The role of proteases of periodontopathogens in iron acquisition

在人类中，细胞外铁通过血清和粘膜表面乳铁蛋白的转移而迅速结合。因此，体内游离铁的浓度太低（10^<-18> M），无法支持微生物的生长。包括牙龈卟啉单胞菌在内的成人牙周病原体缺乏铁离子系统，这是一种铁螯合系统。因此，这些物种必须具有特定的铁获取机制。我们认为微生物利用病区红细胞中的血红蛋白作为铁源，我们的实验证实了这一点。在本研究中，我们观察到肌红蛋白也是体内另一种丰富的血红蛋白，也是铁的来源。发现肌红蛋白以ph依赖的方式可逆地结合到牙龈卟啉卟啉菌的包膜上；结合发生在培养液的中性ph值以下，高ph值时肌红蛋白从包膜中释放。在足够的肌红蛋白存在的情况下，在pH 5.0 / min下，与1mg包膜结合的肌红蛋白量为1.4μg。在pH 5.0时反应的K_d为2.2×10^<-10> m .从斑点斑点法观察，肌红蛋白明显结合于牙龈卟噬菌的血红蛋白结合蛋白（HbBP），但与HbBP结合的肌红蛋白的数量是血红蛋白的一半。通过凝胶过滤分离的肌红蛋白消化物质中含有蛋白酶的洗涤剂溶解包膜的一个组分被发现支持牙龈假单胞菌在缺铁培养基中的生长。

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.