scholarly journals Cleavage of Braun’s lipoprotein Lpp from the bacterial peptidoglycan by a paralog of l,d-transpeptidases, LdtF

2021 ◽  
Vol 118 (19) ◽  
pp. e2101989118
Author(s):  
Raj Bahadur ◽  
Pavan Kumar Chodisetti ◽  
Manjula Reddy
Keyword(s):  
Bacterial Cell ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Hydrolytic Enzyme ◽  
Gram Negative ◽  
E Coli ◽  
Bacterial Cell Envelope ◽  
Cross Links ◽  
Bacterial Peptidoglycan ◽  
Elastic Polymer

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.

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

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.

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

2018 ◽  
Vol 115 (42) ◽  
pp. 10786-10791 ◽  
Author(s):  
Katharina Peters ◽  
Manuel Pazos ◽  
Zainab Edoo ◽  
Jean-Emmanuel Hugonnet ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Bacterial Cell ◽  
Binding Proteins ◽  
Inhibitory Concentration ◽  
Copper Chloride ◽  
Cell Envelope ◽  
Permeability Barrier ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Bacterial Cell Envelope ◽  
Cross Links

The peptidoglycan (PG) layer stabilizes the bacterial cell envelope to maintain the integrity and shape of the cell. Penicillin-binding proteins (PBPs) synthesize essential 4–3 cross-links in PG and are inhibited by β-lactam antibiotics. Some clinical isolates and laboratory strains ofEnterococcus faeciumandEscherichia coliachieve high-level β-lactam resistance by utilizing β-lactam–insensitive LD-transpeptidases (LDTs) to produce exclusively 3–3 cross-links in PG, bypassing the PBPs. InE. coli, other LDTs covalently attach the lipoprotein Lpp to PG to stabilize the envelope and maintain the permeability barrier function of the outermembrane. Here we show that subminimal inhibitory concentration of copper chloride sensitizesE. colicells to sodium dodecyl sulfate and impair survival upon LPS transport stress, indicating reduced cell envelope robustness. Cells grown in the presence of copper chloride lacked 3–3 cross-links in PG and displayed reduced covalent attachment of Braun’s lipoprotein and reduced incorporation of a fluorescentd-amino acid, suggesting inhibition of LDTs. Copper dramatically decreased the minimal inhibitory concentration of ampicillin inE. coliandE. faeciumstrains with a resistance mechanism relying on LDTs and inhibited purified LDTs at submillimolar concentrations. Hence, our work reveals how copper affects bacterial cell envelope stability and counteracts LDT-mediated β-lactam resistance.

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

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.

scholarly journals A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. piscicida Cleaves the Peptidoglycan of Potentially Competing Bacteria

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Johnny Lisboa ◽  
Cassilda Pereira ◽  
Aline Rifflet ◽  
Juan Ayala ◽  
Mateus S. Terceti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Marine Fish ◽  
High Mortality ◽  
Bacterial Cell ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Bacterial Cell Wall ◽  
Gram Negative ◽  
Content Type ◽  
Photobacterium Damselae

ABSTRACT Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-containing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA (Photobacterium NlpC-like protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-d-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and Vibrio vulnificus. This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. IMPORTANCE Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.

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

2021 ◽  
Vol 17 (11) ◽  
pp. e1010051
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 ◽  
Complement Proteins ◽  
Bacterial Cell Envelope ◽  
Complement Resistance

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. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and ‘locked’ C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

scholarly journals A secreted NlpC/P60 endopeptidase from Photobacterium damselae subsp. piscicida cleaves the peptidoglycan of potentially competing bacteria

2020 ◽  
Author(s):  
Johnny Lisboa ◽  
Cassilda Pereira ◽  
Aline Rifflet ◽  
Juan Ayala ◽  
Mateus S. Terceti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Marine Fish ◽  
High Mortality ◽  
Bacterial Cell ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Bacterial Cell Wall ◽  
Warm Water ◽  
Gram Negative ◽  
Photobacterium Damselae

ABSTRACTPeptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. Here, it is described the structural and functional characterization of an NlpC/P60-containing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA (PhotobacteriumNlpC-like Protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-D-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp and neither PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and V. vulnificus. This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA, showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG.IMPORTANCEPeptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, originating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or joint its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either for gaining competitive advantages and/or to get nutrients. The specificity of PnpA to the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.

scholarly journals Peptidoglycan Remodeling EnablesEscherichiacoliTo Survive Severe Outer Membrane Assembly Defect

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
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.

The role of bacterial cell envelope structures in acid stress resistance in E. coli

2020 ◽  
Vol 104 (7) ◽  
pp. 2911-2921 ◽  
Author(s):  
Zhendong Li ◽  
Boyu Jiang ◽  
Xinyi Zhang ◽  
Yang Yang ◽  
Philip R. Hardwidge ◽  
...  
Keyword(s):  
Stress Resistance ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Acid Stress ◽  
E Coli ◽  
Bacterial Cell Envelope
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