ABC transporter-dependent synthesis of the O antigen of lipopolysaccharide in Klebsiella pneumoniae

A crucial virulence determinant for most Gram-negative bacteria is expression of the O-antigenic polysaccharide (O antigen) of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane. O antigen can be synthesized by an ATP-binding cassette (ABC) transporter-dependent pathway, where the O antigen is formed in the cytoplasm and exported to the periplasm by an ABC transporter. There, the O antigen is linked to the lipid A-core oligosaccharide (OS) portion of LPS and the completed molecule is transferred to the. cell surface. In Klebsiella pneumoniae O12, the oligosaccharide repeat unit comprises [3)-beta-D-GlcNAc-(1-4)-alpha-L-Rha-(1], with the rhamnose residue at the non-reducing terminus substituted with beta-(2-3)-Kdo (3-deoxY-D-manno-octulosonic acid). Synthesis of the O12 antigen is thought to follow a mechanism of synthesis elucidated in E. coli O9a, where chain-length regulation is mediated by the addition of a terminating residue (i.e., Kdo) to the growing polymer. In E. coli O9a, the terminating residue is recognized by a specific domain linked to the transporter nucleotide-binding domain (NBD). This specific substrate-binding domain is evident upon alignment to conventional NBDs, and a corresponding region can be identified in the K. pneumoniae O12 NBD. Two glycosyltransferases are required for O12 antigen assembly: WbbL and WbbB. WbbL is a functionally characterized Rha transferase. WbbB contains three predicted conserved domains (CDs): PF05159 (a CD also found in capsule synthesis proteins with unknown functions), glycosyltransferase family 1 (GT-1) and glycosyltransferase family 25 (GT-25). Therefore, WbbB is likely a multi-functional protein that is responsible for GlcNAc and Kdo transfer. Analysis of WbbB truncation derivatives identified independently folding regions of the polypeptide and suggested that the PF05159 motif is not required for O12 polymerization. This research can be integrated into future studies characterizing the potential multiple activities of WbbB.