Serological characterization of the enterobacterial common antigen substitution of the lipopolysaccharide of Yersinia enterocolitica O:3

Enterobacterial common antigen (ECA) is a polysaccharide present in all members of Enterobacteriaceae anchored either via phosphatidylglycerol (PG) or LPS to the outer leaflet of the outer membrane (ECA(PG) and ECA(LPS), respectively). Only the latter form is ECA-immunogenic. We previously demonstrated that Yersinia enterocolitica 0 :3 and its rough (O-specific polysaccharide-negative) mutants were ECA-immunogenic, suggesting that they contained ECA(LPS); however, it was not known which part of the LPS core region was involved in ECA binding. To address this, we used a set of three deep-rough LPS mutants for rabbit immunization. The polyvalent antisera obtained were: (i) analysed for the presence of anti-LPS and anti-ECA antibodies; (ii) treated with caprylic acid (CA) to precipitate IgM antibodies and protein aggregates; and (iii) adsorbed with live ECA-negative bacteria to obtain specific anti-ECA antisera. We demonstrated the presence of antibodies specific for both ECA(PG) and ECA(LPS) in all antisera obtained. Both CA treatment and adsorption with ECA-negative bacteria efficiently removed anti-LPS antibodies, resulting in specific anti-ECA sera. The LPS of the ECA(LPS)-positive deepest-rough mutant contained only lipid A and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residues of the inner core, suggesting that ECA(LPS) was linked to the Kdo region of LPS in Y. enterocolitica O :3.