An Accurate In Vitro Model of the <i>E. coli</i> Envelope

Clifton, Luke A.; Holt, Stephen A.; Hughes, A.; Daulton, Emma L.; Arunmanee, Wanatchaporn; Heinrich, Frank; Khalid, Syma; Jefferies, Damien; Charlton, Timothy; Webster, John R. P.; Kinane, Christian J.; Lakey, Jeremy H.

doi:10.1002/ange.201504287

Cited by 18 publications

(26 citation statements)

References 27 publications

(21 reference statements)

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“…Magnesium has also been shown to stabilize LPS-LPS interactions and thus divalent cations are critical for both LPS-LPS (28) and LPS-porin interactions, explaining why EDTA can destabilize the OM sufficiently for small proteins such as lysozyme to penetrate into the periplasm. The importance of divalent metal ions for stabilization of the LPS monolayer is a logical consequence of the presence of many negatively charged phosphate groups (28), which without neutralization would be repulsive and render the OM highly unstable.…”

Section: Discussionmentioning

confidence: 99%

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Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

Arunmanee

Pathania

Solovyova

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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112

101

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The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin-LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin-LPS interactions and a bridging calcium ion.gram-negative bacteria | lipopolysaccharide | X-ray crystal structure | porin | outer membrane

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Section: Discussionmentioning

confidence: 99%

“…Because divalent cations are known, via interactions with phosphate groups, to stabilize LPS in the OM (28) and in protein complexes (8), we tested whether the complexes here were similarly stabilized. The addition of 5 mM CaCl 2 or MgCl 2 increased the resolution of bands displayed on SDS/PAGE by the complexes formed with a 5:1 ratio of Ra-LPS to OmpF.…”

mentioning

confidence: 99%

Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

Arunmanee

Pathania

Solovyova

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

112

101

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“…24 We used a symmetric bilayer instead of an asymmetric model of the outer membrane purely for reasons of computational pragmatism. We did not want the C 60 to cross the periodic boundaries and insert via the more penetrable inner leaflet given that our main focus is the interaction with the LPS-containing leaflet.…”

Section: Cg Simulation Systems and Protocolsmentioning

confidence: 99%

Molecular Dynamics Simulations Predict the Pathways via Which Pristine Fullerenes Penetrate Bacterial Membranes

2016

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Carbon fullerenes are emerging as effective devices for different biomedical applications, including the transportation of nanosized drugs and the extraction of harmful oxidants and radicals. It has been proposed that fullerenes could be used as novel antibacterial agents, given the realization that the nanoparticles can kill pathogenic Gram-negative bacteria. To explore this at the molecular level, we simulated C 60 fullerenes with bacterial membranes using the coarse-grain (CG) molecular dynamics (MD) Martini force field. We find that pristine C 60 has a limited tendency to penetrate (incomplete core) Re mutant lipopolysaccharide (LPS) leaflets, but the translocation of C 60 fullerenes into (complete core) Ra mutant LPS leaflets is not thermodynamically favored. Moreover, we show that the permeability of Re LPS bilayers depends sensitively on system temperature, the charge of ambient ions, and the prevalence of POPE defect domains. The different permeabilities are rationalized in terms of transitory head group pore formation, which underpins the translocation of C 60 into the lipid core. The Re LPS lipids readily form transient micropores when they are linked with monovalent cations, or when they are heated to a high temperature. POPE lipids are shown to be particularly adept at forming these transient surface cavities, and their inclusion into Re LPS membranes facilitates the formation of particularly large pores that are tunneled by C 60 aggregates of significant size (~ 5 nm wide). After inserting into the lipid core, the aggregates dissociate, and the disbanded nanoparticles migrate to the interface between separate POPE and LPS domains, where they weaken the boundaries between the coexisting lipid fractions and thereby promoting lipid mixing.

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“…(For more recent progress, see Refs. [12][13][14][15][16].) Indeed, increased OM permeability is correlated with enhanced susceptibility of Gram-negative bacteria to AMPs and antibiotics [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

“…A better understanding of how AMPs reduce OM permeability will benefit our effort for combating these bacteria. Indeed, several attempts have recently been made to advance our understanding of OM permeability [12][13][14][15][16]. For instance, an in vitro model of the Escherichia coli cell surface has been proposed and used in further unravelling the effects of divalent cations on OM permeability [12][13][14].…”

Section: Introductionmentioning

confidence: 99%

Opposing effects of cationic antimicrobial peptides and divalent cations on bacterial lipopolysaccharides

et al. 2017

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The permeability of the bacterial outer membrane, enclosing Gram-negative bacteria, depends on the interactions of the outer, lipopolysaccharide (LPS) layer, with surrounding ions and molecules. We present a coarse-grained model for describing how cationic amphiphilic molecules (e.g., antimicrobial peptides) interact with and perturb the LPS layer in a biologically relevant medium, containing monovalent and divalent salt ions (e.g., Mg 2+ ). In our approach, peptide binding is driven by electrostatic and hydrophobic interactions and is assumed to expand the LPS layer, eventually priming it for disruption. Our results suggest that in parameter ranges of biological relevance (e.g., at micromolar concentrations) the antimicrobial peptide magainin 2 effectively disrupts the LPS layer, even though it has to compete with Mg 2+ for the layer. They also show how the integrity of LPS is restored with an increasing concentration of Mg 2+ . Using the approach, we make a number of predictions relevant for optimizing peptide parameters against Gram-negative bacteria and for understanding bacterial strategies to develop resistance against cationic peptides.

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An Accurate In Vitro Model of the E. coli Envelope

Cited by 18 publications

References 27 publications

Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

Molecular Dynamics Simulations Predict the Pathways via Which Pristine Fullerenes Penetrate Bacterial Membranes

Opposing effects of cationic antimicrobial peptides and divalent cations on bacterial lipopolysaccharides

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