Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae

Klebsiella pneumoniae is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing Klebsiella infections. Lipopolysaccharide O antigens are attractive candidates because of the relatively small range of known O-antigen polysaccharide structures, but immunotherapeutic applications require a complete understanding of the structures found in clinical settings. Currently, the precise number of Klebsiella O antigens is unknown because available serological tests have limited resolution, and their association with defined chemical structures is sometimes uncertain. Molecular serotyping methods can evaluate clinical prevalence of Oserotypes but require a full understanding of the genetic determinants for each O-antigen structure. This is problematic with Klebsiella pneumoniae because genes outside the main rfb (O-antigen biosynthesis) locus can have profound effects on the final structure. Here, we report two new loci encoding enzymes that modify a conserved polysaccharide backbone comprising disaccharide repeat units [-> 3)-alpha-D-Galp-( 1 -> 3)-beta-D-Galf-(1 ->] (O2a antigen). We identified in serotype O2aeh a three-component system that modifies completed O2a glycan in the periplasm by adding 1,2-linked alpha-Galp side-group residues. In serotype O2ac, a polysaccharide comprising disaccharide repeat units [-> 5)-beta-D-Galf-(1 -> 3)-beta-D-GlcpNAc-(1 ->] (O2c antigen) is attached to the non-reducing termini of O2a-antigen chains. O2c-polysaccharide synthesis is dependent on a locus encoding three glycosyltransferase enzymes. The authentic O2aeh and O2c antigens were recapitulated in recombinant Escherichia coli hosts to establish the essential gene set for their synthesis. These findings now provide a complete understanding of the molecular genetic basis for the known variations in Klebsiella O-antigen carbohydrate structures based on the O2a backbone.