Biomolecular analysis on iron acquisition mechanism and pathogenicity of periodontopathic bacteria

铁是牙周病细菌的基本营养素，也是大多数生物的必需营养素。我们以前的研究表明，绿色卟啉单胞菌利用血红蛋白作为有效的铁源，其血红蛋白受体与赖氨酸特异性半胱氨酸蛋白酶(KGP)的催化结构域相同。在这项研究中，我们首先研究了血红蛋白对各种牙周病细菌生长的影响。放线菌伴生放线杆菌、中间普氏杆菌和黑色普氏杆菌有效地利用了血红蛋白作为铁源。其中65 kDa的不动杆菌外膜蛋白、60 kDa的中间假单胞菌和41、56和59 kDa的黑色假单胞菌为血红蛋白结合蛋白。然后，用根据DNA序列制备的KGP片段来研究具有血红蛋白结合活性的KGP的活性区域。斑点印迹实验表明，牙龈假单胞菌KGP催化区的氨基末端部分具有较强的结合血红蛋白的活性。Kgp是牙龈假单胞菌的主要蛋白酶之一，在牙周炎的发病机制中起着重要作用。因此，我们构建了针对牙龈假单胞菌Kgp的DNA疫苗，并在小鼠模型中评价了该DNA疫苗作为免疫原对抗P.givalis攻击的效果。DNA疫苗每周被注射到股四头肌，总共接种了四次。免疫后可明显诱导血清中特异性的免疫球蛋白抗体。与对照组动物相比，DNA疫苗免疫对牙龈假单胞菌致死性攻击具有显著的保护作用：这些结果表明KGP作为一种血红蛋白结合蛋白，也可以作为一种免疫原来诱导对牙龈假单胞菌感染的保护性反应。

Iron is an essential nutrient for periodontopathic bacteria as well as it is for most living organisms. Our previous study showed that Porphyromonas gignivalis utilized hemoglobin as an effective iron source and that its hemoglobin receptor was identical to the catalytic domain of lysine-specific cysteine proteinase (KGP). In this study, we first investigated the effects of hemoglobin on the growth of various periodontopathic bacteria. Actinobacillus actinomycetemcomitance, Prevotella intermedia and Prevotella nigrescence effectively utilized hemoglobin as an iron source. 65 kDa outer membrane protein of A. acdnomycetemoomitans, 60 kDa of P.intermedia and 41, 56 and 59 kDa of P.nigrescens were identified as hemoglobin-binding proteins. Next, the active domain of KGP showing hemoglobin-binding activity was investigated with KGP fragments, which were prepared based on the DNA sequence. It was found that amino-terminal portion in catalytic domain of P.gingivalis KGP showed strong -hemoglobin-binding activity by dot blot assay. KGP is one of major proteases of P.gingivais and it is believed to play a critical role in the pathogenesis of P.gingivalis. Therefore, we constructed DNA vaccine targeted for P.gingivalis KGP and evaluated the effect of the DNA vaccine as an immunogen against P.gingivalis challenge in a murine model. The DNA vaccine was injected into the quadriceps muscles weekly for a total of four inoculations. Following immunization, specific IgG antibodies in serum were clearly induced. Immunization of the DNA vaccine conferred a significant amount of protection against P gingivalis lethal challenge compared with that in control animals : These results suggest that KGP acts as a hemoglobin-binding protein and can also be useful as an immunogen to induce a protective response to P.gingivalis infection