Recent advances in understanding of enterobacterial common antigen synthesis and regulation

Abstract

Antibiotic resistance has become one of the most significant global health threats, with nearly three million antibiotic-resistant infections occurring per year in the United States alone. This problem is especially acute in Gram-negative bacteria, which possess an outer membrane (OM) that surrounds the aqueous periplasm and cytoplasmic membrane and acts as a permeability barrier capable of excluding many antibiotics. The OM of Enterobacterales also contains a highly conserved, invariant carbohydrate-derived moiety known as enterobacterial common antigen (ECA), which has been shown to play a significant role in this membrane permeability barrier. Although ECA was first discovered in the 1960s, its precise function and regulation remain largely mysterious. Here, we highlight recent studies that have advanced our understanding of the structure, biosynthesis, regulation and potential functions of ECA. We also review new insights into the complex interactions of the cell envelope biosynthesis pathways which may also play a role in surface antigen biosynthesis.

Keywords: O-antigen; enterobacterial common antigen; gene expression; outer membrane; permeability; surface antigens.

Figure 1.

Schematic representation of ECA Biogenesis in E. coli. ECA biogenesis begins with synthesis of the three amino sugars: N-acetyl-d-glucosamine (G), N-acetyl-d-mannosaminuronic acid (Ma) and 4-acetamido-4,6-dideoxy-d-galactose (Gt). The amino sugars are then loaded sequentially onto the isoprenoid carrier (Und-P) by a series of transferases, dehydrogenases, epimerases, including WecA, WecB, WecC, WecD, WecE, WecF, WecG until it forms a complete ECA subunit. The isoprenoid-linked precursor is then flipped across the IM by the WzxE flippase, and the subunits are polymerized by WzyE which coordinates in a complex with the chain length regulator protein WzzE. The three forms of ECA made with these polymerized subunits are: the surface-exposed, linear ECA_PG, which is transferred off the isoprenoid carrier and attached to phosphatidylglycerol via a phosphodiester linkage before being transferred to the OM via an, as yet, unknown mechanism; the surface-exposed, linear ECA_LPS, which is transferred off the isoprenoid carrier and attached to LPS before being transferred to the OM via what is assumed to be the Lpt pathway; and the periplasmic ECA_CYC, which is transferred off the isoprenoid carrier and retained in the periplasm by a yet unknown mechanism and works in coordination with the IM protein ElyC to regulate ECA_PG levels. Created in BioRender (Bennett, H. (2025) https://BioRender.com/u23n235).

Figure 2.

Function and regulation of the cyclic di-GMP phosphodiesterase PdeL. (a) The wec promoter binding site sequence of PdeL, as determined by [214], in the context of the wec operon in E. coli K 12. (b) PdeL has an autoregulatory function in addition to its function as a transcriptional regulator of genes related to motility, N4 bacteriophage resistance, and ECA biosynthesis and its transcription is also negatively regulated by cdG. Dashed arrow indicates enzymatic regulation of cdG by PdeL. Solid arrow indicates autoregulation of pdeL transcription [215]. Created in BioRender (Bennett, H. (2025) https://BioRender.com/k62c544)