Identification of an iron-regulated hemin-binding outer membrane protein, HupO, in Vibrio fluvialis:: Effects on hemolytic activity and the oxidative stress response
Identification of an iron-regulated hemin-binding outer membrane protein, HupO, in Vibrio fluvialis:: Effects on hemolytic activity and the oxidative stress response
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DOI：
10.1128/iai.73.2.722-729.2005
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发表时间：
2005-02-01
影响因子：
3.1
通讯作者：
Kong, IS
中科院分区：
文献类型：
--
作者：
Ahn, SH;Han, JH;Kong, IS
在病原菌中，铁的获得对于在铁限制条件下在宿主中的定殖和增殖是重要的。Vibrio spp.获得铁通常对它们的毒力至关重要，在人类中引起胃肠炎或过度的水样腹泻。在本研究中，我们克隆了河流弧菌血红素利用蛋白基因hupO。HupO与弧菌属中的铁调节外膜受体蛋白具有高度同源性，并且含有细菌血红素受体所共有的基序，包括共识TonB盒、FRAP结构域和NPNL结构域。为了表征HupO的氯化血红素结合活性，我们使用过表达系统从大肠杆菌中纯化重组HupO蛋白（rHupO）。发现HupO与氯化血红素结合，但不与血红蛋白结合。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质印迹分析表明，在铁限制条件下，河弧菌外膜蛋白HupO的表达量为77 kDa。我们构建了一个hupO突变体HP 1，以研究HupO在河流弧菌中的生化功能。与野生型细胞相比，HP 1的溶血活性降低，并且当暴露于过氧化氢时，存活的HP 1数量显著低于野生型细胞。这些结果表明，HupO通过刺激溶血素产生和对氧化应激的抗性与河流弧菌中的毒力表达相关。在实验感染的小鼠中，野生型的50%致死剂量值低于突变体HP 1。
In pathogenic bacteria, iron acquisition is important for colonization and proliferation in the host under iron-limited conditions. The ability of Vibrio spp. to acquire iron is often critical to their virulence, causing gastroenteritis or excessive watery diarrhea in humans. In the study described here, we cloned the 2,100-bp heme utilization protein gene hupO in Vibrio fluvialis. HupO had high homology to iron-regulated outer membrane receptor proteins in Vibrio sp. and contained motifs that are common to bacterial heme receptors, including a consensus TonB box, a FRAP domain, and an NPNL domain. To characterize the hemin-binding activity of HupO, we purified the recombinant HupO protein (rHupO) from Escherichia coli by using an overexpression system. HupO was found to bind to hemin but not to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting demonstrated that the 77-kDa outer membrane protein HupO of V. fluvialis was induced under iron-restricted conditions. We constructed a hupO mutant, HP1, to investigate the biochemical function of HupO in V. fluvialis. The hemolytic activity of HP1 was reduced compared to that of wild-type cells and, when exposed to hydrogen peroxide, significantly lower numbers of HP1 survived than was the case in the wild type. These results suggest that HupO is associated with virulence expression in V. fluvialis through stimulation of hemolysin production and resistance to oxidative stress. In experimentally infected mice, the 50% lethal dose value of the wild-type was lower than that of the mutant, HP1.