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

Abstract

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, ␣-Man(1-4)[␣-Ko(2-6)]␤-GlcN3N(1-6)␣-GlcN(1-1)␣-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-␣ 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.

Granulibacter bethesdensis, a Pathogen from Patients with Chronic Granulomatous Disease, Produces a Penta-Acylated Hypostimulatory Glycero-D-talo-oct-2-ulosonic Acid–Lipid A Glycolipid (Ko-Lipid A)

“…G. bethesdensis belongs to Acetobacteraceae , the microbial family including bacteria that produce acetic acid from ethanol. Some members of this family, such as Acetobacter , have been shown to synthesize d - glycero - d - talo -oct-2-ulosonic acid (Ko) instead of 3-deoxy- d - manno -oct-2-ulosonic acid (Kdo)—a common core region constituent directly attaching to lipid A in the LPS of many Gram-negative bacteria [ 18 ]. Acid resistance of Ko has been well documented [ 22 , 23 ], and therefore, lipid A release was attempted under more acidic conditions, in the presence of 0.1 M HCl at 100–102 °C for 4.5 h. Although this harsher hydrolysis condition resulted in partial chemical degradation of the polysaccharide portion of the LPS, it substantially increased the yield of lipid A without changing its major matrix assisted laser desorption/ionization- time of flight mass spectrometry (MALDI-TOF MS) signals from those observed after mild 1% HOAc hydrolysis.…”

“…Acetobacter xylinum , a cellulose-producing bacterium, possesses an LPS containing Kdo but was not analyzed immunologically [ 17 ]. Recently, in two independent studies, it was shown that Acetobacter pasteurianus NBRC3283 and A. pasteurianus CIP103108 produce a hexa-acylated lipid A with a Ko (D-gylcero-D-talo-oct-2-ulosonic acid) substitution [ 18 , 19 ]. To better understand how G. bethesdensis avoids activation of immune cells and is resistant to certain antimicrobial peptides, we purified and characterized the structure and bioactivity of the lipid A from this organism.…”

Variation, Modification and Engineering of Lipid A in Endotoxin of Gram-Negative Bacteria

“…Acetobacter species, acetate-forming bacteria that are popular in the food industry, belong to α-Proteobacteria as well. The lipid A of A. pasteurianus was recently investigated, and some features common with those of Rhizobium and Bradyrhizobium species were elucidated ( Figure 6 ) [ 48 , 49 ]. This lipid A lacks phosphate, and the backbone is substituted by glucuronic acid and mannose at the 1 and 4′ positions, respectively.…”

“…This lipid A lacks phosphate, and the backbone is substituted by glucuronic acid and mannose at the 1 and 4′ positions, respectively. The backbone contains GlcN3N as a distal sugar, which was also found in Bradyrhizobium lipid A. Interestingly, the lipid A of A. pasteurianus links to the core oligosaccharide through D- glycero -D- talo -octulosonic acid (Ko) [ 48 ], which was first found in Acinetobacter LPS [ 50 ], and then in other species [ 51 , 52 , 53 ]. This sugar is deduced to stabilize the acid–labile linkage between lipid A and the core to adjust to acidic growth conditions.…”

The Peculiar Structure of Acetobacter pasteurianus CIP103108 LPS Core Oligosaccharide

“…However, the sugar moieties of the lipid A-inner core region were absent in the NMR spectra, likely due to their low flexibility, and impaired the NMR analysis. Nevertheless, the occurrence of a d-glycero-d-talo-oct-2-ulosonic acid (Ko), in place of Kdo, as the first sugar of the core OS directly linked to A. pasteurianus lipid A, [6,7] and the compositional analysis ( Figure S2), showing the presence of a 8-substituted-Kop, suggested the linkage of Kdop (K) to O8 of Ko residue, at its turn connected to the lipid A. Thus, in accordance with compositional analysis, NMR and MS data, the core region from A. pasteurianus CIP103108 ( Figure 5 and S3) possessed the following structure:…”