Matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry of lipopolysaccharide species separated by slab‐polyacrylamide gel electrophoresis: High‐resolution separation and molecular weight determination of lipooligosaccharides from <b><i>Vibrio fischeri</i></b> strain HMK

Pupo, Elder; Phillips, Nancy J.; Gibson, Bradford W.; Apicella, Michael A.; Hardy, Eugenio

doi:10.1002/elps.200405980

Cited by 17 publications

(20 citation statements)

References 45 publications

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“…In addition to the lipid A studies, our group also examined the whole LPS structure by silver-stained SDS-PAGE. These data demonstrated that unlike traditional LPS, which generates a ladder-like banding pattern on the gel due to the O-antigen repeat units, the V. fischeri LPS migrated as two low molecular weight bands, likely corresponding to the core and the core plus one O-antigen repeat unit as observed previously (7).…”

supporting

confidence: 81%

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O-antigen and Core Carbohydrate of Vibrio fischeri Lipopolysaccharide

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Krasity

et al. 2012

Journal of Biological Chemistry

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Background:The structure and function of the Vibrio fischeri O-antigen were unknown. Results: The O-antigen structure was determined using an O-antigen ligase mutant. Conclusion: This mutant had a motility defect, and comparison of its LPS with wild-type LPS showed that the O-antigen contains some unusual sugars. Significance: The O-antigen mutation results in a significantly slower rate of bacterial colonization of the squid light organ.

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supporting

confidence: 81%

“…The absence of the O-antigen ladder from the LPS, when examined by SDS-PAGE, has been previously observed for V. fischeri strain HMK as well as other Vibrio spp. (7,46,57,(61)(62)(63). In addition, both NMR and MS analyses (data not shown) suggested that the LPS from V. fischeri strain ES114 lacks repeating units of the O-antigen.…”

Section: Discussionmentioning

confidence: 94%

O-antigen and Core Carbohydrate of Vibrio fischeri Lipopolysaccharide

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Krasity

et al. 2012

Journal of Biological Chemistry

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“…1 LPS of V. fischeri showed only two bands rather than the ladder-like banding pattern typical of LPS with large O-antigen polysaccharides composed of multiple repeat units (7). Mass spectrometric analysis suggested that the higher molecular weight LPS species bore a single O-antigen repeat unit, whereas the lower molecular weight forms contained only core sugars (7). The details of these carbohydrate structures are currently being investigated.…”

mentioning

confidence: 98%

The Lipid A from Vibrio fischeri Lipopolysaccharide

Phillips

Adin

Stabb

et al. 2011

Journal of Biological Chemistry

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Vibrio fischeri, a bioluminescent marine bacterium, exists in an exclusive symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, whose light organ it colonizes. Previously, it has been shown that the lipopolysaccharide (LPS) or free lipid A of V. fischeri can trigger morphological changes in the juvenile squid's light organ that occur upon colonization. To investigate the structural features that might be responsible for this phenomenon, the lipid A from V. fischeri ES114 LPS was isolated and characterized by multistage mass spectrometry (MS n ). A microheterogeneous mixture of mono-and diphosphorylated diglucosamine disaccharides was observed with variable states of acylation ranging from tetra-to octaacylated forms. All lipid A species, however, contained a set of conserved primary acyl chains consisting of an N- The bioluminescent bacterium Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes. Colonization of the juvenile squid's light organ by V. fischeri begins within hours of hatching (1). From the complex bacterial community present in seawater, V. fischeri, which represents less than 1% of the bacterial population, is exclusively recruited by E. scolopes in a multistep winnowing process (2). As the colonization of the juvenile's light organ progresses, a series of developmental changes occur in the tissues. The most dramatic of these morphogenetic events is the loss of a superficial ciliated field of cells important for the initial recruitment of V. fischeri. This process occurs over the ϳ96 -120 h following initial colonization by the symbiont (2) and does not occur without interactions with the symbiont in the deep crypt regions of the organ. Once the colonization is established, the squid continues to maintain a population of V. fischeri in its light organ, with cycles of daily flushing and repopulation by residual bacteria.The selection process that leads to the exclusive symbiotic relationship between V. fischeri and E. scolopes involves the interaction of bacterial surface components with host tissues. Previously, we showed that bacterial lipopolysaccharide (LPS) and lipid A can induce early stage apoptosis in the cells of the ciliated field of the juvenile squid's light organ (3). However, the effect was not species-specific, suggesting that a conserved portion of the lipid A may be the responsible component of the LPS structure (3). In later stages of the colonization process, bacterial peptidoglycan acts synergistically with LPS to induce most, if not all, of light organ morphogenesis (4).How the developmental signals of bacterial LPS and peptidoglycan, which are presented by the symbionts in the deep crypts of the light organ, are conveyed to the superficial tissue remains unknown, but one piece of the puzzle has recently been discovered. At hatching, the light organ has high levels of nitric oxide (NO). Following symbiont colonization of the crypts, the levels of both NO and the enzyme that catalyzes its production, nitric-o...

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“…Alternatively, an increased resolution of LPS can be obtained with SDS-PAGE using a Tricine buffer system [53], or by employing polyacrylamide gel concentration gradients within the gel [54,55]. Accordingly there are numerous ways of dissociating LPS glycoform aggregates into individual glycoforms and reproducibly separating the native LPS glycoforms with high resolution by slab-PAGE [56,57].…”

Section: Compositional Analysis Of Lps Aggregates Using Slab-pagementioning

confidence: 99%

“…B) Detection of lysozyme-LPS interaction and complex formation by double staining with imidazole-zinc/Coomassie brilliant blue R-250 [37]. C) Schematic representation of slab PAGE-mass spectrometry [56,57]. This technology allows assigning molecular structures to the LPS species recovered from PAGE gels by high-efficiency passive elution [60].…”

Section: Introductionmentioning

confidence: 99%

Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

Hardy¹,

Rodríguez²

2016

Biol Med (Aligarh)

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A prerequisite to the discovery and characterization of lipopolysaccharide (LPS) interaction with specific receptors and the resulting pathophysiological effects is the comprehensive structural analysis of LPS species. This brief review is aimed to summarize the use of gel electrophoresis linked to other biochemical technologies for detecting and characterizing LPSs. Lipopolysaccharide aggregates alone or mixtures containing LPSs and proteins/peptides can be separated by native agarose gel electrophoresis (NAGE), after which LPSs are detected with imidazole and zinc salts. A double-staining process with Coomassie brilliant blue R-250 enables the use of NAGE for detecting and studying protein-LPS interactions. For compositional analysis, the LPS aggregates are separated, with high resolution, by surfactant-polyacrylamide gel electrophoresis. After reverse staining with zinc-imidazole and elution from gel microparticles, glycoform-specific LPSs are ready for structural and biological analysis. For sequence analysis based on tandem electrospray ionization mass spectrometry (ESI-MS/MS) of oligosaccharides, LPSs are subjected to mild acid hydrolysis, dephosphorylation and permethylation. Also, O-deacylated LPS forms can be analyzed by matrix-assisted laser desorption/ionization-time of flight-MS. By comparison to spectra of unpurified LPSs, mass spectra of the micropurified LPSs show reduce heterogeneity and increased signal-to-noise ratios. Furthermore, the micropurification of LPSs prior to MS allows a higher sensitivity of detection for less abundant LPS glycoforms. The micropurified LPS fractions can be used to form self-assembled nanoaggregates which may be detected by dynamic light scattering. The effect of the O-side chain length on the Z-potential of LPS aggregates may be estimated by measurements based on laser Doppler electrophoresis. The thus obtained glycoform-specific LPSs are not only intact chemically but also biologically active as tested by e.g. Limulus amoebocyte lysate test, TNF-α assay and agonistic effect on human Toll-like receptor 4.

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Matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry of lipopolysaccharide species separated by slab‐polyacrylamide gel electrophoresis: High‐resolution separation and molecular weight determination of lipooligosaccharides from Vibrio fischeri strain HMK

Cited by 17 publications

References 45 publications

O-antigen and Core Carbohydrate of Vibrio fischeri Lipopolysaccharide

O-antigen and Core Carbohydrate of Vibrio fischeri Lipopolysaccharide

The Lipid A from Vibrio fischeri Lipopolysaccharide

Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

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