A novel peptidoglycan deacetylase modulates daughter cell separation in E. coli.

Peptidoglycan hydrolases facilitate bacterial cell wall growth by creating space for insertion of new material and allowing physical separation of daughter cells. In Escherichia coli, three peptidoglycan amidases, AmiA, AmiB and AmiC, cleave septal peptidoglycan during cell division. The LytM-domain proteins EnvC, NlpD and ActS activate these amidases either from inside the cell or the outer membrane: EnvC binds to the cytoplasmic membrane-anchored divisome components FtsEX, while NlpD and ActS are outer membrane-anchored lipoproteins.

A novel peptidoglycan deacetylase modulates daughter cell separation in .

Peptidoglycan hydrolases facilitate bacterial cell wall growth by creating space for insertion of new material and allowing physical separation of daughter cells. In , three peptidoglycan amidases, AmiA, AmiB and AmiC, cleave septal peptidoglycan during cell division. The LytM-domain proteins EnvC and NlpD activate these amidases either from inside the cell or the outer membrane: EnvC binds to the cytoplasmic membrane-anchored divisome components FtsEX, and NlpD and ActS are outer membrane lipoproteins.

Targeting the LPS export pathway for the development of novel therapeutics.

The rapid rise of multi-resistant bacteria is a global health threat. This is especially serious for Gram-negative bacteria in which the impermeable outer membrane (OM) acts as a shield against antibiotics. The development of new drugs with novel modes of actions to combat multi-drug resistant pathogens requires the selection of suitable processes to be targeted.

Affinity Purification and Coimmunoprecipitation of Transenvelope Protein Complexes in Gram-Negative Bacteria.

Multiprotein complexes are important machineries that organize a large number of different proteins into functional units. Studying protein-protein interactions in the complexes, rather than individual proteins, is a fundamental step to gaining functional insights into a biological process. Here, we present the sequential affinity purification and coimmunoprecipitation system that was applied to enable the efficient purification of all the proteins that compose the Lpt system complex in Escherichia coli and their identification by western blotting and mass spectrometry (MS).

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

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) (LptBCFG), 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.

DpaA Detaches Braun's Lipoprotein from Peptidoglycan.

Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. such as covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun's lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB, and LdtC.

ActS activates peptidoglycan amidases during outer membrane stress in Escherichia coli.

The integrity of the cell envelope of E. coli relies on the concerted activity of multi-protein machineries that synthesize the peptidoglycan (PG) and the outer membrane (OM). Our previous work found that the depletion of lipopolysaccharide (LPS) export to the OM induces an essential PG remodeling process involving LD-transpeptidases (LDTs), the glycosyltransferase function of PBP1B and the carboxypeptidase PBP6a.

The Landscape of Membrane-Associated Proteins.

Background: cell envelope-associated proteins play a relevant role in infection mechanisms. They can contribute to the antibiotic resistance of the bacterial cells and be involved in the interaction with host cells. Thus, studies contributing to elucidating these key molecular elements are of great importance to find alternative therapeutics.

The Lpt ABC transporter for lipopolysaccharide export to the cell surface.

The surface of the outer membrane of Gram-negative bacteria is covered by a tightly packed layer of lipopolysaccharide molecules which provide a barrier against many toxic compounds and antibiotics. Lipopolysaccharide, synthesized in the cytoplasm, is assembled in the periplasmic leaflet of the inner membrane where the intermembrane Lpt system mediates its transport to the cell surface. The first step of lipopolysaccharide transport is its extraction from the outer leaflet of inner membrane powered by the atypical LptBFGC ABC transporter.

Lipopolysaccharide Biosynthesis and Transport to the Outer Membrane of Gram-Negative Bacteria.

Gram-negative bacteria have an outer membrane that is positioned at the frontline of the cell's interaction with the environment and that serves as a barrier against noxious molecules including many antibiotics. This protective function mainly relies on lipopolysaccharide, a complex glycolipid located in the outer leaflet of the outer membrane. In this chapter we will first summarize lipopolysaccharide structure, functions and biosynthetic pathway and then we will discuss how it is transported and assembled to the cell surface.

Peptidoglycan Remodeling Enables Escherichia coli To Survive Severe Outer Membrane Assembly Defect.

Gram-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.

Pseudomonas aeruginosa LptE is crucial for LptD assembly, cell envelope integrity, antibiotic resistance and virulence.

Lipopolysaccharide (LPS) is an essential structural component of the outer membrane (OM) of most Gram-negative bacteria. In the model organism Escherichia coli, LPS transport to the OM requires seven essential proteins (LptABCDEFG) that form a continuous bridge across the cell envelope. In Pseudomonas aeruginosa the recently-demonstrated essentiality of LptD and LptH, the P.

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

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 of and achieve 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.

Solid State NMR Studies of Intact Lipopolysaccharide Endotoxin.

Lipopolysaccharides (LPS) are complex glycolipids forming the outside layer of Gram-negative bacteria. Their hydrophobic and heterogeneous nature greatly hampers their structural study in an environment similar to the bacterial surface. We have studied LPS purified from E.

Skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems.

Nuclear factor of activated T cells (NFAT) is activated in innate immune cells downstream of pattern recognition receptors, but little is known about NFAT's functions in innate immunity compared with adaptive immunity. We show that early activation of NFAT balances the two major phases of the innate response to skin infections: the protective containment (abscess) and the elimination (expulsion) phases. During the early containment phase, transforming growth factor- (TGF-) induces the deposit of collagen around newly recruited polymorphonuclear cells to prevent microbial spreading.

The lipopolysaccharide transport (Lpt) machinery: A nonconventional transporter for lipopolysaccharide assembly at the outer membrane of Gram-negative bacteria.

The outer membrane (OM) of Gram-negative is a unique lipid bilayer containing LPS in its outer leaflet. Because of the presence of amphipathic LPS molecules, the OM behaves as an effective permeability barrier that makes Gram-negative bacteria inherently resistant to many antibiotics. This review focuses on LPS biogenesis and discusses recent advances that have contributed to our understanding of how this complex molecule is transported across the cellular envelope and is assembled at the OM outer leaflet.

Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer containing a unique glycolipid, lipopolysaccharide (LPS) in its outer leaflet. LPS molecules confer to the OM peculiar permeability barrier properties enabling Gram-negative bacteria to exclude many toxic compounds, including clinically useful antibiotics, and to survive harsh environments. Transport of LPS poses several problems to the cells due to the amphipatic nature of this molecule.

Differential Proteomics Based on Multidimensional Protein Identification Technology to Understand the Biogenesis of Outer Membrane of Escherichia coli.

Cell envelope proteins in bacteria are typically difficult to characterize due to their low abundance, poor solubility, and the problematic isolation of pure surface fraction with only minimal contamination. Here we describe a method for cell membrane fractionation followed by mass spectrometry-based proteomics to analyze and determine protein abundance in bacterial membranes.

Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli.

Unlabelled: The assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made in Escherichia coli of seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood.

An induced folding process characterizes the partial-loss of function mutant LptAI36D in its interactions with ligands.

Lipopolysaccharide (LPS) is an essential glycolipid of the outer membrane (OM) of Gram-negative bacteria with a tripartite structure: lipid A, oligosaccharide core and O antigen. Seven essential LPS-transport proteins (LptABCDEFG) move LPS to the cell surface. Lpt proteins are linked by structural homology, featuring a β-jellyroll domain that mediates protein-protein interactions and LPS binding.

Crystal structure of LptH, the periplasmic component of the lipopolysaccharide transport machinery from Pseudomonas aeruginosa.

Unlabelled: Lipopolysaccharide (LPS) is the main glycolipid present in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria, where it modulates OM permeability, therefore preventing many toxic compounds from entering the cell. LPS biogenesis is an essential process in Gram-negative bacteria and thus is an ideal target pathway for the development of novel specific antimicrobials. The lipopolysaccharide transport (Lpt) system is responsible for transporting LPS from the periplasmic surface of the inner membrane, where it is assembled, to the cell surface where it is then inserted in the OM.

Dissecting Escherichia coli outer membrane biogenesis using differential proteomics.

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device.

The Escherichia coli Lpt transenvelope protein complex for lipopolysaccharide export is assembled via conserved structurally homologous domains.

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain.