scholarly journals How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

BioEssays ◽  
2019 ◽  
Vol 41 (10) ◽  
pp. 1900074 ◽  
Author(s):  
Dennis J. Doorduijn ◽  
Suzan H. M. Rooijakkers ◽  
Dani A. C. Heesterbeek
Keyword(s):  
Bacterial Cell ◽  
Cell Envelope ◽  
Membrane Attack Complex ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bacterial Cell Envelope

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 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 Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes

2021 ◽  
Vol 17 (1) ◽  
pp. e1009227
Author(s):  
Dani A. C. Heesterbeek ◽  
Remy M. Muts ◽  
Vincent P. van Hensbergen ◽  
Pieter de Saint Aulaire ◽  
Tom Wennekes ◽  
...  
Keyword(s):  
Cell Wall ◽  
Immune System ◽  
Outer Membrane ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Membrane Attack Complex ◽  
Human Neutrophils ◽  
Gram Negative Bacteria ◽  
Antimicrobial Proteins ◽  
Gram Negative

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.

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 Extracellular Vesicles: An Overlooked Secretion System in Cyanobacteria

Life ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 129 ◽  
Author(s):  
Steeve Lima ◽  
Jorge Matinha-Cardoso ◽  
Paula Tamagnini ◽  
Paulo Oliveira
Keyword(s):  
Extracellular Vesicles ◽  
Bacterial Cell ◽  
Cell Biology ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Formation Processes ◽  
Gram Negative ◽  
Nanoscale Structures ◽  
Membrane Components

In bacteria, the active transport of material from the interior to the exterior of the cell, or secretion, represents a very important mechanism of adaptation to the surrounding environment. The secretion of various types of biomolecules is mediated by a series of multiprotein complexes that cross the bacterial membrane(s), each complex dedicated to the secretion of specific substrates. In addition, biological material may also be released from the bacterial cell in the form of vesicles. Extracellular vesicles (EVs) are bilayered, nanoscale structures, derived from the bacterial cell envelope, which contain membrane components as well as soluble products. In cyanobacteria, the knowledge regarding EVs is lagging far behind compared to what is known about, for example, other Gram-negative bacteria. Here, we present a summary of the most important findings regarding EVs in Gram-negative bacteria, discussing aspects of their composition, formation processes and biological roles, and highlighting a number of technological applications tested. This lays the groundwork to raise awareness that the release of EVs by cyanobacteria likely represents an important, and yet highly disregarded, survival strategy. Furthermore, we hope to motivate future studies that can further elucidate the role of EVs in cyanobacterial cell biology and physiology.

Bacterial cell damage by the complement system of channel catfish, Ictalurus punctatus

1987 ◽  
Vol 45 ◽  
pp. 888-889
Author(s):  
Rosemarie Rosell-Davis ◽  
Jill A. Jenkins ◽  
Lewis B. Coons ◽  
Donald D. Ourth
Keyword(s):  
Ictalurus Punctatus ◽  
Complement System ◽  
Channel Catfish ◽  
Bacterial Cell ◽  
Cell Damage ◽  
Membrane Attack Complex ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
The Complement System

The alternative complement pathway (ACP) provides the non-immune channel catfish with protection against many Gram-negative bacteria. The role of serum complement against Gram-negative bacteria is death of cells by insertion of the membrane attack complex (C5b-9) into the cell membrane. The assembly of the membrane attack complex is generated by the ACP and is activated by bacterial cell wall components. Pseudomonas fluorescens is a pathogen of channel catfish. In this study, bacteria were examined after incubation with catfish serum by scanning and transmission electron microscopy (SEM and TEM) for ultrastructural evidence of cell envelope damage by the complement system.A percent bactericidal assay determined that catfish plasma was 99% bactericidal against a 24 h culture of P. fluorescens (ATCC 13525). Following a 1 h incubation at 30°C of bacterial dilutions with equal volumes of serum, heat-inactivated serum, zymosan-adsorbed serum, or saline, the bacterial cells were filtered onto 0.22 um nuclepore filters, fixed in glutaraldehyde, dehydrated in ethanol, critical point dried, sputter coated with 15 nm gold and imaged using a JEOL SEM.

scholarly journals The Peptidoglycan Sacculus of Myxococcus xanthus Has Unusual Structural Features and Is Degraded during Glycerol-Induced Myxospore Development

2008 ◽  
Vol 191 (2) ◽  
pp. 494-505 ◽  
Author(s):  
Nhat Khai Bui ◽  
Joe Gray ◽  
Heinz Schwarz ◽  
Peter Schumann ◽  
Didier Blanot ◽  
...  
Keyword(s):  
Fruiting Body ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Myxococcus Xanthus ◽  
Structural Features ◽  
Diaminopimelic Acid ◽  
Gram Negative Bacteria ◽  
Structural Elements ◽  
Vegetative Cells ◽  
Gram Negative

ABSTRACT Upon nutrient limitation cells of the swarming soil bacterium Myxococcus xanthus form a multicellular fruiting body in which a fraction of the cells develop into myxospores. Spore development includes the transition from a rod-shaped vegetative cell to a spherical myxospore and so is expected to be accompanied by changes in the bacterial cell envelope. Peptidoglycan is the shape-determining structure in the cell envelope of most bacteria, including myxobacteria. We analyzed the composition of peptidoglycan isolated from M. xanthus. While the basic structural elements of peptidoglycan in myxobacteria were identical to those in other gram-negative bacteria, the peptidoglycan of M. xanthus had unique structural features. meso- or ll-diaminopimelic acid was present in the stem peptides, and a new modification of N-acetylmuramic acid was detected in a fraction of the muropeptides. Peptidoglycan formed a continuous, bag-shaped sacculus in vegetative cells. The sacculus was degraded during the transition from vegetative cells to glycerol-induced myxospores. The spherical, bag-shaped coats isolated from glycerol-induced spores contained no detectable muropeptides, but they contained small amounts of N-acetylmuramic acid and meso-diaminopimelic acid.

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.

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