Copper inhibits peptidoglycan LD-transpeptidases suppressing β-lactam resistance due to bypass of penicillin-binding proteins

The gram‐negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM, protecting cells from turgor and environmental stress conditions. In several bacteria, including Escherichia coli, the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun’s lipoprotein), that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery, Lpp has been studied extensively, and although l,d-transpeptidases, the enzymes that catalyze the formation of PG−Lpp linkages, have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK), a newly identified paralog of l,d-transpeptidases in E. coli, is a murein hydrolytic enzyme that catalyzes cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF was earlier presumed to be an l,d-transpeptidase; however, our results show that it is indeed an l,d-endopeptidase that hydrolyzes the products generated by the l,d-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG−Lpp cross-links, suggesting a role for LdtF in the regulation of PG–OM linkages to maintain the structural integrity of the bacterial cell envelope.

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Cleavage of Braun lipoprotein Lpp from the bacterial peptidoglycan by a paralog of L,D-transpeptidases, LdtF

10.1101/2021.02.24.432682 ◽

2021 ◽

Author(s):

Raj Bahadur ◽

Pavan Kumar Chodisetti ◽

Manjula Reddy

Keyword(s):

Outer Membrane ◽

Bacterial Cell ◽

Drug Targets ◽

Structural Integrity ◽

Cell Envelope ◽

Hydrolytic Enzyme ◽

Permeability Barrier ◽

Gram Negative ◽

E Coli ◽

Bacterial Cell Envelope

AbstractGram-negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM protecting cells from turgor and environmental stress conditions. In several bacteria including E. coli, the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun lipoprotein) that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery Lpp has been studied extensively and although L,D-transpeptidases, the enzymes that catalyse the formation of PG–Lpp linkages have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK), a newly-identified paralog of L,D-transpeptidases in E. coli is a murein hydrolytic enzyme that catalyses cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF is earlier presumed to be an L,D-transpeptidase; however, our results show that it is indeed an L,D-endopeptidase that hydrolyses the products generated by the L,D-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG–Lpp cross-links suggesting a role for LdtF in regulation of PG-OM linkages to maintain the structural integrity of the bacterial cell envelope.Significance statementBacterial cell walls contain a unique protective exoskeleton, peptidoglycan, which is a target of several clinically important antimicrobials. In Gram-negative bacteria, peptidoglycan is covered by an additional lipid layer, outer membrane that serves as permeability barrier against entry of toxic molecules. In some bacteria, an extremely abundant lipoprotein, Lpp staples outer membrane to peptidoglycan to maintain the structural integrity of the cell envelope. In this study, we identify a previously unknown peptidoglycan hydrolytic enzyme that cleaves Lpp from the peptidoglycan sacculus and show how the outer membrane-peptidoglycan linkages are modulated in Escherichia coli. Overall, this study helps in understanding the fundamental bacterial cell wall biology and in identification of alternate drug targets for development of new antimicrobials.

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Analysis of the interactions of a novel cephalosporin derivative with its potential targets penicillin-binding proteins from different sources using a covalent docking approac

10.7287/peerj.preprints.2270v1 ◽

2016 ◽

Author(s):

Anna Verdino ◽

Margherita De Rosa ◽

Annunziata Soriente ◽

Anna Marabotti

Keyword(s):

Binding Proteins ◽

Rational Approach ◽

Beta Lactam ◽

Penicillin Binding Proteins ◽

E Coli ◽

Beta Lactam Antibiotics ◽

Linear Chains ◽

Lactam Ring ◽

Covalent Docking ◽

Cross Links

Motivation. Cephalosporins are a class of beta-lactam antibiotics widely used in clinics for their antibacterial activity. Their mode of action, common to other beta lactam antibiotics such as penicillins, is the impairment of the synthesis of the peptidoglycan forming the bacterial cell wall. This polymer, essential for bacterium survival, is made by aminosugars connected by glycosidic bonds to form linear chains, and by short peptides forming cross-links between the linear chains. The enzymes catalyzing the creation of these cross-links are transpeptidases, also called penicillin binding proteins (PBPs) for their ability to interact with penicillins and other beta lactam antibiotics. These molecules mimic the D-Ala-D-Ala terminus of the peptides, therefore they competitively inactivate the PBPs by binding covalently to the Ser residue responsible for the catalysis and stopping the transpeptidation. This results in cell lysis and bacterial death. One of the main problems to face when using cephalosporins is the development of several mechanisms of resistance, either for the reduced affinity of PBPs to the beta lactams, or for the selection of new beta-lactam-insensitive PBPs, or for the production of beta lactamases, enzymes able to hydrolyze the beta lactam ring, thus deactivating the antibiotics. Additionally, most cephalosporins have a limited spectrum of action, against only Gram+ or Gram- bacteria. Therefore, during the time, many new beta lactam antibiotics have been synthesized with the aim of broadening the spectrum of action and/or overcoming the resistance. The prototype of a new group of cephalosporins is AMA-10, in which another beta lactam ring bound to a short alkyl chain has been linked to the aminocephalosporanic ring by means of an amidic bond. In order to develop other molecules, however, it is essential to understand how they interact with their target. Therefore, to apply a rational approach for the design of new derivatives, we have performed a computational study by simulating the binding of AMA-10 to selected PBPs of different species, whose crystallographic structures were available, using a particular approach, covalent docking, able to take into account the covalent bond formed between the antibiotic and the enzyme. Methods. The structures of PBP3 and PBP4 from both Gram+ (S. aureus, B. subtilis) and Gram- (E. coli, P. aeruginosae) organisms were downloaded from Protein Data Bank (PDB) database, as well as the structures of beta-lactamase from S. aureus and from E. coli. The representative structures were selected on the basis of their quality. Then, covalent docking was made by using a modified version of the program AutoDock 4.2, using the flexible side chain method [Bianco et al, 2016]. [Abstract truncated at 3,000 characters - the full version is available in the pdf file].

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Analysis of the interactions of a novel cephalosporin derivative with its potential targets penicillin-binding proteins from different sources using a covalent docking approac

10.7287/peerj.preprints.2270 ◽

2016 ◽

Author(s):

Anna Verdino ◽

Margherita De Rosa ◽

Annunziata Soriente ◽

Anna Marabotti

Keyword(s):

Binding Proteins ◽

Rational Approach ◽

Beta Lactam ◽

Penicillin Binding Proteins ◽

E Coli ◽

Beta Lactam Antibiotics ◽

Linear Chains ◽

Lactam Ring ◽

Covalent Docking ◽

Cross Links

Motivation. Cephalosporins are a class of beta-lactam antibiotics widely used in clinics for their antibacterial activity. Their mode of action, common to other beta lactam antibiotics such as penicillins, is the impairment of the synthesis of the peptidoglycan forming the bacterial cell wall. This polymer, essential for bacterium survival, is made by aminosugars connected by glycosidic bonds to form linear chains, and by short peptides forming cross-links between the linear chains. The enzymes catalyzing the creation of these cross-links are transpeptidases, also called penicillin binding proteins (PBPs) for their ability to interact with penicillins and other beta lactam antibiotics. These molecules mimic the D-Ala-D-Ala terminus of the peptides, therefore they competitively inactivate the PBPs by binding covalently to the Ser residue responsible for the catalysis and stopping the transpeptidation. This results in cell lysis and bacterial death. One of the main problems to face when using cephalosporins is the development of several mechanisms of resistance, either for the reduced affinity of PBPs to the beta lactams, or for the selection of new beta-lactam-insensitive PBPs, or for the production of beta lactamases, enzymes able to hydrolyze the beta lactam ring, thus deactivating the antibiotics. Additionally, most cephalosporins have a limited spectrum of action, against only Gram+ or Gram- bacteria. Therefore, during the time, many new beta lactam antibiotics have been synthesized with the aim of broadening the spectrum of action and/or overcoming the resistance. The prototype of a new group of cephalosporins is AMA-10, in which another beta lactam ring bound to a short alkyl chain has been linked to the aminocephalosporanic ring by means of an amidic bond. In order to develop other molecules, however, it is essential to understand how they interact with their target. Therefore, to apply a rational approach for the design of new derivatives, we have performed a computational study by simulating the binding of AMA-10 to selected PBPs of different species, whose crystallographic structures were available, using a particular approach, covalent docking, able to take into account the covalent bond formed between the antibiotic and the enzyme. Methods. The structures of PBP3 and PBP4 from both Gram+ (S. aureus, B. subtilis) and Gram- (E. coli, P. aeruginosae) organisms were downloaded from Protein Data Bank (PDB) database, as well as the structures of beta-lactamase from S. aureus and from E. coli. The representative structures were selected on the basis of their quality. Then, covalent docking was made by using a modified version of the program AutoDock 4.2, using the flexible side chain method [Bianco et al, 2016]. [Abstract truncated at 3,000 characters - the full version is available in the pdf file].

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Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores

10.1101/2021.05.12.443779 ◽

2021 ◽

Author(s):

Dennis J. Doorduijn ◽

Dani A.C. Heesterbeek ◽

Maartje Ruyken ◽

Carla J.C. de Haas ◽

Daphne A.C. Stapels ◽

...

Keyword(s):

Escherichia Coli ◽

Bacterial Cell ◽

Cell Envelope ◽

Transmembrane Helix ◽

Membrane Attack Complex ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

Complement Proteins ◽

Bacterial Cell Envelope

Complement proteins can form Membrane Attack Complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria, because a robust way to specifically prevent polymerization of C9 has been lacking. In this paper, polymerization of C9 was prevented without affecting the binding of C9 to C5b-8 by locking the first transmembrane helix domain of C9. We show that polymerization of C9 strongly enhanced bacterial cell envelope damage and killing by MAC pores for several Escherichia coli and Klebsiella strains. Moreover, we show that polymerization of C9 is impaired on complement-resistant E. coli strains that survive killing by MAC pores. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

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Peptidoglycan Remodeling EnablesEscherichiacoliTo Survive Severe Outer Membrane Assembly Defect

mBio ◽

10.1128/mbio.02729-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 30

Author(s):

Niccolò Morè ◽

Alessandra M. Martorana ◽

Jacob Biboy ◽

Christian Otten ◽

Matthias Winkle ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Bacterial Cell ◽

Cell Envelope ◽

Permeability Barrier ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Cross Links ◽

Peptidoglycan Layer

ABSTRACTGram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show thatEscherichia colicells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCEIn Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show thatEscherichia colicells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.

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