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

从龈下菌斑中分离出15株β溶血性链球菌（5株来自成人牙周病患者，10株来自健康成人）。其中13株链球菌的链图编码为0110434，表明它们与血管链球菌NCTC 10713的参考菌株具有相近的性质。此外，13株菌株中有12株在LA链球菌滑片胶乳凝集试验中与抗G组链球菌血清反应。这些观察结果表明，分离的β溶血微生物与广泛分布于口腔的血管链球菌相同。在细胞外培养液中检测到该菌株的溶血素，在一般厌氧培养基（GAM）中观察到的溶血素比在脑心输注或托德休伊特肉汤中观察到的要多。对培养上清溶血素采用硫酸铵（60%饱和度）浓缩、离子交换层析、凝胶过滤、羟基磷灰石层析等方法进行625倍的纯化，回收率为33.6%。纯化的溶血素在SDS-PAGE上形成50 ~ 60kda的弥漫性条带。等电点为4.2，经70℃加热10min灭活。溶血的最适pH为7.5。巯基乙醇和二硫苏糖醇可使活性提高2 ~ 3倍。红细胞溶血谱较宽；人、牛、马、羊、豚鼠和兔的红细胞被溶血素溶解的程度几乎相同。其他性状与s.a anginosus NCTC 10713非常相似。从分离菌株和参考菌株（NCTC 10713）中纯化的溶血素样品均可诱导BALB/c小鼠腹腔巨噬细胞产生TNF-α和IL-6。

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.