Characterization of outer membrane vesicles of Acetobacter pasteurianus NBRC3283.

Acetobacter pasteurianus is characterized as a fermenting bacterium of kurozu, which is a common traditional Japanese black vinegar. Recently, we separated acid-resistant and low Toll-like receptor 4 (TLR4)-stimulatory lipopolysaccharides (LPS) from A. pasteurianus. We also showed that their lipid A parts possessed a novel sugar backbone that is responsible for the low TLR4-stimulatory and acid-resistant properties of the LPS. Outer membrane vesicles (OMVs) are nano-sized spherical structures secreted from many gram-negative bacteria. OMVs contain LPS and act as immunomodulants such as vaccines. In this study, we investigated OMVs secreted from A. pasteurianus. OMV secretion from A. pasteurianus NBRC 3283 cells was observed after 2 days in culture by transmission electron microscopy imaging. Thus OMVs were separated from the culture supernatants by ultracentrifugation and then purified by OptiPrep density gradient centrifugation. The OMVs contained several proteins including outer membrane proteins, and several sugars as components of LPS. The OMVs weakly stimulated TLR4 in accordance with the activity of A. pasteurianus LPS. Additionally, the TLR2-stimulating activity of the OMVs was significantly potent, indicating the existence of lipoproteins. Furthermore OMV-like spherical particles were observed in kurozu. Some of these particles are probably derived from A. pasteurianus. These data suggest that A. pasteurianus produce OMVs that contain LPS and probably lipoproteins, and can modulate the innate immune system.

Characterization of the O-antigen polysaccharide derived from Pantoea agglomerans IG1 lipopolysaccharide.

A polysaccharide fraction was isolated from the Pantoea agglomerans IG1 lipopolysaccharide (IP-PA1), and its O-antigenic polysaccharide was characterized by chemical analyses and 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharide is composed of linear tetrasaccharide repeating units, consisting of glucose and rhamnose, where 40% of one of the rhamnose residues is substituted with glucose: →2)-α-l-Rhap-(1→6)-α-d-Glcp-(1→2)-[ß-d-Glcp-(1→3)]0.4-α-l-Rhap-(1→2)-α-l-Rhap-(1→.

TLR2-stimulating contaminants in glycoconjugate fractions prepared from Bacteroides fragilis.

Bacteroides fragilis is a member of the normal intestinal flora and is involved in host immunostimulation via TLR2. On the bacterial cell surface, glycoconjugates, such as LPS and capsular polysaccharide A (PSA), have been reported to participate in host immunostimulation via TLR2. Previously, we identified a TLR2-stimulating lipoprotein in B. fragilis cells. In this study, we demonstrated that TLR2-stimulating principal molecules in glycoconjugate fractions prepared from B. fragilis are contaminating proteinous molecules, which may also be lipoproteins. The glycoconjugate fractions were prepared by phenol-hot water extraction of B. fragilis wild type and PSA-deficient strains, followed by hydrophobic interaction chromatography. TLR2-stimilating activities of the fractions were not affected by PSA deficiency. By in-gel TLR2-stimulation assay, molecules in high-molecular-mass area, where capsular polysaccharides were migrated, were found not to stimulate TLR2, but those in the range of 15-40 kDa were active. Further, proteinase K could digest the latter molecules and the TLR2-stimulating activities were migrated to the area of below 15 kDa. These results support that proteinous molecules, which are estimated to be lipoproteins, are responsible for almost all TLR2-stimulating activity in the glycoconjugate fractions prepared from B. fragilis.

Characterization of a Novel d-Glycero-d-talo-oct-2-ulosonic acid-substituted Lipid A Moiety in the Lipopolysaccharide Produced by the Acetic Acid Bacterium Acetobacter pasteurianus NBRC 3283.

Acetobacter pasteurianus is an aerobic Gram-negative rod that is used in the fermentation process used to produce the traditional Japanese black rice vinegar kurozu. Previously, we found that a hydrophobic fraction derived from kurozu stimulates Toll-like receptors to produce cytokines. LPSs, particularly LPS from A. pasteurianus, are strong candidates for the immunostimulatory component of kurozu. The LPS of A. pasteurianus remains stable in acidic conditions during the 2 years of the abovementioned fermentation process. Thus, we hypothesized that its stability results from its structure. In this study, we isolated the LPS produced by A. pasteurianus NBRC 3283 bacterial cells and characterized the structure of its lipid A component. The lipid A moiety was obtained by standard weak acid hydrolysis of the LPS. However, the hydrolysis was incomplete because a certain proportion of the LPS contained acid-stable d-glycero-d-talo-oct-2-ulosonic acid (Ko) residues instead of the acid-labile 3-deoxy-d-manno-oct-2-ulosonic acid residues that are normally found in typical LPS. Even so, we obtained a Ko-substituted lipid A with a novel sugar backbone, α-Man(1-4)[α-Ko(2-6)]ß-GlcN3N(1-6)α-GlcN(1-1)α-GlcA. Its reducing end GlcN(1-1)GlcA bond was also found to be quite acid-stable. Six fatty acids were attached to the backbone. Both the whole LPS and the lipid A moiety induced TNF-α production in murine cells via Toll-like receptor 4, although their activity was weaker than those of Escherichia coli LPS and lipid A. These results suggest that the structurally atypical A. pasteurianus lipid A found in this study remains stable and, hence, retains its immunostimulatory activity during acetic acid fermentation.

Separation and characterization of the immunostimulatory components in unpolished rice black vinegar (kurozu).

Unpolished rice black vinegar (kurozu), a traditional Japanese vinegar, is considered to have beneficial health effects. Kurozu is produced via a static fermentation process involving the saccharification of rice by Aspergillus oryzae, alcohol fermentation by Saccharomyces cerevisiae, and the oxidation of ethanol to acetic acid by acetic acid bacteria such as Acetobacter pasteurianus. Since this process requires about 6 months' fermentation and then over a year of aging, most of these organisms die during the production process and so microbial components, which might stimulate the innate immune system, are expected to be present in the vinegar. In this study, we investigated whether microbial components are present in kurozu, and after confirming this we characterized their immunostimulatory activities. Lyophilized kurozu stimulated murine spleen cells to produce tumor necrosis factor (TNF)-α, at least in part, via Toll-like receptor (TLR) 2 and the Nod-like receptors NOD1 and 2. The active components associated with TLR2 activation were concentrated by Triton X-114-water phase partitioning and hydrophobic interaction chromatography on Octyl Sepharose. TLR4-activating components were also enriched by these methods. The concentrated preparation stimulated murine spleen cells to produce TNF-α and interferon (IFN)-γ. These results indicate that long-term fermented kurozu contains immunostimulatory components and that the TLR2 and TLR4-activating immunostimulatory components of kurozu are hydrophobic. These components might be responsible for the beneficial health effects of kurozu.