Analysis of the virulence factors from periodontopathic bacteria

牙周病菌毒力因子分析

• 批准号: 08672095
• 负责人: KATO Tetsuo
• 金额: $ 1.66万
• 依托单位: TOKYO DENTAL COLLEGE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672095/
• 关键词: periodontitis; cytokines; Actinobacillus actinomycetemcomitans; Porphyromonas gingivalis; hemolytic activity; cell surface polysaccharide; endotoxin; monoclonal antibody; Actinobacillus actinomycelemcomitans; 菌体表層多糖; 外膜タンパク; 溶血素; 遺伝子クローニング; 歯

Actinobacillus actinomycetemcomitans, a gram-negative capnophlic rod, has been implicated in human periodontal diseases. This microorganism produces a number of virulence factors. Recently several investigators have been reported that strains of A.actinomycetemcomitans express hemolytic activity. In order to examine the potential role of the. hemolytic activity in the process of periodontal diseases, we have isolated a hemolytic gene from strain ATCC 43718. DNA hybridization analysis indicates that the hemolytic gene is conserved among all A.actinomycetemcomitans strains tested. The. complete nucleotide sequence of the gene has been determined. The deduced amino acid sequence. corresponds to a basic protein of 9.5 kDa. Sonicated extract from Escherichia coli clone possessed hemolytic activity on horse, sheep and human erythrocytes but not those of rabbit. To determine whether the antiserum against A.actinomycetemcomitans reacted with a protein of the sonic extract of the E.coli clone w … More as analyzed by Western blot analysis. Rabbit antiserum to A.actinomycetemcomitans ATCC 43718 whole cells reacted with sonic extract of E.coli clone at Ca. lOkDa which was dependent upon insert DNA of A.Actinobacillus actinomycetemcomitans wornztans on the plasmid. Also the rabbit antiserum against ATCC 43718 inhibited the hemolytic activity of the clone. These results indicate that a gene coding hemolytic activity has been isolated from Actinobacillus actinomycetemcomitans.Two polysaccharide fractions, A and B, were prepared from autoclaved extract of A.actinomycetemcomitans ATCC 43718 (serotype. b) by DEAB anion-exchange chromatography. LPS was extracted by hot phenol- water procedure. Ouchterlony analysis demonstrated that LPS and fraction A reacted with rabbit antisera against A.actinomycetemcomitans serotypes a and b but not with antiserum to serotype c. Fraction B was found to react with antiserum to serotype b but not with antisera specific for other serotypes, suggesting that fraction consists of a serotype b specific polysaccharide antigen.Porphyromanas gingivalis, a gram-negative anaerobic rod, has been implicated as a periodontal pathogen. Interleukin-6 (IL-6) is a multifunctional cytokine that is produced by numerous cell types, including macrophages, fibroblasts and endothelial cells, and play a central role in host defense mechanisms. In the present study, we investigated the effect of cell surface components of P.gingivalis on IL-6 production by human gingival fibroblasts. The release of IL-6 by these cells was measured using ELISA systems specific for IL-6 according to the manufacture's recommended procedures. Cell surface polysacebaride and outer membrane proteins upregulated the IL-6 production by human gingival fibroblast cell lines as well as LPS.These activities were dependent on the increase of the preparations dose. Less

伴放线放线杆菌是一种革兰氏阴性嗜二氧化碳杆菌，与人类牙周疾病有关。这种微生物产生许多毒力因子。最近有研究者报道伴放线菌菌株具有溶血活性。为了研究的潜在作用。溶血活性在牙周病的过程中，我们已经从菌株ATCC 43718中分离出溶血基因。DNA杂交分析表明，溶血基因在所有供试的伴放线菌菌株中是保守的。的.该基因的完整核苷酸序列已经确定。推导的氨基酸序列。对应于9.5kDa的碱性蛋白。大肠杆菌克隆的超声提取物对马、羊和人红细胞有溶血活性，但对兔红细胞无溶血活性。为了确定抗伴放线菌的抗血清是否与大肠杆菌克隆的声波提取物的蛋白质反应， ...更多信息 如通过蛋白质印迹分析所分析的。兔抗伴放线菌ATCC 43718全细胞抗血清与大肠杆菌克隆的声波提取物在Ca. 10 kDa，依赖于伴放线放线杆菌的插入DNA。抗ATCC 43718的兔抗血清也抑制克隆的溶血活性。从伴放线放线杆菌ATCC 43718（血清型）的高压灭菌提取物中制备了两个多糖组分A和B。B）通过DEAB阴离子交换色谱法。采用热酚-水法提取LPS. Ouchterlony分析表明，LPS和组分A与抗伴放线菌血清型a和B的兔抗血清反应，但与抗血清型c的抗血清不反应。部分B被发现与血清型B的抗血清反应，但不与其他血清型的特异性抗血清反应，表明该部分由血清型B特异性多糖抗原组成。牙龈卟啉瘤菌，一种革兰氏阴性厌氧杆菌，被认为是牙周病原体。白细胞介素-6（IL-6）是一种多功能细胞因子，由多种细胞类型（包括巨噬细胞、成纤维细胞和内皮细胞）产生，并在宿主防御机制中发挥核心作用。在本研究中，我们研究了牙龈卟啉单胞菌细胞表面成分对人牙龈成纤维细胞产生IL-6的影响。根据制造商推荐的程序，使用IL-6特异性ELISA系统测量这些细胞释放的IL-6。细胞表面多糖和外膜蛋白上调人牙龈成纤维细胞系和脂多糖产生IL-6，这些活性依赖于制剂剂量的增加。少

项目成果

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Saeki, Y.: "Inhibitory effects of funoran on the adherence and colonization of mutans streptococci" Caries Res.30. 119-125 (1996)

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Honma, K.: "Genetic control of immune responses to a synthetic fimbrial antigen of Actinobacillus actinomycetem" Microbiol.Immunol.41. 609-614 (1997)

Honma，K.：“放线杆菌合成菌毛抗原免疫反应的遗传控制”Microbiol.Immunol.41。

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Ishihara, K.、Honma, K.、Miura, T.、Kato, T. 和 Okuda, K.：“Actinobacillus actinomycetemcomitans 菌毛相关蛋白 (fap) 基因的克隆和序列分析”微生物 Patho.23。