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

Abstract

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 6C and 37 6C 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 ECAspecific 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 6C and also the general reduction in wild-type bacteria of ECA-specific monovalentreactive material at 37 6C suggested that temperature regulates the expression of ECA. Indeed, RNAsequencing analysis showed significant downregulation of the ECA biosynthetic gene cluster at 37 6C.

First Evidence for a Covalent Linkage between Enterobacterial Common Antigen and Lipopolysaccharide in Shigella sonnei Phase II ECALPS

“…Crossreactivity of absorbed anti-ECA sera provided preliminary evidence for the presence of low molecular mass forms of ECA (ECA LPS ) in Y. enterocolitica O:3 ECA LPS . This finding was supported by more recent, extensive serological studies on Y. enterocolitica smooth and rough mutants (21). In this latter study, the authors used high-resolution SDS-PAGE to separate LPS and LOS preparations for subsequent immunoblotting with the above described detection antibodies and concluded that both low molecular mass (one to three repeating units) and high molecular mass forms of ECA LPS were present.…”

“…This species has been extensively investigated in studies designed to identify ECA LPS (21)(22)(23)(24). In these studies, researchers used two types of antibodies to track different forms of ECA: (i) a monoclonal anti-ECA antibody specific for ECA PG and (ii) a monovalent anti-ECA LPS antiserum produced by absorption of serum obtained against ECA-positive strain with ECA-negative bacteria (specific for ECA LPS ).…”

“…In subsequent studies, Kunin et al (9) and Mayer et al (20) identified the presence of such an immunogenic form of ECA in rough mutants of E. coli Ra, R1, R4, and K-12 that express a complete core OS region of LOS. More recent and extensive serological studies have addressed the occurrence of ECA LPS in Yersinia enterocolitica (21)(22)(23)(24) and Proteus mirabilis. ECA immunogenicity has also been demonstrated for several wild-type strains and mutants of Y. enterocolitica O:3 and O:9 as well as P. mirabilis (25).…”

“…The authors of these latter studies utilized polyclonal and monoclonal antibodies against different forms of ECA to screen SDS-PAGE-separated LPS/LOS isolated from a broad range of wildtype and mutant bacteria to show the coexistence of ECA and LPS epitopes. Recent serological studies of Y. enterocolitica LPS pointed to the core OS of LPS as the probable location of the ECA and its coexistence with O-specific polysaccharide (21). The only attempted structural analyses of ECA LPS were reported for E. coli strain F470 (R1 core type) (26) and S. sonnei phase II (27).…”

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

“…One important virulence factor of this bacterium is LPS that covers *70 % of the bacterial surface. It consists of three structural components: distal O polysaccharide (O-antigen), core region and lipid A, which anchors LPS to the membrane (Muszyń ski et al, 2013). The core region is divided into the inner core (IC) and outer core (OC).…”