Rhodobacter azotoformans LPS (RAP99-LPS) Is a TLR4 Agonist That Inhibits Lung Metastasis and Enhances TLR3-Mediated Chemokine Expression.

The lipopolysaccharides (LPSs) of Rhodobacter are reported to be TLR4 antagonists. Accordingly, the extract of Rhodobacter azotoformans (RAP99) is used as a health supplement for humans and animals in Japan to regulate immune responses in vivo. We previously analyzed the LPS structure of RAP99 (RAP99-LPS) and found it is different from that of E. coli-LPS but similar to lipid A from Rhodobacter sphaeroides (RSLA), a known antagonist of TLR4, with both having three C14 fatty acyl groups, two C10 fatty acyl groups, and two phosphates. Here we show that RAP99-LPS has an immune stimulatory activity and acts as a TLR4 agonist. Pretreatment of RAP99-LPS suppressed E. coli-LPS-mediated weight loss, suggesting it is an antagonist against E. coli-LPS like other LPS isolated from Rhodobacter. However, injections of RAP99-LPS caused splenomegaly and increased immune cell numbers in C57BL/6 mice but not in C3H/HeJ mice, suggesting that RAP99-LPS stimulates immune cells via TLR4. Consistently, RAP99-LPS suppressed the lung metastasis of B16F1 tumor cells and enhanced the expression of TLR3-mediated chemokines. These results suggest that RAP99-LPS is a TLR4 agonist that enhances the activation status of the immune system to promote anti-viral and anti-tumor activity in vivo.

Structural analysis of a novel lipooligosaccharide (LOS) from Rhodobacter azotoformans.

Lipopolysaccharides (LPS) are components of the Gram-negative bacterial cell surface that stimulate the host innate immune system through the Toll-like receptor (TLR) 4-MD-2 complex. Rhodobacter sp. have been reported to produce LPS that lack endotoxic activity, and instead act as antagonists of other endotoxins. In this report, we focused on LPS, especially the lipooligosaccharide (LOS) fraction produced by Rhodobacter azotoformans that shows production of IL-8, but has an inverse correlation with IL-6 production. We analyzed their molecular structure by using mass spectrometry and nuclear magnetic resonance spectroscopy and report a novel LOS consisting of a shorter glycan structure containing glucuronic acid but not heptoses. A novel glycan structure, Glcα(1 → 4)GlcAα(1 → 4)KDOα(2 → 4)[Glcα(1 → 5)]KDOα(2 → 6)[4-phosphate]GlcNß(1 → 6) GlcNα1-phosphate, was proposed using NMR methods. The structure was consistent with one obtained based on MS. The MS analysis further revealed the existence of structural variation caused by extension with hexoses. The acyl composition in lipid A was suggested to contain three C14 fatty acyl chains (3-OH-14:0 or 3-oxo-14:0 at N2 of GlcN-1, 3-OH-14:0 at N2 of GlcN-2, that carried another 14:1 Δ7 on its ß-hydroxyl group) and two C10 fatty acyl chains (3-OH-10:0 at O3 of both GlcN), which are same as those found in lipid A from Rhodobacter sphaeroides.