A mutant Escherichia coli that attaches peptidoglycan to lipopolysaccharide and displays cell wall on its surface

The lipopolysaccharide (LPS) forms the surface-exposed leaflet of the outer membrane (OM) of Gram-negative bacteria, an organelle that shields the underlying peptidoglycan (PG) cell wall. Both LPS and PG are essential cell envelope components that are synthesized independently and assembled by dedicated transenvelope multiprotein complexes. We have identified a point-mutation in the gene for O-antigen ligase (WaaL) in Escherichia coli that causes LPS to be modified with PG subunits, intersecting these two pathways. Synthesis of the PG-modified LPS (LPS*) requires ready access to the small PG precursor pool but does not weaken cell wall integrity, challenging models of precursor sequestration at PG assembly machinery. LPS* is efficiently transported to the cell surface without impairing OM function. Because LPS* contains the canonical vancomycin binding site, these surface-exposed molecules confer increased vancomycin-resistance by functioning as molecular decoys that titrate the antibiotic away from its intracellular target. This unexpected LPS glycosylation fuses two potent pathogen-associated molecular patterns (PAMPs).

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Degradation of Components of the Lpt Transenvelope Machinery Reveals LPS-Dependent Lpt Complex Stability in Escherichia coli

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.758228 ◽

2021 ◽

Vol 8 ◽

Author(s):

Alessandra M. Martorana ◽

Elisabete C. C. M. Moura ◽

Paola Sperandeo ◽

Flavia Di Vincenzo ◽

Xiaofei Liang ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Envelope ◽

Barrier Properties ◽

Permeability Barrier ◽

Gram Negative Bacteria ◽

Complex Stability ◽

Gram Negative ◽

Essential Proteins ◽

Assembly Result ◽

The Stability

Lipopolysaccharide (LPS) is a peculiar component of the outer membrane (OM) of many Gram-negative bacteria that renders these bacteria highly impermeable to many toxic molecules, including antibiotics. LPS is assembled at the OM by a dedicated intermembrane transport system, the Lpt (LPS transport) machinery, composed of seven essential proteins located in the inner membrane (IM) (LptB2CFG), periplasm (LptA), and OM (LptDE). Defects in LPS transport compromise LPS insertion and assembly at the OM and result in an overall modification of the cell envelope and its permeability barrier properties. LptA is a key component of the Lpt machine. It connects the IM and OM sub-complexes by interacting with the IM protein LptC and the OM protein LptD, thus enabling the LPS transport across the periplasm. Defects in Lpt system assembly result in LptA degradation whose stability can be considered a marker of an improperly assembled Lpt system. Indeed, LptA recruitment by its IM and OM docking sites requires correct maturation of the LptB2CFG and LptDE sub-complexes, respectively. These quality control checkpoints are crucial to avoid LPS mistargeting. To further dissect the requirements for the complete Lpt transenvelope bridge assembly, we explored the importance of LPS presence by blocking its synthesis using an inhibitor compound. Here, we found that the interruption of LPS synthesis results in the degradation of both LptA and LptD, suggesting that, in the absence of the LPS substrate, the stability of the Lpt complex is compromised. Under these conditions, DegP, a major chaperone–protease in Escherichia coli, is responsible for LptD but not LptA degradation. Importantly, LptD and LptA stability is not affected by stressors disturbing the integrity of LPS or peptidoglycan layers, further supporting the notion that the LPS substrate is fundamental to keeping the Lpt transenvelope complex assembled and that LptA and LptD play a major role in the stability of the Lpt system.

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Core oligosaccharide portion of lipopolysaccharide plays important roles on multiple antibiotic resistance in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00341-21 ◽

2021 ◽

Author(s):

Jianli Wang ◽

Wenjian Ma ◽

Yu Fang ◽

Hao Liang ◽

Huiting Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Cell Envelope ◽

Gene Clusters ◽

Gram Negative Bacteria ◽

Multiple Antibiotic Resistance ◽

Core Oligosaccharide ◽

Gram Negative ◽

The Core ◽

K 12

Gram-negative bacteria are intrinsically resistant to antibiotics due to the presence of the cell envelope, but mechanisms are still not fully understood. In this study, a series of mutants that lack one or more major components associated with the cell envelope were constructed from Escherichia coli K-12 W3110. WJW02 can only synthesize Kdo 2 -lipid A which lacks the core oligosaccharide portion of lipopolysaccharide. WJW04, WJW07 and WJW08 were constructed from WJW02 by deleting the gene clusters relevant to the biosynthesis of exopolysaccharide, flagella and fimbria, respectively. WJW09, WJW010 and WJW011 cells cannot synthesize exopolysaccharide, flagella and fimbria, respectively. Comparing to the wild type W3110, mutants WJW02, WJW04, WJW07 and WJW08 cells showed decreased resistance to more than 10 different antibacterial drugs, but not the mutants WJW09, WJW010 and WJW011. This indicates that the core oligosaccharide portion of lipopolysaccharide plays important roles on multiple antibiotic resistance in E. coli and the 1 st heptose in core oligosaccharide portion is critical. Furthermore, the removal of the core oligosaccharide of LPS leads to influences on cell wall morphology, cell phenotypes, porins, efflux systems, and the respond behaviors to antibiotic stimulation. The results demonstrated the important role of lipopolysaccharide on the antibiotic resistance of Gram-negative bacteria.

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Bacterial Metal Resistance: Coping with Copper without Cooperativity?

mBio ◽

10.1128/mbio.00653-21 ◽

2021 ◽

Author(s):

Nicholas P. Greene ◽

Vassilis Koronakis

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Cell Envelope ◽

Metal Resistance ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Rnd Transporter ◽

Entire Cell

In Escherichia coli and other Gram-negative bacteria, tripartite efflux pumps (TEPs) span the entire cell envelope and serve to remove noxious molecules from the cell. CusBCA is a TEP responsible for copper and silver detoxification in E. coli powered by the resistance-nodulation-cell division (RND) transporter, CusA.

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LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization

Frontiers in Microbiology ◽

10.3389/fmicb.2021.676596 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xudong Tian ◽

Guillaume Manat ◽

Elise Gasiorowski ◽

Rodolphe Auger ◽

Samia Hicham ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Surface ◽

Lipid A ◽

Negative Charge ◽

Polymyxin B ◽

Gram Negative Bacteria ◽

Bacterial Cells ◽

Gram Negative ◽

E Coli ◽

Gut Colonization

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella, EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli.

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Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2015.0029 ◽

2015 ◽

Vol 370 (1679) ◽

pp. 20150029 ◽

Cited By ~ 35

Author(s):

Brent W. Simpson ◽

Janine M. May ◽

David J. Sherman ◽

Daniel Kahne ◽

Natividad Ruiz

Keyword(s):

Cell Surface ◽

Outer Membrane ◽

Cell Envelope ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Final Destination ◽

Outer Leaflet ◽

Hydrophobic Compounds ◽

Animal Hosts

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370 , 20150027. ( doi:10.1098/rstb.2015.0027 )) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.

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THE LOCATION OF THE MUCOPEPTIDE IN SECTIONS OF THE CELL WALL OF ESCHERICHIA COLI AND OTHER GRAM-NEGATIVE BACTERIA

Canadian Journal of Microbiology ◽

10.1139/m65-072 ◽

1965 ◽

Vol 11 (3) ◽

pp. 547-560 ◽

Cited By ~ 180

Author(s):

R. G. E. Murray ◽

Pamela Steed ◽

H. E. Elson

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Outer Cell ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

Innermost Layer ◽

Wall Profile ◽

Double Track ◽

Outer Cell Wall

Electron micrographs of sections of Escherichia coli have shown that the wall has an extra component 20–30 Å in thickness on the inside of the usual double-track profile. Demonstration was aided by treating the sections with uranium, lanthanum, thallium, or lead salts. This innermost layer alone was lost in spheroplasts produced by penicillin poisoning or treatment with lysozyme-EDTA, and was removed from isolated cell walls by lysozyme. The innermost layer is considered, therefore, to contain the mucopeptide characteristic of bacteria. The inner taut layer (or "intermediate layer") of Spirillum serpens, Vitreoscilla sp., and Simonsiella sp. was also found to be lysozyme sensitive. In the latter species this layer was the sole component of the septum, so that the outer cell wall components enclosed the elements of the trichoma. Other components were less easily localized but it was considered that the lipoprotein layer was outside of the limits of the wall profile usually visualized in sections. The outer layers generally loosened during embedding, but in E. coli and some others the layers all stayed tightly adherent to each other. The Gram-negative bacteria seem to have the double-track layer and the mucopeptide as a basic complement for the cell wall.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1009227 ◽

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.

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Restoring Balance to the Outer Membrane: YejM’s Role in LPS Regulation

mBio ◽

10.1128/mbio.02624-20 ◽

2020 ◽

Vol 11 (6) ◽

pp. e02624-20

Author(s):

Brent W. Simpson ◽

Martin V. Douglass ◽

M. Stephen Trent

Keyword(s):

Membrane Protein ◽

Cell Surface ◽

Outer Membrane ◽

Strong Evidence ◽

Cell Envelope ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Multiple Functions ◽

Inner Membrane Protein

ABSTRACTGram-negative bacteria produce an asymmetric outer membrane (OM) that is particularly impermeant to many antibiotics and characterized by lipopolysaccharide (LPS) exclusively at the cell surface. LPS biogenesis remains an ideal target for therapeutic intervention, as disruption could kill bacteria or increase sensitivity to existing antibiotics. While it has been known that LPS synthesis is regulated by proteolytic control of LpxC, the enzyme that catalyzes the first committed step of LPS synthesis, it remains unknown which signals direct this regulation. New details have been revealed during study of a cryptic essential inner membrane protein, YejM. Multiple functions have been proposed over the years for YejM, including a controversial hypothesis that it transports cardiolipin from the inner membrane to the OM. Strong evidence now indicates that YejM senses LPS in the periplasm and directs proteolytic regulation. Here, we discuss the standing literature of YejM and highlight exciting new insights into cell envelope maintenance.

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Membrane Topology of the Escherichia coli AmpG Permease Required for Recycling of Cell Wall Anhydromuropeptides and AmpC β-Lactamase Induction

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.3.1145-1149.2005 ◽

2005 ◽

Vol 49 (3) ◽

pp. 1145-1149 ◽

Cited By ~ 20

Author(s):

Aicha Chahboune ◽

Marc Decaffmeyer ◽

Robert Brasseur ◽

Bernard Joris

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Membrane Topology ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Transmembrane Segments ◽

Induction Mechanism ◽

Proposed Model ◽

Cytoplasmic Loops ◽

Inducible Gene

ABSTRACT Escherichia coli, and presumably most other gram-negative bacteria, possesses an efficient protein machinery for recycling its peptidoglycan during cell growth. The major recycled peptidoglycan product is N-acetylglucosamine-1,6-anhydro-N-acetylmuramic acid-tetrapeptide. Its uptake from the periplasm into the cytoplasm is carried out via the AmpG protein, an intrinsic membrane protein. In gram-negative bacteria carrying an ampC β-lactamase-inducible gene on their chromosomes, the induction mechanism is directly linked to peptidoglycan recycling. After identification of the different putative hydrophobic segments by computing, the AmpG topology was experimentally determined by using β-lactamase fusion. In the proposed model, AmpG contains 10 transmembrane segments and two large cytoplasmic loops.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1010051 ◽

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.

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