James DeAnna, Shao Hanjuan, Lamont Richard J, Demuth Donald R
Department of Periodontics, Endodontics and Dental Hygiene, University of Louisville School of Dentistry, 501 South Preston Street, Room 209, Louisville, KY 40292, USA.
Infect Immun. 2006 Jul;74(7):4021-9. doi: 10.1128/IAI.01741-05.
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. actinomycetemcomitans 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.
由口腔病原体伴放线放线杆菌产生的自诱导物2（AI-2）在铁限制条件下影响该菌的生长，并调节铁摄取基因的表达。然而，介导伴放线放线杆菌对AI-2作出反应的细胞成分尚未完全明确。对伴放线放线杆菌完整基因组序列（www.oralgen.lanl.gov）的分析表明，RbsB蛋白与哈氏弧菌的AI-2受体LuxP相关。为确定RbsB是否与AI-2相互作用，在用来自伴放线放线杆菌的部分纯化AI-2或哈氏弧菌培养物的条件培养基刺激后，在有和没有纯化的六组氨酸标签RbsB存在的情况下，测定报告菌株哈氏弧菌BB170（传感器1-，传感器2+）的生物发光。RbsB以剂量依赖的方式有效抑制由伴放线放线杆菌AI-2和哈氏弧菌AI-2诱导的哈氏弧菌生物发光，表明RbsB与LuxP竞争AI-2。对于伴放线放线杆菌AI-2，约0.3 nM的RbsB可产生50%的抑制，对于哈氏弧菌AI-2，15 nM的RbsB可产生50%的抑制。添加50 mM核糖可逆转RbsB介导的对哈氏弧菌生物发光的抑制，表明伴放线放线杆菌AI-2和核糖结合在RbsB的同一部位。RbsB/AI-2复合物是热稳定的，因为加热后无法回收伴放线放线杆菌AI-2。这不是由于伴放线放线杆菌AI-2的热失活，因为在没有RbsB的情况下，加热不影响信号活性。此外，与野生型菌株相比，不能表达rbsB的同基因伴放线放线杆菌突变体在从溶液中消耗伴放线放线杆菌AI-2方面存在缺陷。rbsB的失活也影响了该菌在铁限制条件下的生长能力。在铁限制条件下，突变菌株达到的细胞密度约为野生型菌株的30%。此外，实时PCR显示，编码主要铁离子转运蛋白的afuABC在rbsB突变体中的表达降低了约八倍。这种表型与不能产生AI-2的伴放线放线杆菌LuxS缺陷突变体相似。总之，我们的结果表明RbsB可能在伴放线放线杆菌对AI-2的反应中起作用。
