CHARACTERIZATION OF THE HEMOLYTIC-ACTIVITY OF STREPTOCOCCUS-EQUI

The haemolytic activity of Streptococcus equi, the cause of equine str angles, was characterized. Production of haemolysin in Todd Hewitt bro th was dependent on an equine serum supplement and the logarithmic pha se of growth after which activity declined sharply. RNA core also indu ced haemolysin production from cells harvested at the end of the logar ithmic Phase of growth. Haemolysis was not affected by cholesterol, wa s only slightly increased in reducing conditions and was completely in activated by trypan blue, identifying the haemolytic activity as strep tolysin S-like (SLS-like). Purification by hydroxyapatite and Sephacry l column chromatography yielded proteins of molecular weights of appro ximately 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 Tn91 6. Southern bits of HindIII digests of DNA revealed that one of the mu tants contained three transposon insertions and the other just one. A lambda phage library of S. equi contained plaques whose haemolytic act ivity was enhanced by reducing conditions and inhibited by cholesterol , suggesting a streptolysin O-like (SLO-like) activity. However, haemo lysin in culture sonicates of host E. coli in which the lambda phage i nsert was subcloned into plasmid (pUC18), was not affected by these co nditions. Seven isolates of S. equi in medium without SLS-like inducer s showed no SLO-like activity and no evidence for an SLO-like toxin co uld be found by immunoblotting with pneumolysin antiserum and monoclon al antibodies or by polymerase chain reaction with primers derived fro m sequences conserved between the SLO genes of Lancefield group A, C a nd G streptococci. S. equi does not appear to possess a streptolysin O but does make a streptolysin S-like toxin whose production can be int errupted at just one genetic locus. (C) 1998 Academic Press Limited.