Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis

Lipopolysaccharide (LPS) is the major component of the surface of Gram-negative bacteria and its polysaccharide portion is situated at the outermost region. We investigated the relationship between the polysaccharide portion of LPS and biofilm formation using a series of Escherichia coli mutants defective in genes earlier shown to affect the LPS sugar compositions. Biofilm formation by a deep rough LPS mutant, the hldE strain, was strongly enhanced in comparison with the parental strain and other LPS mutants. The hldE strain also showed a phenotype of increased auto-aggregation and stronger cell surface hydrophobicity compared to the wild-type. Similar results were obtained with another deep rough LPS mutant, the waaC strain whose LPS showed same molecular mass as that of the hldE strain. Confocal laser scanning microscopy (CLSM) analysis and biofilm formation assay using DNase I revealed that biofilm formation by the hldE strain was dependent on extracellular DNA. Furthermore, a loss of flagella and an increase in amount of outer membrane vesicles in case of the hldE strain were also observed by transmission electron microscopy and atomic force microscopy, respectively. In addition, we demonstrated that a mutation in the hldE locus, which alters the LPS structure, caused changes in both expression and properties of several surface bacterial factors involved in biofilm formation and virulence. We suggest that the implication of these results should be considered in the context of biofilm formation on abiotic surfaces, which is frequently associated with nosocominal infections such as the catheter-associated infections.脂多糖（Lipopolysaccharide，LPS）是革兰氏阴性菌（Gram-negative bacteria）表面的主要组成成分，其多糖组分位于最外层区域。本研究通过一系列此前被证实可影响脂多糖糖组成的基因缺陷型大肠杆菌（Escherichia coli）突变株，探究了脂多糖多糖组分与生物被膜形成之间的关联。与亲本菌株及其他脂多糖突变株相比，深度粗糙型脂多糖突变株hldE菌株的生物被膜形成能力显著增强。与野生型菌株相比，hldE菌株还表现出自身凝集能力增强以及细胞表面疏水性提升的表型。另一株深度粗糙型脂多糖突变株waaC菌株（其脂多糖分子质量与hldE菌株一致）也得到了相似的实验结果。共聚焦激光扫描显微镜（Confocal laser scanning microscopy，CLSM）分析与脱氧核糖核酸酶I（DNase I）介导的生物被膜形成实验结果显示，hldE菌株的生物被膜形成依赖于胞外DNA。此外，分别通过透射电子显微镜与原子力显微镜观察发现，hldE菌株存在鞭毛缺失以及外膜囊泡数量增加的现象。此外，本研究证实，改变脂多糖结构的hldE基因座突变，会导致多种参与生物被膜形成与毒力的细菌表面因子的表达水平与特性发生改变。本研究认为，在非生物表面的生物被膜形成（该过程常与导管相关感染等医院感染相关）这一背景下，应考虑本研究结果的实际意义。