Interactions of Lipopolysaccharide and Polymyxin Studied by NMR Spectroscopy

Mareš, Jiřı́ J.; Kumaran, Sowmini; Gobbo, Marina; Zerbe, Oliver

doi:10.1074/jbc.m806587200

Cited by 97 publications

(122 citation statements)

References 46 publications

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“…Using this strategy, we obtained de novo ssNMR assignments of LPS and PG glucosamine units and some of their substituents (Table S1). Solid-state NMR chemical shifts were compatible with the presence of polysubstituted glucosamine units within the lipid A of LPS and backbone PG moieties, as previously deduced from the analysis of purified molecules (21)(22)(23), even when part of cell envelope preparations. However, carbohydrates from the core of the LPS could not be identified unambiguously due to the large overlap of 13 C and 1 H resonances and the inherent structural heterogeneity of the LPS core region (Fig.…”

Section: Resultssupporting

confidence: 79%

Cellular solid-state nuclear magnetic resonance spectroscopy

Renault¹,

Boxtel²,

Bos³

et al. 2012

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

178

220

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Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a wellestablished imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution. cellular envelope | Escherichia coli | lipoprotein | PagL | magic angle spinning P hysiological processes rely on the concerted action of molecular entities in and across different cellular compartments. Whereas advancements in molecular imaging have provided unprecedented insights into the macromolecular organization in the subnanometer range (1), studying atomic structure and motion in situ has been challenging for structural biology. NMR has provided insight into cellular processes (2-4) and can determine entire 3D molecular structures inside living cells (5) provided that molecular entities tumble rapidly in a cellular setting. In principle, solid-state NMR (ssNMR) spectroscopy offers a complementary spectroscopic tool to monitor molecular structure and dynamics at atomic resolution in a complex setting (see ref. 6 for a recent review). Indeed, ssNMR has already been used to study individual molecular components in the context of natural bilayers (7,8), bacterial cell walls (9), and cellular organelles (10).Here, we introduce a general approach to investigate structure and dynamics of an arbitrary molecular target and its potential molecular partners in a cellular setting. Our studies focuses on the Gram-negative bacterial cell that is characterized by a molecularly complex but architecturally unique envelope, consisting of two lipid bilayers, the inner and outer membrane (IM, OM), separated by the periplasm containing the peptidoglycan (PG) layer (Fig. 1A). The IM is a phospholipid bilayer and harbors α-helical proteins, whereas the OM is asymmetrical and consists of phospholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrelfold integral membrane proteins. LPS forms the outermost layer of the OM and protects the cell against harmful compounds from the environment. PG is a large macromolecule that gives the cell its shape and rigidity.Using uniformly 13 C, 15 N-labeled cellular preparations of Escherichia coli, we characterized the structure and dynamics of a recombinant integral membrane protein (PagL) and other major endogenous molecular components of the cell envelope in...

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Section: Resultssupporting

confidence: 79%

Cellular solid-state nuclear magnetic resonance spectroscopy

Renault¹,

Boxtel²,

Bos³

et al. 2012

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

178

220

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“…Colistin is a polypeptide antibiotic belonging to the polymyxin family, and it is now increasingly being used to treat Gram-negative bacterial infections as last-line salvage therapy. In addition to its bactericidal effect, colistin has also been shown to bind to LPS and prevent the pathophysiologic effects of the endotoxin in the circulation (23). On the basis of this knowledge, the hypotheses of this study are (i) that LPSs from S. enterica and P. aeruginosa exhibit species-dependent effects on BBB dynamics and (ii) that coadministration of colistin prevents LPSinduced BBB disruption through sequestration of free LPS in the systemic circulation.…”

mentioning

confidence: 99%

Species-Dependent Blood-Brain Barrier Disruption of Lipopolysaccharide: Amelioration by ColistinIn VitroandIn Vivo

Jin

Nation

et al. 2013

Antimicrob Agents Chemother

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The aim of this study was to use in vitro and in vivo models to assess the impact of lipopolysaccharide (LPS) from two different bacterial species on blood-brain barrier (BBB) integrity and brain uptake of colistin. Following repeated administration of LPS from Pseudomonas aeruginosa, the brain-to-plasma ratio of [ 14 C]sucrose in Swiss outbred mice was not significantly increased. Furthermore, while the brain uptake of colistin in mice increased 3-fold following administration of LPS from Salmonella enterica, LPS from P. aeruginosa had no significant effect on colistin brain uptake. This apparent species-dependent effect did not appear to correlate with differences in plasma cytokine levels, as the concentrations of tumor necrosis factor alpha and interleukin-6 following administration of each LPS were not different (P > 0.05). To clarify whether this species-specific effect of LPS was due to direct effects on the BBB, human brain capillary endothelial (hCMEC/D3) cells were treated with LPS from P. aeruginosa or S. enterica and claudin-5 expression was measured by Western blotting. S. enterica LPS significantly (P < 0.05) reduced claudin-5 expression at a concentration of 7.5 g/ml. In contrast, P. aeruginosa LPS decreased (P < 0.05) claudin-5 expression only at the highest concentration tested (i.e., 30 g/ml). Coadministration of therapeutic concentrations of colistin ameliorated the S. enterica LPS-induced reduction in claudin-5 expression in hCMEC/D3 cells and the perturbation in BBB function in mice. This study demonstrates that BBB disruption induced by LPS is species dependent, at least between P. aeruginosa and S. enterica, and can be ameliorated by colistin.T he blood-brain barrier (BBB) is formed by specialized endothelial cells of cerebral microvessels. Unlike endothelial cells in other organs, the layer of endothelial cells lining the cerebral microvessels constitutes a physical barrier between blood and brain tissue by a complex network of tight junctions (TJs) (1). The TJs consist of transmembrane proteins spanning the intercellular cleft; these proteins include occludin, claudins (particularly claudin-5) (2), and several cytoplasmic proteins, such as zonula occludens (3). Under normal conditions, these TJ proteins seal the paracellular route of the BBB, thus preventing the movement of relatively small hydrophilic molecules from the blood into the brain, ensuring homeostatic regulation of the central nervous system (CNS) (4).Alterations to TJ proteins and increases in BBB permeability are observed during various disease states, including peripheral hyperalgesia, bacterial meningitis, systemic inflammation, and sepsis (5-8). Indeed, we have demonstrated that systemic administration to mice of lipopolysaccharide (LPS), a key component of the outer membrane of Gram-negative bacteria, from Salmonella enterica (as a model of bacterium-induced inflammation) leads to BBB dysfunction (9). One proposed mechanism for the decreased paracellular integrity of the BBB in response to LPS involves elevated plasma c...

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“…Sepsis has been a serious source of mortality in many clinical cases, but no effective medical therapy has been established. To overcome sepsis, antimicrobial peptides (AMPs) that interact with LPS have recently received increasing attention in the field of drug discovery [4,5].…”

Section: Introductionmentioning

confidence: 99%

Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide

Kushibiki

Kamiya

Aizawa

et al. 2014

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program.CD and NMR measurements revealed that binding to LPS slightly extends the two β-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS.

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Interactions of Lipopolysaccharide and Polymyxin Studied by NMR Spectroscopy

Cited by 97 publications

References 46 publications

Cellular solid-state nuclear magnetic resonance spectroscopy

Cellular solid-state nuclear magnetic resonance spectroscopy

Species-Dependent Blood-Brain Barrier Disruption of Lipopolysaccharide: Amelioration by ColistinIn VitroandIn Vivo

Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide

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