Identification of the Linkage between A-Polysaccharide and the Core in the A-Lipopolysaccharide of Porphyromonas gingivalis W50

Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. The structure of the core oligosaccharide (OS) of O-LPS and the attachment site of the O-polysaccharide (O-PS) repeating unit [-> 3)-alpha-D-Galp-(1 -> 6)-alpha-D-Glcp-(1 -> 4)-alpha-L-Rhap-(1 -> 3)-beta-D-GalNAcp-(1 ->] to the core have been elucidated using the Delta PG1051 (WaaL, O-antigen ligase) and Delta PG1142 (Wzy, O-antigen polymerase) mutant strains, respectively. The core OS occurs as an "uncapped" glycoform devoid of O-PS and a "capped" glycoform that contains the attachment site of O-PS via beta-D-GalNAc at position O-3 of the terminal alpha-(1 -> 3)-linked mannose (Man) residue. In this study, the attachment site of A-PS to the core OS was determined based on structural analysis of SR-type LPS (O-LPS and A-LPS) isolated from a P. gingivalis Delta PG1142 mutant strain by extraction with aqueous hot phenol to minimize the destruction of A-LPS. Application of one-and two-dimensional nuclear magnetic resonance (NMR) spectroscopy in combination with methylation analysis showed that the A-PS repeating unit is linked to a nonterminal alpha-(1 -> 3)-linked Man of the "capped core" glycoform of outer core OS at position O-4 via a -> 6)-[alpha-D-Man-alpha-(1 -> 2)-alpha-D-Man-1-phosphate -> 2]-alpha-D-Man-(1 -> motif. In order to verify that O-PS and A-PS are attached to almost identical core glycoforms, we identified a putative alpha-mannosyltransferase (PG0129) in P. gingivalis W50 that may be involved in the formation of core OS. Inactivation of PG0129 led to the synthesis of deep-R-type LPS with a truncated core that lacks alpha-(1 -> 3)-linked mannoses and is devoid of either O-PS or A-PS. This indicated that PG0129 is an alpha-1,3-mannosyltransferase required for synthesis of the outer core regions of both O-LPS and A-LPS in P. gingivalis. IMPORTANCE Porphyromonas gingivalis, a Gram-negative anaerobe, is considered to be an important etiologic agent in periodontal disease, and among the virulence factors produced by the organism are two lipopolysaccharides (LPSs), O-LPS and A-LPS. The structures of the O-PS and A-PS repeating units, the core oligosaccharide (OS), and the linkage of the O-PS repeating unit to the core OS in O-LPS have been elucidated by our group. It is important to establish whether the attachment site of the A-PS repeating unit to the core OS in A-LPS is similar to or differs from that of the O-PS repeating unit in O-LPS. As part of understanding the biosynthetic pathway of the two LPSs in P. gingivalis, PG0129 was identified as an alpha-mannosyltransferase that is involved in the synthesis of the outer core regions of both O-LPS and A-LPS.