Studies on hemolysin of Anginosus group streptococci isolated from the human oral cavity

人口腔分离的心绞痛群链球菌溶血素的研究

• 批准号: 11671826
• 负责人: NAKAMURA Takeshi
• 金额: $ 1.22万
• 依托单位: Matsumoto Dental University, School of Dentistry
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671826/
• 关键词: Oral streptococci; Anginosus group; S.anginosus; Hemolysin; Cytokine; 精製

Total 15 β hemolytic streptococcal strains were isolated from subgingival plaque samples (5 from patients with adult periodontal disease and 10 from healthy adults). Of these strains, 13 strains exhibited streptogram code of 0110434, indicating that they have close properties of reference strain of Streptococcus anginosus NCTC 10713. Furthermore, 12 strains out of the 13 ones reacted with anti G group streptococcal serum in slide latex agglutination test of Strept LA.These observations suggest that the isolated β hemolytic microorganisms are identical to Streptococcus anginosus which are considered to distribute widely in the oral cavity. The hemolysin of this strain was detected in the extracellular culture fluids and the much more production was observed in general anaerobic medium (GAM) than in brain heart infusion or Todd Hewitt broth.Purification of the hemolysin in the culture supernatant was carried out by concentration by ammonium sulfate (60% saturation), ion-exchange chromatography, gel filtration, and hydroxyapatite chromatography by 625 fold with the recovery of 33.6%. The purified hemolysin formed diffuse bands between 50 to 60 kDa in SDS-PAGE.Isoelectric point was 4.2 and it was inactivated by heating at 70℃ for 10 min. The optimum pH for hemolysis was 7.5. The activity was enhanced 2-3 fold by 2 mercaptoethanol or dithiothreitol. The hemolytic spectrum of erythrocytes was found to be wide ; human, bovine, horse, sheep, guinea pig, and rabbit erythrocytes were lyzed at almost the same degree by this hemolysin. The other properties were quite similar to those of S.anginosus NCTC 10713.Both purified hemolysin samples from the isolated strain and reference strain (NCTC 10713) induced the production of TNF-α and IL-6 by peritoneal macrophages of BALB/c mice.

从龈下菌斑标本中分离到15株β溶血性链球菌，其中5株来自成人牙周病患者，10株来自健康成人。在这些菌株中，有13株的链菌谱编码为0110434，表明它们具有与血管紧张链球菌NCTC 10713参考菌株相近的特性。此外，13株β中有12株在玻片乳胶凝集试验中与抗G群链球菌血清反应，这些观察结果表明，分离到的溶血微生物与广泛分布于口腔的血管紧张素链球菌相同。在细胞外培养液中检测到该菌株的溶血素，在普通厌氧培养基(GAM)中的溶血素产量远高于脑心输液和Todd Hewitt发酵液，经60%饱和硫酸铵浓缩、离子交换层析、凝胶过滤和羟基磷灰石层析纯化625倍，回收率为33.6%。纯化后的溶血素在50~60 kDa之间形成弥散带，等电点为4.2，在70℃加热10min后失活。溶血的最适pH为7.5。2-巯基乙醇或二硫苏糖醇可使酶活提高2-3倍。该溶血素对人、牛、马、羊、豚鼠和兔红细胞的裂解程度几乎相同。分离株和参比株(NCTC 10713)的纯化溶血素均能诱导小鼠巨噬细胞产生肿瘤坏死因子α和IL 6。