The role of proteases of periodontopathogens in iron acquisition

在人类中，细胞外铁通过转移到粘膜表面的血清和乳铁蛋白中而迅速结合。因此，游离铁的体内浓度太低（10 μ <-18>M），无法支持微生物的生长。人类成年牙周病原体，包括牙龈卟啉单胞菌缺乏铁载体系统，铁螯合系统。因此，这些物种必须具有特定的铁获取机制。我们曾认为该微生物以病区红细胞中的血红蛋白为铁源，实验证实了这一点。在本研究中，我们观察到肌红蛋白（体内另一种丰富的血红素蛋白）也作为铁源，发现肌红蛋白以pH依赖的方式可逆地结合牙龈卟啉单胞菌的包膜;结合发生在低于中性pH的孵育混合物中，而结合的肌红蛋白在高pH时从包膜中释放。在有足够肌红蛋白存在的情况下，在pH 5.0下每分钟与1 mg包膜结合的肌红蛋白量为1.4μg。在pH5.0条件下，反应的Kd为2.2×10 ~（-1）<-10>M。斑点杂交结果表明，肌红蛋白与牙龈卟啉单胞菌血红蛋白结合蛋白（HbBP）有明显的结合，但结合量只有血红蛋白的一半。发现通过凝胶过滤分离的肌红蛋白被含有蛋白酶的洗涤剂溶解的包膜消化的材料的级分之一支持牙龈卟啉单胞菌在铁源耗尽的培养基中的生长。

In humans, extracellular irons are rapidly bound by transferring in serum and lactoferrin at mucosal surfaces. Thus the in vivo concentration of free iron is too low (10^<-18> M) to support the growth of microorganisms. Human adult periodontal pathogens including Porphyromonas gingivalis lack siderophore system, an iron chelate system. Therefore, these species must possess the specific mechanism for iron acquisition. We had considered that the microorganisms utilize hemoglobin of the erythrocytes in the diseased area as iron source, which was confirmed by our experiments. In this study, we observed that myoglobin another abundant hemoprotein in the body also serve as iron source.Myoglobin was found to bind reversibly to the envelope of P.gingivalis in a pH-dependent manner ; the binding took place below neutral pHs of the incubation mixtures and myoglobin bound released from the envelope at high pHs. The amounts of myoglobin bound to 1 mg of the envelope at pH 5.0 per min under the presence of sufficient myoglobin were 1.4μg. K_d for the reaction at pH 5.0 was 2.2×10^<-10> M.From the dot blot assay, myoglobin obviously bound to hemoglobin-binding protein (HbBP) of P.gingivalis, however, the amounts of myoglobin that bound to HbBP were half those of hemoglobin. One of the fractions, separated by gel filtration, of the digested materials of myoglobin by the detergent-solubilized envelope containing proteases was found to support the growth of P.gingivalis in the iron source-depleted medium.