Identification of the Shigella flexneri Wzy Domain Modulating WzzpHs-2 Interaction and Detection of the Wzy/Wzz/Oag Complex
Identification of the Shigella flexneri Wzy Domain Modulating WzzpHs-2 Interaction and Detection of the Wzy/Wzz/Oag Complex
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DOI：
10.1128/jb.00224-22
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发表时间：
2022-08-18
影响因子：
3.2
通讯作者：
Morona, Renato
中科院分区：
文献类型：
--
作者：
Ascari, Alice;Tran, Elizabeth Ngoc Hoa;Morona, Renato
福氏志贺菌通过Wzy依赖途径生物合成其表面脂多糖的O抗原(OAG)组分。内膜聚合酶Wzy(SF)催化十一烯醇-二磷酸连接的OAG(und-PP-RUS)的重复加成生成多糖，其长度受两个竞争的共聚酶蛋白WZZ(SF)(短型OAG；10到17RU)和WZZ(PHS-2)(超长型OAG；>；90RU)的严格调控。在OAG聚合过程中，Wzy(SF)和WZZ(SF)/WZZ(PHS-2)之间的相互作用的性质仍然没有得到很好的描述，大多数文献描述了Wzy依赖的途径的单个蛋白质成分。在这里，我们报告了一项关于Wzy(SF)与其共聚酶对应物的特定结合作用的主要研究。首次确定了Wzy(S)(F)的一个区域，该区域形成了WZZ(PHS-2)的唯一结合位点。具体地说，本工作阐明了N-末端和C-末端结构域(NTD和CTD)上的关键Wzy(SF)部分，这些部分形成了调节WZZ(PHS-2)相互作用的分子内口袋。新的交配数据突出表明，这个NTD-CTD口袋中残基的破坏会削弱与WZZ(PHS-2)的相互作用，而不会影响WZZ(S)(F)的结合，从而特异性地扰乱较长多糖链的聚合。本研究对Wzy依赖途径中Wzy(SF)与WZZ(SF)/WZZ(PHS-2)的分子相互作用提供了新的认识，并首次检测到Wzy/WZZ/und-PP-OAG复合体。除了福氏志贺氏菌，这项工作还可以扩展到深入了解相关物种，特别是肠杆菌科成员表达的蛋白同源物之间的相互作用，这些蛋白同源物产生双重OAG链长度决定因素。导致福氏杆菌致病的一个主要毒力因子是其表面脂多糖，它由三个结构域组成：脂类A、核心寡糖和O抗原(OAG)。依赖于Wzy的途径是革兰氏阴性菌合成OAG最常见的生物合成机制，包括志贺氏菌。在OAG聚合中，聚合酶Wzy(S)(F)与多糖共聚酶WZZ(S)(F)和WZZ(P)(HS-2)之间的相互作用性质尚不清楚。本研究研究了Wzy(SF)及其共聚酶之间的分子相互作用，破译了Wzy依赖的途径中的关键相互作用，这些相互作用可能延伸到福氏葡萄球菌之外，为了解革兰氏阴性菌中OAG的生物合成提供了洞察力。
Shigella flexneri implements the Wzy-dependent pathway to biosynthesize the O antigen (Oag) component of its surface lipopolysaccharide. The inner membrane polymerase Wzy(SF) catalyzes the repeat addition of undecaprenol-diphosphate-linked Oag (Und-PP-RUs) to produce a polysaccharide, the length of which is tightly regulated by two competing copolymerase proteins, Wzz(SF) (short-type Oag; 10 to 17 RUs) and Wzz(pHS-2) (very-long-type Oag; >90 RUs). The nature of the interaction between Wzy(SF) and Wzz(SF)/Wzz(pHS-2) in Oag polymerization remains poorly characterized, with the majority of the literature characterizing the individual protein constituents of the Wzy-dependent pathway. Here, we report instead a major investigation into the specific binding interactions of Wzy(SF) with its copolymerase counterparts. For the first time, a region of Wzy(S)(F) that forms a unique binding site for Wzz(pHS-2) has been identified. Specifically, this work has elucidated key Wzy(SF) moieties at the N- and C-terminal domains (NTD and CTD) that form an intramolecular pocket modulating the Wzz(pHS-2) interaction. Novel copurification data high-light that disruption of residues within this NTD-CTD pocket impairs the interaction with Wzz(pHS-2) without affecting Wzz(S)(F) binding, thereby specifically disrupting polymerization of longer polysaccharide chains. This study provides a novel understanding of the molecular interaction of Wzy(SF) with Wzz(SF)/Wzz(pHS-2) in the Wzy-dependent pathway and, furthermore, detects the Wzy/Wzz/Und-PP-Oag complex for the first time. Beyond S. flexneri, this work may be extended to provide insight into the interactions between protein homologues expressed by related species, especially members of Enterobacteriaceae, that produce dual Oag chain length determinants.IMPORTANCE Shigella flexneri is a pathogen causing significant morbidity and mortality, predominantly devastating the pediatric age group in developing countries. A major virulence factor contributing to S. flexneri pathogenesis is its surface lipopolysaccharide, which is comprised of three domains: lipid A, core oligosaccharide, and O antigen (Oag). The Wzy-dependent pathway is the most common biosynthetic mechanism implemented for Oag biosynthesis by Gram-negative bacteria, including S. flexneri. The nature of the interaction between the polymerase, Wzy(S)(F), and the polysaccharide copolymerases, Wzz(S)(F) and Wzz(p)(HS-2), in Oag polymerization is poorly characterized. This study investigates the molecular interplay between Wzy(SF) and its copolymerases, deciphering key interactions in the Wzy-dependent pathway that may be extended beyond S. flexneri, providing insight into Oag biosynthesis in Gram-negative bacteria.