Biomolecular analysis on iron acquisition mechanism and pathogenicity of periodontopathic bacteria

牙周病细菌铁获取机制及致病性的生物分子分析

• 批准号: 11307050
• 负责人: SHIZUKUISHI Satoshi
• 金额: $ 23.07万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11307050/
• 关键词: periodontopathic bacteria; Porphyromonas gingivalis; Actinobacillus actinomycetemcomitans; Prevotella; Iron; hemoglobin; Virulence factor; hemoglobin-binding protein; Actionbacillus actinomycetemcomitans; Prevotella nigrescens; ヘモグロビン結合蛋白質; 鉄源

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

铁是牙周病细菌以及大多数生物体的必需营养素。我们以前的研究表明，Gignivalis利用血红蛋白作为一个有效的铁源，其血红蛋白受体是相同的赖氨酸特异性半胱氨酸蛋白酶（KGP）的催化结构域。在这项研究中，我们首先调查了血红蛋白对各种牙周病细菌生长的影响。放线共生放线杆菌、中间普雷沃菌和黑色普雷沃菌能有效利用血红蛋白作为铁源。65 kDa外膜蛋白。嗜酸性粒菌的60 kDa、中间嗜酸性粒菌的60 kDa和变黑嗜酸性粒菌的41、56和59 kDa被鉴定为血红蛋白结合蛋白。接下来，用基于DNA序列制备的KGP片段研究显示血红蛋白结合活性的KGP活性结构域。斑点杂交实验发现牙龈卟啉单胞菌KGP催化结构域的氨基端部分具有较强的α-血红蛋白结合活性。KGP是牙龈卟啉单胞菌的主要蛋白酶之一，被认为在牙龈卟啉单胞菌的发病机制中起关键作用。因此，我们构建了针对牙龈卟啉单胞菌KGP的DNA疫苗，并在小鼠模型中评估了DNA疫苗作为免疫原对抗牙龈卟啉单胞菌攻击的效果。每周将DNA疫苗注射到四头肌中，总共接种四次。免疫后，血清中特异性IgG抗体明显诱导。与对照动物相比，DNA疫苗的免疫对牙龈卟啉单胞菌致死性攻击具有显著的保护作用：这些结果表明KGP作为血红蛋白结合蛋白，也可以作为免疫原诱导对牙龈卟啉单胞菌感染的保护性反应

项目成果

Masse Kuboniwa: "Specific antibodies to Porphyromonas gingivalis Lys-gingipain by DNA vaccination inhibit bacterial binding to hemoglobin and protect mice from infection"Infection and Immunity. 69. 2972-2979 (2001)

Masse Kuboniwa：“通过 DNA 疫苗接种针对牙龈卟啉单胞菌 Lys-gingipain 的特异性抗体可抑制细菌与血红蛋白的结合，并保护小鼠免受感染”感染和免疫。

Masae Kuboniwa: "Specific Autibodies to Porphyromonas gingivalis Lys-Gingipain by DNA Vaccination Inhibit Bacterial Binding to Hemoglobin and Protect Mice from Infection"Infection and Immunity. 69・5(in press). (2001)

Masae Kuboniwa：“通过 DNA 疫苗接种对牙龈卟啉单胞菌 Lys-Gingipain 的特异性抗体抑制细菌与血红蛋白的结合并保护小鼠免受感染”感染和免疫（2001 年）。

Hideki Nagata: "Characterization of hemoglobin binding to Actinobacillus actinomycetemcomitans"Anaerobe. 8. 109-114 (2002)

Hideki Nagata：“血红蛋白与放线杆菌伴放线杆菌结合的特征”厌氧菌。

Masae Kuboniwa: "Specific antibodies to Porphyromonas gingivalis Lys-gingipain by DNA vaccination inhibit bacterial binding to hemoglobin and protect mice from infection"Infection and Immunity. 69. 2972-2979 (2001)

Masae Kuboniwa：“通过 DNA 疫苗接种针对牙龈卟啉单胞菌 Lys-gingipain 的特异性抗体可抑制细菌与血红蛋白的结合，并保护小鼠免受感染”感染和免疫。

Miki Ojima: "Relation of motility of subgingival microflora as a clinical parameter to periodontal disease status in human subjects"Journal of clinical Periodontology. 27. 405-410 (2000)

Miki Ojima：“作为临床参数的龈下微生物群运动与人类受试者牙周疾病状态的关系”临床牙周病学杂志。