Structural analysis and immunomodulatory properties of lipopolysaccharide from the gut symbiont Phocaeicola dorei

Lipopolysaccharide (LPS) in Bacteroidetes engages in intimate crosstalk with host immunity, and although LPS from Phocaeicola dorei (LPS_Pd) exhibits immunomodulatory properties, its chemical structure has been poorly characterized. Here, we prepared intact LPS_Pd with high purity, and determined its structural regions (saccharide chains and lipid A) using HPGPC, GC-MS, MALDI-TOF MS/MS and 1D/2D NMR. The lipid A component consists of a mixture of tetra- and penta-acylated species exhibiting monophosphorylated patterns. The saccharide chains could be divided into two distinct types: the O-specific chain comprised of disaccharide repeating unit (RU) [→1)-α-l-Rhap-(4→1)-β-d-Manp-(3→], with phosphorylated Rhap as end group. The average RU number of the O-specific chain was approximately 18. The core OS structure is represented by the following chemical formula: α-d-GlcpNAc-(1→3)-β-d-Glcp-(1→6)-[β-d-Galf-(1→2)]-β-d-Galp-(1→4)-[β-l-Fucp-(1→3)/β-l-Rhap-(1→3)]-α-l-Rhap-(1→4)-α/β-l-Rhap(3P)-(1→5)-α-D-Kdop. The immunological properties of LPS_Pd and its structural regions were evaluated in vitro using RAW264.7 macrophages. LPS_Pd is a weak activator of TLR4-mediated signaling that inhibits the pro-inflammatory activity of E.coli LPS. Even though the lipid A portion of LPS_Pd is essential for its function, the presence of its saccharide portion provides a synergistic functional effect. Our results provide the first comprehensive structural elucidation of LPS_Pd along with crucial information on its structure-activity relationships.