O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification.

A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-d-Gal-(1→3)-β-d-Gal-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-d-Gal-(1→3)-β-d-Gal-(1→] or O2c [→3)-β-d-GlcNAc-(1→5)-β-d-Gal-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Gal-dependent GT-A galactosyltransferase adding β-(1→3)-linked d-Gal, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)-linked d-Gal These activities build the O1 antigen on a terminal Gal in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Gal-dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Gal Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria.