In the context of this underlying bacterial community, typical CF pathogens appear in a succession; with Staphylococcus aureus and Haemophilus influenzae dominating early in life, followed by chronic colonization by Pseudomonas aeruginosa and members of the Burkholderia cepacia complex 11, Unbiased molecular detection methods, based on bacterial 16S rRNA gene sequencing or phylogenetic microarrays, have shed new light on the complexity of the CF microbiome and implicate anaerobic bacterial species as dominant members of the CF airway microbiota 11, These findings have been confirmed by parallel anaerobic culture based studies Among the anaerobic species, Prevotella melaninogenica has emerged as a frequent and abundant member of the CF airway microbiota 14, , 23, 29, Additionally, P.
In the context of CF airway disease, the potential role of P. In particular, the structure and stimulatory. The goal of this study was to determine the P. We found that there are two dominant forms of P. Bacterial strains and growth conditions: P. The mixture was cooled on ice and centrifuged at 10, rpm at room temperature for 30 minutes. The aqueous phase was collected and an equal volume of endotoxin-free water was added to the organic phase. The sample was treated as above and aqueous phases were combined and dialyzed against Milli-Q purified water to remove residual phenol and then freeze-dried.
Proteinase K was added and. The solution was extracted with an equal volume of water-saturated phenol. The aqueous phase was collected, dialyzed against Milli-Q purified water and freeze-dried as above.
Rapid microextraction Lipid A isolation from whole cells. The P. After cooling,. Samples were. Data was acquired in reflectron mode with a Smartbeam laser with 1 kHz repetition rate and up to shots were accumulated for each spectrum.
Data was acquired with a Smartbeam laser with 2 kHz repetition rate and up to shots were accumulated for each spectrum. Instrument calibration and tuning parameters were optimized using the recommended Bruker calibration standard mixture Bruker Daltonics, Bremen, Germany in negative ion mode.
Tandem mass spectrometry experiments involved isolation and collision induced fragmentation of the precursor ion in the front end quadruple. Precursor isolation was set to 5 Da and fragmentation energy was adjusted accordingly to maximize observation of product ions. LPS was diluted in endotoxin free H2O. Statistical differences were considered significant for P values of less than 0.
All reported experiments were performed at least twice in triplicate and each graph represents standard deviation of averages. The lipid A structure of P. To determine lipid A structure, the typed P. These ion series corresponded to singly deprotonated lipid A structures that contained one phosphate group monophosphoryl or two phosphates diphosphoryl and five acyl chains penta-acylated , respectively. Within the ion series, each ion peak.
Tandem mass spectrometric experiments on penta-acylated lipid A isolated from P. In order to determine the positioning of the five fatty acids, we conducted tandem mass spectrometric experiments aimed at highlighting diagnostic cross-ring and glycosidic cleaveage product ions that provided decisive evidence for pinpointing acyl chain positions Figure 1B.
The penta-acylated lipid A structure was determined to have the following configuration: the C-2 position contained a primary amide-linked C16 3-OH , the C-3 position contained a primary ester-linked C16 3-OH , the C-2' position contained a primary amide-linked C17 3-OH isomethyl and a secondary ester-linked C16, and the C-3' position contained a primary ester-linked C15 3- OH isomethyl.
The acyl chain configuration as outlined above held true for diphosphoryl penta-acylated lipid A extracted from P.
To test the ability of P. LPS from P. To determine whether the weak cytokine response to P. In our studies, TLR4. As expected the isotype mAb control did not reduce P. We also tested the ability of P.
Pam3CSK4, a. The TLR2 neutralizing antibody reduced P. The isotype mAb control did not reduce P. This study represents the first investigation into lipid A structure and inflammatory response to P. The aqueous phase was collected, dialyzed against Milli-Q purified water and freeze-dried as above. Rapid microextraction Lipid A isolation from whole cells. The P. After cooling,. Samples were. Data was acquired in reflectron mode with a Smartbeam laser with 1 kHz repetition rate and up to shots were accumulated for each spectrum.
Data was acquired with a Smartbeam laser with 2 kHz repetition rate and up to shots were accumulated for each spectrum. Instrument calibration and tuning parameters were optimized using the recommended Bruker calibration standard mixture Bruker Daltonics, Bremen, Germany in negative ion mode. Tandem mass spectrometry experiments involved isolation and collision induced fragmentation of the precursor ion in the front end quadruple.
Precursor isolation was set to 5 Da and fragmentation energy was adjusted accordingly to maximize observation of product ions. LPS was diluted in endotoxin free H2O. Statistical differences were considered significant for P values of less than 0. All reported experiments were performed at least twice in triplicate and each graph represents standard deviation of averages. The lipid A structure of P. To determine lipid A structure, the typed P. These ion series corresponded to singly deprotonated lipid A structures that contained one phosphate group monophosphoryl or two phosphates diphosphoryl and five acyl chains penta-acylated , respectively.
Within the ion series, each ion peak. Tandem mass spectrometric experiments on penta-acylated lipid A isolated from P. In order to determine the positioning of the five fatty acids, we conducted tandem mass spectrometric experiments aimed at highlighting diagnostic cross-ring and glycosidic cleaveage product ions that provided decisive evidence for pinpointing acyl chain positions Figure 1B.
The penta-acylated lipid A structure was determined to have the following configuration: the C-2 position contained a primary amide-linked C16 3-OH , the C-3 position contained a primary ester-linked C16 3-OH , the C-2' position contained a primary amide-linked C17 3-OH isomethyl and a secondary ester-linked C16, and the C-3' position contained a primary ester-linked C15 3- OH isomethyl.
The acyl chain configuration as outlined above held true for diphosphoryl penta-acylated lipid A extracted from P. To test the ability of P. LPS from P. To determine whether the weak cytokine response to P. In our studies, TLR4. As expected the isotype mAb control did not reduce P. We also tested the ability of P. Pam3CSK4, a. The TLR2 neutralizing antibody reduced P. The isotype mAb control did not reduce P. This study represents the first investigation into lipid A structure and inflammatory response to P.
In studying the structure of lipid A there are four main areas of potential variation: the number of phosphates attached to the glucosamine backbone, the number and length of acyl chains and whether the acyl chains are branched Contrasting E. These key differences could explain the striking difference in IL Lipid A molecules that have two phosphates attached to the glucosamine background are known to be more potent activators of TLR4 than lipid A molecules with a single phosphate or no phosphate Additionally, lipid A containing 5 or 7 acyl chains is fold less active compared to structures with 6 acyl chains In our experiments with purified LPS, we established that P.
The low stimulatory effect of P. Structure and innate immune response to In document Structure and innate immune response to lipopolysaccharide lipid A of Prevotella melaninogenica Abstract: Prevotella melaninogenica, regarded as a commensal oral bacterium, is frequently isolated from extra-oral polymicrobial infections, including the airways of individuals with the genetic disease cystic fibrosis CF.
Lipopolysaccharide LPS , the primary constituent of the outer membrane of Gram-negative bacteria, has the potential to be an important proinflammatory mediator.
Introduction: Bacterial lipopolysaccharide LPS comprises the outer layer of the outer membrane of Gram-negative bacteria. In particular, the structure and stimulatory properties of P. Materials and Methods: Bacterial strains and growth conditions: P. The samples were subsequently frozen and lyophilized overnight.
Results: Characterization of P.
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