Flanagan J, Collin N, Timoney J, Mitchell T, Mumford J A, Chanter N
Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU, U.K.
Microb Pathog. 1998 Apr;24(4):211-21. doi: 10.1006/mpat.1997.0190.
The haemolytic activity of Streptococcus equi, the cause of equine strangles, was characterized. Production of haemolysin in Todd Hewitt broth was dependent on an equine serum supplement and the logarithmic phase of growth after which activity declined sharply. RNA core also induced haemolysin production from cells harvested at the end of the logarithmic phase of growth. Haemolysis was not affected by cholesterol, was only slightly increased in reducing conditions and was completely inactivated by trypan blue, identifying the haemolytic activity as streptolysin S-like (SLS-like). Purification by hydroxyapatite and Sephacryl column chromatography yielded proteins of molecular weights of approximately 6000 and 17 000-22 000 Da with a 64-fold increase in specific activity. Low molecular weight proteins from the RNA core were still present in the purified toxin. Two non-haemolytic mutants were derived by conjugation with an Enterococcus faecalis-carrying transposon Tn916. Southern blots of HindIII digests of DNA revealed that one of the mutants contained three transposon insertions and the other just one. A lambda phage library of S. equi contained plaques whose haemolytic activity was enhanced by reducing conditions and inhibited by cholesterol, suggesting a streptolysin O-like (SLO-like) activity. However, haemolysin in culture sonicates of host E. coli in which the lambda phage insert was subcloned into plasmid (pUC18), was not affected by these conditions. Seven isolates of S. equi in medium without SLS-like inducers showed no SLO-like activity and no evidence for an SLO-like toxin could be found by immunoblotting with pneumolysin antiserum and monoclonal antibodies or by polymerase chain reaction with primers derived from sequences conserved between the SLO genes of Lancefield group A, C and G streptococci. S. equi does not appear to possess a streptolysin O but does make a streptolysin S-like toxin whose production can be interrupted at just one genetic locus.
马腺疫的病原体马链球菌的溶血活性得到了表征。在托德-休伊特肉汤中溶血素的产生依赖于马血清补充剂以及生长对数期，此后活性急剧下降。RNA核心也能诱导对数生长期末收获的细胞产生溶血素。溶血不受胆固醇影响，在还原条件下仅略有增加，并且被台盼蓝完全灭活，确定该溶血活性为类链球菌溶血素S（SLS样）。通过羟基磷灰石和Sephacryl柱色谱法纯化得到分子量约为6000以及17000 - 22000 Da的蛋白质，比活性提高了64倍。来自RNA核心的低分子量蛋白质仍存在于纯化毒素中。通过与携带转座子Tn916的粪肠球菌进行接合得到了两个非溶血突变体。DNA经HindIII酶切后的Southern印迹显示，其中一个突变体含有三个转座子插入，另一个仅含有一个。马链球菌的λ噬菌体文库包含一些噬菌斑，其溶血活性在还原条件下增强，在胆固醇存在下受到抑制，表明具有类链球菌溶血素O（SLO样）活性。然而，将λ噬菌体插入片段亚克隆到质粒（pUC18）中的宿主大肠杆菌培养超声裂解物中的溶血素不受这些条件影响。在没有SLS样诱导剂的培养基中培养的七株马链球菌分离株未显示出SLO样活性，并且通过用肺炎球菌溶血素抗血清和单克隆抗体进行免疫印迹或用源自兰斯菲尔德A、C和G组链球菌SLO基因之间保守序列的引物进行聚合酶链反应，均未发现SLO样毒素的证据。马链球菌似乎不具有链球菌溶血素O，但确实产生一种类链球菌溶血素S样毒素，其产生可在仅一个基因位点被中断。
