Structural and Biochemical Analysis of a Single Amino-Acid Mutant of WzzBSF That Alters Lipopolysaccharide O-Antigen Chain Length in Shigella flexneri

Lipopolysaccharide (LPS), a surface polymer of Gram-negative bacteria, helps bacteria survive in different environments and acts as a virulence determinant of host infection. The O-antigen (Oag) component of LPS exhibits a modal chain-length distribution that is controlled by polysaccharide co-polymerases (PCPs). The molecular basis of the regulation of Oag chain-lengths remains unclear, despite extensive mutagenesis and structural studies of PCPs from Escherichia coli and Shigella. Here, we identified a single mutation (A107P) of the Shigella flexneri WzzBSF, by a random mutagenesis approach, that causes a shortened Oag chain-length distribution in bacteria. We determined the crystal structures of the periplasmic domains of wild-type WzzBSF and the A107P mutant. Both structures form a highly similar open trimeric assembly in the crystals, and show a similar tendency to self-associate in solution. Binding studies by bio-layer interferometry reveal cooperative binding of very short (VS)-core-plus-O-antigen polysaccharide (COPS) to the periplasmic domains of both proteins, but with decreased affinity for the A107P mutant. Our studies reveal that subtle and localized structural differences in PCPs can have dramatic effects on LPS chain-length distribution in bacteria, for example by altering the affinity for the substrate, which supports the role of the structure of the growing Oag polymer in this process.

脂多糖（Lipopolysaccharide, LPS）是革兰氏阴性菌的表面聚合物，能够帮助细菌在不同环境中存活，同时作为宿主感染的毒力决定因子。LPS的O抗原（O-antigen, 简称Oag）组分呈现出受多糖共聚合酶（polysaccharide co-polymerases, 简称PCPs）调控的模态链长分布。尽管针对大肠杆菌（Escherichia coli）和志贺氏菌（Shigella）的PCPs已开展了大量诱变及结构研究，但Oag链长调控的分子基础仍未明确。本研究通过随机诱变方法，鉴定出福氏志贺氏菌（Shigella flexneri）WzzBSF的单一位点突变A107P，该突变可导致细菌内Oag链长分布缩短。我们解析了野生型WzzBSF与A107P突变体的周质结构域晶体结构。两种结构在晶体中均形成高度相似的开放三聚体组装体，且在溶液中表现出相近的自聚集倾向。生物层干涉术结合实验显示，极短型核心加O抗原多糖（very short core-plus-O-antigen polysaccharide, 简称VS-COPS）可与两种蛋白的周质结构域发生协同结合，但A107P突变体对该底物的亲和力有所降低。本研究揭示，多糖共聚合酶中细微的局部结构差异可对细菌内LPS链长分布产生显著影响，例如通过改变对底物的亲和力，这一结果支持了正在延伸的Oag聚合物的结构在该调控过程中发挥作用的观点。