The Actinobacillus actinomycetemcomitans ribose binding protein RbsB interacts with cognate and heterologous autoinducer 2 signals
The Actinobacillus actinomycetemcomitans ribose binding protein RbsB interacts with cognate and heterologous autoinducer 2 signals
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DOI：
10.1128/iai.01741-05
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发表时间：
2006-07-01
影响因子：
3.1
通讯作者：
Demuth, Donald R.
中科院分区：
文献类型：
--
作者：
James, DeAnna;Shao, HanJuan;Demuth, Donald R.
由口腔病原体伴放线放线杆菌产生的自诱导子 2 (AI-2) 在铁限制下影响生物体的生长并调节铁吸收基因的表达。然而，介导 A. actinomycetemcomitans 对 AI-2 反应的细胞成分尚未完全表征。 A. actinomycetemcomitans 的完整基因组序列分析 (www.oralgen.lanl.gov) 表明 RbsB 蛋白与哈维氏弧菌的 AI-2 受体 LuxP 相关。为了确定 RbsB 是否与 AI-2 相互作用，在存在和不存在纯化的六组氨酸标签 RbsB 的情况下，用来自 A. actinomycetemcomitans 的部分纯化的 AI-2 或来自 V. harveyi 培养物的条件培养基刺激后，测定报告菌株 V. harveyi BB170（传感器 1-、传感器 2+）的生物发光。 RbsB 以剂量依赖性方式有效抑制由 A. actinomycetemcomitans AI-2 和 V harveyi AI-2 诱导的 V. harveyi 生物发光，表明 RbsB 与 LuxP 竞争 AI-2。对于 A. actinomycetemcomitans AI-2，大约 0.3 nM RbsB 产生 50% 的抑制作用；对于 V. harveyi AI-2，大约 15 nM RbsB 产生 50% 的抑制作用。添加 50 mM 核糖可逆转 RbsB 介导的哈维弧菌生物发光抑制，表明放线放线菌 AI-2 和核糖在 RbsB 的同一位点结合。 RbsB/AI-2 复合物是热稳定的，因为 A. actinomycetemcomitans AI-2 无法通过加热回收。这不是由于 A. actinomycetemcomitans AI-2 的热灭活，因为在不存在 RbsB 的情况下信号活性不受加热影响。此外，相对于野生型生物体，不能表达rbsB的同基因A. actinomycetemcomitans突变体在从溶液中消耗A. actinomycetemcomitans AI-2方面存在缺陷。 rbsB 失活也会影响生物体在铁限制条件下生长的能力。在铁限制下，突变株的细胞密度约为野生型生物体的30%。此外，实时PCR表明，编码主要铁离子转运蛋白的afuABC的表达在rbsB突变体中减少了大约八倍。该表型与 A. actinomycetemcomitans 的 LuxS 缺陷突变体相似，该突变体无法产生 AI-2。总之，我们的结果表明 RbsB 可能在 A. actinomycetemcomitans 对 AI-2 的反应中发挥作用。
Autoinducer 2 (AI-2) produced by the oral pathogen Actinobacillus actinomycetemcomitans influences growth of the organism under iron limitation and regulates the expression of iron uptake genes. However, the cellular components that mediate the response of A. actinomycetemcomitans to AI-2 have not been fully characterized. Analysis of the complete genome sequence of A. actinomycetemcomitans (www.oralgen.lanl.gov) indicated that the RbsB protein was related to LuxP, the AI-2 receptor of Vibrio harveyi. To determine if RbsB interacts with AI-2, the bioluminescence of the reporter strain V. harveyi BB170 (sensor 1-, sensor 2+) was determined after stimulation with partially purified AI-2 from A. actinomycetemcomitans or conditioned medium from V. harveyi cultures in the presence and absence of purified six-His-tagged RbsB. RbsB efficiently inhibited V. harveyi bioluminescence induced by both A. actinomycetemcomitans AI-2 and V harveyi AI-2 in a dose-dependent manner, suggesting that RbsB competes with LuxP for AI-2. Fifty percent inhibition occurred with approximately 0.3 nM RbsB for A. actinomycetemcomitans AI-2 and 15 nM RbsB for V. harveyi AI-2. RbsB-mediated inhibition of V. harveyi bioluminescence was reversed by the addition of 50 mM ribose, suggesting that A. actinomycetenicomitans AI-2 and ribose bind at the same site of RbsB. The RbsB/AI-2 complex was thermostable since A. actinomycetemcomitans AI-2 could not be recovered by heating. This was not due to heat inactivation of A. actinomycetemcomitans AI-2 since signal activity was unaffected by heating in the absence of RbsB. Furthermore, an isogenic A. actinomycetemcomitans mutant that was unable to express rbsB was deficient in depleting A. actinomycetemcomitans AI-2 from solution relative to the wild-type organism. Inactivation of rbsB also influenced the ability of the organism to grow under iron-limiting conditions. The mutant strain attained a cell density of approximately 30% that of the wild-type organism under iron limitation. In addition, real-time PCR showed that the expression of afuABC, encoding a major ferric ion transporter, was reduced by approximately eightfold in the rbsB mutant. This phenotype was similar to that of a LuxS-deficient mutant of A. actinomycetemcomitans that is unable to produce AI-2. Together, our results suggest that RbsB may play a role in the response of A. actinomycetemcomitans to AI-2.