 20230610 1

The peptidoglycan (PG) cell wall protects bacteria against osmotic lysis and determines cell shape, making this structure a key antibiotic target. Peptidoglycan is a polymer of glycan chains connected by peptide crosslinks, and its synthesis requires precise spatiotemporal coordination between glycan polymerization and crosslinking. However, the molecular mechanism by which these reactions are initiated and coupled is unclear. Here we use single-molecule FRET and cryo-EM to show that an essentia ...[more]

PMID: 37301887

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Project description: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.

Project description:BackgroundIn osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages.MethodsMonocyte-derived M1-like and M2-like macrophages were polarized with LPS?+?IFN-?, L-MTP-PE +/- IFN-? or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab.ResultsM1-like macrophages inhibited osteosarcoma cell growth when activated with LPS?+?IFN-?. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-?. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-? and IL-1?. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS?+?IFN-?-activated M2-like macrophages had low anti-tumor activity, IL-10-polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis.ConclusionThis study demonstrates that human macrophages can be induced to exert direct anti-tumor activity against osteosarcoma cells. Our observation that the induction of macrophage anti-tumor activity by L-MTP-PE required IFN-? may be of relevance for the optimization of L-MTP-PE therapy in osteosarcoma patients.

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Allosteric activation of cell wall synthesis during bacterial growth.

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Allosteric activation of cell wall synthesis during bacterial growth.

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