A hexasaccharide from capsular polysaccharide of carbapenem-resistant Klebsiella pneumoniae KN2 is a ligand of Toll-like receptor 4.

Klebsiella pneumoniae serotype KN2 is a carbapenem-resistant strain and leads to the health care-associated infections, such as bloodstream infections. Its capsular polysaccharide (CPS) was isolated and cleaved by a specific enzyme from a bacteriophage into a hexasaccharide-repeating unit. With GC-MS, NMR, and Mass analyses, the structure of KN2 CPS was determined to be {→3)-β-D-Glcp-(1→3)-[α-D-GlcpA-(1→4)-β-D-Glcp-(1→6)]-α-D-Galp-(1→6)-β-D-Galp-(1→3)-β-D-Galp-(1→}n. We demonstrated that 1 μg/mL CPS could stimulate J774A.1 murine macrophages to release tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in vitro. Also, we proved that KN2 CPS induced the immune response through Toll-like receptor 4 (TLR4) in the human embryonic kidney (HEK)-293 cells. Strikingly, the hexasaccharide alone shows the same immune response as the CPS, suggesting that the hexasaccharide can shape the adaptive immunity to be a potential vaccine adjuvant. The glucuronic acid (GlcA) on other polysaccharides can affect the immune response, but the GlcA-reduced KN2 CPS and hexasaccharide still maintain their immunomodulatory activities.