Identification of a second lipopolysaccharide in Porphyromonas gingivalis

Objectives: We previously described a surface anionic polysaccharide (APS) in P.gingivalis which is required for serum resistance. APS is a phosphorylated-branched mannan which shares a common epitope with the post-translational additions to the Arg-gingipains. This study aimed to determine the mechanism of anchoring of APS to the cell surface.

Methods: APS was purified on concanavalin A -affinity columns to minimise the loss of the anchoring system which occurred during chemical extraction. Analysis of the resultant polysaccharide was determined by NMR spectroscopy, mass spectrometry and fatty acid analysis. Biological activity was assessed following stimulation of MonoMac-6 cells and ELISA's for IL-1á, IL-1â, IL-6 and IL-8.

Results: Proton-NMR spectroscopy of lectin-purified APS confirmed the previous structure but also revealed additional signals which suggested the presence of a lipid A. This was confirmed by fatty-acid analysis of the APS and MALDI-ToF MS of lipid A released by treatment with sodium acetate buffer. Hence, P.gingivalis produces two distinct LPS macromolecules containing different glycan repeating units: O-LPS (with O-antigen tetrasaccharide repeating units) and A-LPS (with APS repeating units). Non-phosphorylated penta-acylated and non-phosphorylated tetra-acylated species were detected in the 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 pro-inflammatory activity of A-LPS compared to 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.

Conclusions: These data suggest that the APS of P. gingivalis is anchored through lipid A and hence this macromolecule may represent a second lipopolysaccharide species in this organism.