10 research outputs found
Conformationally Constrained Lipid A Mimetics for Exploration of Structural Basis of TLR4/MD‑2 Activation by Lipopolysaccharide
Recognition of the lipopolysaccharide
(LPS), a major component
of the outer membrane of Gram-negative bacteria, by the Toll-like
receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex
is essential for the control of bacterial infection. A pro-inflammatory
signaling cascade is initiated upon binding of membrane-associated
portion of LPS, a glycophospholipid Lipid A, by a coreceptor protein
MD-2, which results in a protective host innate immune response. However,
activation of TLR4 signaling by LPS may lead to the dysregulated immune
response resulting in a variety of inflammatory conditions including
sepsis syndrome. Understanding of structural requirements for Lipid
A endotoxicity would ensure the development of effective anti-inflammatory
medications. Herein, we report on design, synthesis, and biological
activities of a series of conformationally confined Lipid A mimetics
based on β,α-trehalose-type scaffold. Replacement of the
flexible three-bond β(1→6) linkage in diglucosamine backbone
of Lipid A by a two-bond β,α(1↔1) glycosidic linkage
afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated
Lipid A mimetics based on a β–GlcN(1↔1)α–GlcN
scaffold selectively block the LPS binding site on both human and
murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory
signaling, thereby serving as antisepsis drug candidates. In contrast
to their natural counterpart lipid IVa, conformationally constrained
Lipid A mimetics do not activate mouse TLR4. The structural basis
for high antagonistic activity of novel Lipid A mimetics was confirmed
by molecular dynamics simulation. Our findings suggest that besides
the chemical structure, also the three-dimensional arrangement of
the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic
effects on TLR4
B. longum 35624 EPS characterization.
<p>(A) The 600 MHz <sup>1</sup>H NMR proton spectrum of the acid-treated <b>35624</b> EPS (D<sub>2</sub>O, 338 K) is illustrated. A part of the high-field region is displayed in the insert. <b>(B)</b> Expansion plot of the 150 MHz 13C NMR spectrum of the acid-treated <b>35624</b> exopolysaccharide. The anomeric signals on the left confirmed the presence of a hexasaccharide repeat unit.</p
<sup>1</sup>H and <sup>13</sup>C NMR chemical shifts (δ, ppm) of the exopolysaccharide (recorded at 338 K) and the tetrasaccharide os211 (recorded at 300 K) from <i>B</i>. <i>longum</i> 35624.
<p><sup>1</sup>H and <sup>13</sup>C NMR chemical shifts (δ, ppm) of the exopolysaccharide (recorded at 338 K) and the tetrasaccharide os211 (recorded at 300 K) from <i>B</i>. <i>longum</i> 35624.</p
<i>Bifidobacterium longum</i> general genome features.
<p><i>Bifidobacterium longum</i> general genome features.</p
B. longum 35624 EPS proton and carbon signals.
<p>(<b>A</b>) A selected region of the multiplicity-edited, gradient enhanced <sup>1</sup>H, <sup>13</sup>C-HSQC NMR spectrum of the exopolysaccharide. Letters denote the residues as given in the structural formula and arabic numerals denote the respective pyranose position. Resonances from anomeric carbons/protons, glycosylation sites and resolved signals are annotated. (<b>B</b>) Selected region of the <sup>1</sup>H, <sup>13</sup>C-HSQC-TOCSY NMR spectrum (600 MHz) of the acid-treated <b>35624</b> EPS. Arabic numerals before and after oblique stroke denote carbons and protons, respectively.</p
Phylogenetic tree based on the B. longum core-genome.
<p>(A) The <i>B</i>. <i>longum</i> subsp. <i>longum</i> phylogenetic group. (B) The <i>B</i>. <i>longum</i> subsp. <i>infantis</i> phylogenetic group. <i>B</i>. <i>longum</i> subsp. <i>longum</i> and <i>B</i>. <i>longum</i> subsp. <i>infantis</i> type strains are indicated in blue text. <i>Lactobacillus salivarius</i> was included as an outlier.</p
B. longum 35624 electron microscopy.
<p>(A) A layer of extracellular polysaccharide is clearly visible by electronic microscopy of the <b>35624</b> strain. (B) The isolated and purified EPS is illustrated.</p
Mild acid hydrolysis of EPS.
<p>(A) Separation of EPS fragments by PGC HPLC with MS/MS detection. The extracted ion chromatogram for mass 1008.39 Da shows four peaks. Their reducing end sugar was clearly revealed by ESI-MS/MS. Their assignment as either Gal or Glc and the interpretation in terms of fragment structures was done <i>a posteriori</i> based on MALDI-TOF data and on knowledge of the EPS structure. (B) Example of a MALDI-TOF/TOF fragment spectrum showing b-ions from the non-reducing and y- and y´ (= <sup>1,5</sup>x) -ions from the reducing end.</p
EPS gene cluster.
<p>Illustration of the EPS cluster located in the <i>B</i>. <i>longum</i> <b>35624</b> genome and comparison to similar clusters located in <i>B</i>. <i>longum</i> 105-A, <i>B</i>. <i>longum</i> subsp. <i>longum</i> JCM1217 and <i>B</i>. <i>longum</i> subsp. <i>longum</i> NCC2705. Each gene is colour-coded according to function which is indicated in the legend located at the end of the page. Percentages represent the percent of sequence similarity at the protein level with corresponding genes in the <i>B</i>. <i>longum</i> <b>35624</b> genome. The locus tags of the first and last genes located in the EPS clusters of <i>B</i>. <i>longum</i> 105-A, <i>B</i>. <i>longum</i> subsp. <i>longum</i> JCM1217 and <i>B</i>. <i>longum</i> subsp. <i>longum</i> NCC2705 are also indicated in the illustration.</p
B. longum 35624 EPS composition and structure.
<p>The structure is annotated as the chemical formula and in condensed form. Capital letters denote the residues as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162983#pone.0162983.g006" target="_blank">6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162983#pone.0162983.g007" target="_blank">7</a>.</p