Cytotoxicity of the Vibrio vulnificus MARTX toxin effector DUF5 is linked to the C2A subdomain.
Cytotoxicity of the Vibrio vulnificus MARTX toxin effector DUF5 is linked to the C2A subdomain.
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DOI：
10.1002/prot.24628
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发表时间：
2014-10
影响因子：
2.9
通讯作者：
Satchell, Karla J. F.
中科院分区：
文献类型：
--
作者：
Antic, Irena;Biancucci, Marco;Satchell, Karla J. F.
多功能自动处理毒素重复序列（MARTX）对细菌蛋白毒素进行毒素处理，这些毒素充当细胞毒性效应子结构域的递送平台。DUF 5效应子结构域存在于至少六种不同物种的MARTX毒素中，并且作为一种假设的蛋白存在于光杆状菌属中。它的存在增加了小鼠毒力研究中创伤弧菌MARTX毒素的效力，表明DUF 5直接导致发病。在这项工作中，DUF 5被证明是细胞毒性时，瞬时表达在HeLa细胞。DUF 5依赖于其C1结构域定位于质膜，并且依赖于其C2结构域使细胞变圆。当在酿酒酵母中表达时，全长DUF 5和C2结构域都引起生长抑制。DUF 5的结构模型是基于多杀性巴氏杆菌毒素的结构产生的，其有助于将细胞毒活性定位于称为C2 A的186个氨基酸的亚结构域。在该亚结构域内，丙氨酸扫描诱变揭示天冬氨酸-3721和精氨酸-3841作为细胞毒性的关键残基。这些残基也是必不可少的HeLa细胞中毒时，纯化的DUF 5融合炭疽毒素致死因子的胞质传递。热位移实验表明，这些保守的残基是重要的，以维持蛋白质的结构，而不是催化。气单胞菌MARTX毒素DUF 5Ah结构域也具有细胞毒性，而来自多杀性巴氏杆菌毒素的弱保守C1-C2结构域则没有。总的来说，这项研究是第一次证明，DUF 5中发现的MARTX毒素具有细胞毒性活性，这取决于保守的残基在C2 A亚结构域。
The multifunctional-autoprocessing repeats-in-toxin (MARTX) toxins bacterial protein toxins that serve as delivery platforms for cytotoxic effector domains. The domain of unknown function in position 5 (DUF5) effector domain is present in at least six different species’ MARTX toxins and as a hypothetical protein in Photorhabdus spp. Its presence increases the potency of the Vibrio vulnificus MARTX toxin in mouse virulence studies, indicating DUF5 directly contributes to pathogenesis. In this work, DUF5 is shown to be cytotoxic when transiently expressed in HeLa cells. DUF5 localized to the plasma membrane dependent upon its C1 domain and the cells become rounded dependent upon its C2 domain. Both full-length DUF5 and the C2 domain caused growth inhibition when expressed in Saccharomyces cerevisiae. A structural model of DUF5 was generated based on the structure of Pasteurella multocida toxin facilitating localization of the cytotoxic activity to a 186 amino acid subdomain termed C2A. Within this subdomain, an alanine scanning mutagenesis revealed aspartate-3721 and arginine-3841 as residues critical for cytotoxicity. These residues were also essential for HeLa cell intoxication when purified DUF5 fused to anthrax toxin lethal factor was delivered cytosolically. Thermal shift experiments indicated that these conserved residues are important to maintain protein structure, rather than for catalysis. The Aeromonas hydrophila MARTX toxin DUF5Ah domain was also cytotoxic, while the weakly conserved C1-C2 domains from P. multocida toxin were not. Overall, this study is the first demonstration that DUF5 as found in MARTX toxins has cytotoxic activity that depends on conserved residues in the C2A subdomain.
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