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

Tomasz K Gozdziewicz,Jolanta Lukasiewicz,Czeslaw Lugowski

doi:10.1074/jbc.m113.512749

Tomasz K Gozdziewicz, Jolanta Lukasiewicz + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m113.512749

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Abstract

Enterobacterial common antigen (ECA) is expressed by Gram-negative bacteria belonging to Enterobacteriaceae, including emerging drug-resistant pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus spp. Recent studies have indicated the importance of ECA for cell envelope integrity, flagellum expression, and resistance of enteric bacteria to acetic acid and bile salts. ECA, a heteropolysaccharide built from the trisaccharide repeating unit, →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→, occurs as a cyclic form (ECA(CYC)), a phosphatidylglycerol (PG)-linked form (ECA(PG)), and an endotoxin/lipopolysaccharide (LPS)-associated form (ECA(LPS)). Since the discovery of ECA in 1962, the structures of ECA(PG) and ECA(CYC) have been completely elucidated. However, no direct evidence has been presented to support a covalent linkage between ECA and LPS; only serological indications of co-association have been reported. This is paradoxical, given that ECA was first identified based on the capacity of immunogenic ECA(LPS) to elicit antibodies cross-reactive with enterobacteria. Using a simple isolation protocol supported by serological tracking of ECA epitopes and NMR spectroscopy and mass spectrometry, we have succeeded in the first detection, isolation, and complete structural analysis of poly- and oligosaccharides of Shigella sonnei phase II ECA(LPS). ECA(LPS) consists of the core oligosaccharide substituted with one to four repeating units of ECA at the position occupied by the O-antigen in the case of smooth S. sonnei phase I. These data represent the first structural evidence for the existence of ECA(LPS) in the half-century since it was first discovered and provide insights that could prove helpful in further structural analyses and screening of ECA(LPS) among Enterobacteriaceae species.

Highlights

Enterobacterial common antigen (ECA) is a surface antigen of all enteric bacteria
Isolation and Purification of S. sonnei Phase II ECA covalently associated with LPS (ECALPS)—The LOS of S. sonnei phase II was extracted from bacterial cells by a hot phenol/water method and was obtained with a yield of 2.11% of dry bacterial mass
Five fractions were eluted and analyzed for the presence of ECA epitopes by dot-blotting using polyclonal rabbit anti-ECACYC and anti-E. coli R1 core OS sera

Summary

Background

Enterobacterial common antigen (ECA) is a surface antigen of all enteric bacteria. Results: ECA polysaccharide substitutes the outer core region of Shigella sonnei lipopolysaccharide (LPS). This suggested that, unlike most E. coli strains, a few, especially O14, express this antigen in an immunogenic form, the presumptive ECALPS, capable of eliciting cross-reactive anti-ECA antibodies [1, 13,14,15] Following this initial discovery of ECA, ECAPG was identified [4, 9, 10], and its chemical structure and covalent linkage to PG were elucidated for E. coli and Salmonella typhimurium [11, 12]. The only attempted structural analyses of ECALPS were reported for E. coli strain F470 (R1 core type) [26] and S. sonnei phase II [27] These analytical strategies utilized chromatography to separate different fractions of degraded LPS and characterize them for the presence of ECA constituents. The data presented here, coming a half-century after the initial discovery of ECA, represent the first structural evidence for the existence of ECALPS and provide insights that could prove helpful in further structural analyses and screening of ECALPS among Enterobacteriaceae species

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION