title: Identification of a second lipopolysaccharide in Porphyromonas gingivalis W50.
abstract: We previously described a cell surface anionic polysaccharide (APS) in Porphyromonas gingivalis that is required for cell integrity and serum resistance. APS is a phosphorylated branched mannan that shares a common epitope with posttranslational additions to some of the Arg-gingipains. This study aimed to determine the mechanism of anchoring of APS to the surface of P. gingivalis. APS was purified on concanavalin A affinity columns to minimize the loss of the anchoring system that occurred during chemical extraction. (1)H nuclear magnetic resonance spectroscopy of the lectin-purified APS confirmed the previous structure but also revealed additional signals that suggested the presence of a lipid A. This was confirmed by fatty acid analysis of the APS and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the lipid A released by treatment with sodium acetate buffer (pH 4.5). Hence, P. gingivalis synthesizes two distinct lipopolysaccharide (LPS) macromolecules containing different glycan repeating units: O-LPS (with O-antigen tetrasaccharide repeating units) and A-LPS (with APS repeating units). Nonphosphorylated penta-acylated and nonphosphorylated tetra-acylated species were detected in lipid A from P. gingivalis total LPS and in lipid A from A-LPS. These lipid A species were unique to lipid A derived from A-LPS. Biological assays demonstrated a reduced proinflammatory activity of A-LPS compared to that of total LPS. Inactivation of a putative O-antigen ligase (waaL) at PG1051, which is required for the final step of LPS biosynthesis, abolished the linkage of both the O antigen and APS to the lipid A core of O-LPS and A-LPS, respectively, suggesting that WaaL in P. gingivalis has dual specificity for both O-antigen and APS repeating units.
authors.affiliation: Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and The London Queen Mary's School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, United Kingdom. m.rangarajan@qmul.ac.uk
authors.forename: Minnie
authors.initials: M
authors.lastname: Rangarajan
authors.forename: Joseph
authors.initials: J
authors.lastname: Aduse-Opoku
authors.forename: Nikolay
authors.initials: N
authors.lastname: Paramonov
authors.forename: Ahmed
authors.initials: A
authors.lastname: Hashim
authors.forename: Nagihan
authors.initials: N
authors.lastname: Bostanci
authors.forename: Owen P
authors.initials: OP
authors.lastname: Fraser
authors.forename: Edward
authors.initials: E
authors.lastname: Tarelli
authors.forename: Michael A
authors.initials: MA
authors.lastname: Curtis
mesh_terms: D002241:Carbohydrates / Q000032:analysis; D019729:Carbon-Oxygen Ligases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity; D008401:Gas Chromatography-Mass Spectrometry; D017353:Gene Deletion; D006461:Hemolysis; D006801:Humans; D008050:Lipid A / Q000737:chemistry / Q000302:isolation & purification; D008055:Lipids / Q000032:analysis; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D009682:Magnetic Resonance Spectroscopy; D046529:Microscopy, Electron, Transmission; D019081:O Antigens / Q000737:chemistry; D016966:Porphyromonas gingivalis / Q000737:chemistry* / Q000378:metabolism / Q000472:pathogenicity / Q000648:ultrastructure; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002241:Carbohydrates; D008050:Lipid A; D008055:Lipids; D008070:Lipopolysaccharides; D019081:O Antigens; D019729:Carbon-Oxygen Ligases