A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification.

Tomasz K Gozdziewicz,Czeslaw Lugowski,Alona Tsybulska,Anna Maciejewska,Jolanta Lukasiewicz

doi:10.3390/ijms22020701

Abstract

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide − endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.

Highlights

Enterobacterial common antigen (ECA), described for the first time in the 1960s by Calvin M
ECA occurs in three different forms: as surface-bound linear polysaccharide linked to a phosphoglyceride (ECAPG ), cyclic oligosaccharide composed of 3–6 trisaccharide subunits (ECACYC ) [5,7,8,9,10], and as polysaccharide linked to lipopolysaccharide (ECALPS ) [1,11,12]
All S. sonnei phase II, E. coli R1, and K12 LPS-derived supernatants were hydrolyzed with 1.5% acetic acid in 100 ◦ C for 1 h to degrade or denaturate nucleic acids and proteins and further purified by gel filtration chromatography on the Bio-Gel P-30 yielding from one to two high molecular weight fractions (I-II) and two low molecular weight fractions (III and IV) (Figure 1)

Summary

Introduction

Enterobacterial common antigen (ECA), described for the first time in the 1960s by Calvin M. ECA occurs in three different forms: as surface-bound linear polysaccharide linked to a phosphoglyceride (ECAPG ), cyclic oligosaccharide composed of 3–6 trisaccharide subunits (ECACYC ) [5,7,8,9,10], and as polysaccharide linked to lipopolysaccharide (ECALPS ) [1,11,12]. ECA was discovered by observation of broad cross-reactivity between strains of E. coli causing urinary tract infections and rabbit antisera generated against the strains and 102 homologous and heterologous E. coli strains [1,3]. ECAPG represents a major form of ECA and together with lipopolysaccharide (LPS) and ECALPS , is located on the cell surface, contributing to antigenicity and outer membrane integrity. ECACYC is located in the periplasm and has been recently pointed out as an important factor maintaining the outer membrane permeability barrier [15]

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Discussion

Conclusion