Yamaguchi Saki, Ishikawa Kazuya, Furuta Kazuyuki, Kaito Chikara
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
Appl Environ Microbiol. 2025 May 21;91(5):e0259524. doi: 10.1128/aem.02595-24. Epub 2025 Apr 10.
Colanic acid and enterobacterial common antigen (ECA) are cell-surface polysaccharides that are produced by many isolates. Colanic acid is induced under acidic, low temperature, and high-salt conditions and is important for resistance to these stresses; however, the role of ECA in these stresses is less clear. Here, we observed that knockout of flippase , which translocates lipid-linked ECA repeat units from the cytoplasmic side of the inner membrane to the periplasmic side, resulted in the sensitivity of BW25113 to acidic conditions. The -knockout mutant showed reduced growth potential and viable counts in vegetable extracts with acidic environments, including cherry tomatoes, carrots, celery, lettuce, and spinach. A double-knockout strain of and (glycosyltransferase that adds the third-and-final sugar of the lipid-linked ECA repeat unit) was not sensitive to acidic conditions, with similar results obtained for a double-knockout strain of and (glycosyltransferase that initiates colanic acid lipid-linked repeat-unit biosynthesis). The -knockout mutant was sensitive to low temperatures or high-salt conditions, which induced colanic acid synthesis, and these sensitivities were abolished by the additional knockout of . These results suggest that lipid-linked ECA repeat units confer susceptibility to acidic, low temperatures, and high-salt conditions in a colanic acid-dependent manner and that suppresses this negative effect.IMPORTANCEThe role of the common enterobacterial antigen, a polysaccharide that is conserved throughout enterobacteria, in stress resistance is unclear. Our results suggest that lipid-linked enterobacterial common antigen repeat units (which are typically translocated across the inner membrane by the flippase WzxE) cause sensitivity of to acidic, low-temperature, and high-salt conditions in a manner dependent on colanic acid. The -knockout mutant was sensitive to crude vegetable extracts, suggesting that the development of WzxE inhibitors could lead to novel food poisoning prevention agents. Considering previous findings that lipid-linked ECA repeat units are flipped by both WzxE and the flippase for colanic acid lipid-linked repeat-unit, the colanic acid-dependence of the -knockout phenotype proposes a model in which a large amount of colanic acid under stress conditions occupies the flippase for colanic acid lipid-linked repeat unit, leading to accumulation of lipid-linked ECA repeat units on the inner membrane.
结肠酸和肠杆菌共同抗原（ECA）是许多菌株产生的细胞表面多糖。结肠酸在酸性、低温和高盐条件下被诱导产生，对抵抗这些胁迫很重要；然而，ECA在这些胁迫中的作用尚不清楚。在这里，我们观察到，负责将脂质连接的ECA重复单元从内膜细胞质侧转运到周质侧的翻转酶缺失，导致BW25113对酸性条件敏感。该缺失突变体在包括樱桃番茄、胡萝卜、芹菜、生菜和菠菜在内的酸性环境蔬菜提取物中生长潜力和活菌数降低。 和 （添加脂质连接的ECA重复单元第三个也是最后一个糖的糖基转移酶）的双缺失菌株对酸性条件不敏感， 和 （启动结肠酸脂质连接重复单元生物合成的糖基转移酶）的双缺失菌株也得到了类似结果。该缺失突变体对低温或高盐条件敏感，这些条件会诱导结肠酸合成，而通过额外缺失 可消除这些敏感性。这些结果表明，脂质连接的ECA重复单元以结肠酸依赖的方式使细胞对酸性、低温和高盐条件敏感，而 可抑制这种负面影响。
常见肠杆菌抗原是一种在所有肠杆菌中都保守的多糖，其在抗胁迫中的作用尚不清楚。我们的结果表明，脂质连接的肠杆菌共同抗原重复单元（通常由翻转酶WzxE跨内膜转运）以依赖结肠酸的方式使细胞对酸性、低温和高盐条件敏感。该缺失突变体对粗制蔬菜提取物敏感，这表明开发WzxE抑制剂可能会产生新型食物中毒预防剂。考虑到之前的研究结果，即脂质连接的ECA重复单元可被WzxE和结肠酸脂质连接重复单元的翻转酶翻转，该缺失表型对结肠酸的依赖性提出了一个模型，即在胁迫条件下大量的结肠酸占据了结肠酸脂质连接重复单元的翻转酶，导致脂质连接的ECA重复单元在内膜上积累。
