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首页> 外文期刊>Molecular Microbiology >Functional complementation reveals the importance of intermolecular monomer interactions for Helicobacter pylori VacA vacuolating activity.
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Functional complementation reveals the importance of intermolecular monomer interactions for Helicobacter pylori VacA vacuolating activity.

机译：功能互补揭示了分子间单体相互作用对幽门螺杆菌VacA空泡活性的重要性。

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摘要

The Helicobacter pylori vacuolating cytotoxin (VacA) induces degenerative vacuolation of sensitive mammalian cell lines. Although evidence is accumulating that VacA enters cells and functions from an intracellular site of action, the biochemical mechanism by which VacA mediates cellular vacuolation has not been established. In this study, we used functional complementation and biochemical approaches to probe the structure of VacA. VacA consists of two discrete fragments, p37 and p58, that are both required for vacuolating activity. Using a transient transfection system, we expressed genetically modified forms of VacA and identified mutations in either p37 or p58 that inactivated the toxin. VacA with an inactivating single-residue substitution in the p37 domain [VacA (P9A)] functionally complemented a second mutant form of VacA with an inactivating two-residue deletion in the p58 domain [VacA Delta(346-347)]. VacA (P9A) and VacA Delta(346-347) also co-immunoprecipitated from vacuolated monolayers, supporting the hypothesis that these two inactive mutants associate directly to function in trans. p37 and p58 interact directly when expressed as separate fragments within HeLa cells, suggesting that p37-p58 inter-actions facilitate VacA monomer associations. Collectively, these results support a model in which the active form of VacA requires assembly into a complex of two or more monomers to elaborate toxin function.
机译：幽门螺杆菌空泡细胞毒素（VacA）诱导敏感的哺乳动物细胞系的变性空泡。尽管有越来越多的证据表明VacA从细胞内的作用位点进入细胞并发挥功能，但尚未确定VacA介导细胞空泡化的生化机制。在这项研究中，我们使用功能互补和生化方法来探测VacA的结构。 VacA由两个离散的片段p37和p58组成，这两个片段都是空泡活性所必需的。使用瞬时转染系统，我们表达了VacA的基因修饰形式，并鉴定了使毒素失活的p37或p58中的突变。在p37域[VacA（P9A）]中具有失活的单残基取代的VacA在功能上与在p58域[VacA Delta（346-347）]中具有失活的两个残基缺失的VacA的第二种突变形式互补。 VacA（P9A）和VacA Delta（346-347）也可从空泡的单层膜上共免疫沉淀，支持以下假设：这两个无活性的突变体直接与反式功能相关。 p37和p58在HeLa细胞中表达为单独的片段时直接相互作用，这表明p37-p58的相互作用促进了VacA单体缔合。总的来说，这些结果支持了一个模型，在该模型中，活性形式的VacA需要组装成两种或多种单体的复合物才能发挥毒素功能。

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