Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O: 3

Yersinia enterocolitica serotype O : 3 produces two types of lipopolysaccharide (LPS) molecules to its surface. In both types the lipid A (LA) structure is substituted by inner core (IC) octasaccharide to which either outer core (OC) hexasaccharide or homopolymeric O-polysaccharide (OPS) is linked. In addition, enterobacterial common antigen (ECA) can be covalently linked to LPS, however, via an unknown linkage. To elucidate the relationship between ECA and LPS in Y enterocolitica O : 3 and the effect of temperature on their expression, LPS was isolated from bacteria grown at 22 degrees C and 37 degrees C by consequent hot phenol/water and phenol-chloroform-light petroleum extractions to obtain LPS preparations free of ECA linked to glycerophospholipid. In immunoblotting, monoclonal antibodies TomA6 and 898, specific for OPS and ECA, respectively, reacted both with ladder-like bands and with a slower-migrating smear suggesting that the ECA and OPS epitopes coexist on the same molecules. These results were supported by immunoblotting with a monovalent Y. enterocolitica O : 3 ECA-specific rabbit antiserum. Also, two or three 898-positive (and monovalent-positive) TomA6-negative bands migrated at the level of the LA IC band in LPS samples from certain OC mutants, most likely representing LA IC molecules carrying 1-3 ECA repeat units but no OPS. These bands were also present in Y. enterocolitica O : 9 OC mutants; however, coexistence of ECA and OPS in the same molecules could not be detected. Finally, the LA IC ECA bands were missing from LPS of bacteria grown at 37 degrees C and also the general reduction in wild-type bacteria of ECA-specific monovalent-reactive material at 37 degrees C suggested that temperature regulates the expression of ECA. Indeed, RNA-sequencing analysis showed significant downregulation of the ECA biosynthetic gene cluster at 37 degrees C.