Characterization of the haemolytic activity of Streptococcus equi.

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.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  I. Ofek,et al.  Insertional inactivation of streptolysin S expression is associated with altered riboflavin metabolism in Streptococcus pyogenes. , 1997, Microbial pathogenesis.

[3]  P. Andrew,et al.  Streptococcus pneumoniae produces a second haemolysin that is distinct from pneumolysin. , 1997, Microbial pathogenesis.

[4]  J. R. de los Toyos,et al.  Functional analysis of pneumolysin by use of monoclonal antibodies , 1996, Infection and immunity.

[5]  J. Timoney,et al.  The protective M proteins of the equine group C streptococci. , 1993, Veterinary microbiology.

[6]  S. Michalek,et al.  Cloning of a locus involved in Streptococcus mutans intracellular polysaccharide accumulation and virulence testing of an intracellular polysaccharide-deficient mutant , 1992, Infection and immunity.

[7]  J. Timoney.  Protecting against 'strangles': a contemporary view. , 1988, Equine veterinary journal.

[8]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[9]  P. Quie,et al.  DNA fingerprinting of Streptococcus zooepidemicus (Lancefield group C) as an aid to epidemiological study. , 1987, The Journal of infectious diseases.

[10]  J. Alouf,et al.  Purification of RNA-core induced streptolysin S, and isolation and haemolytic characteristics of the carrier-free toxin. , 1986, Journal of general microbiology.

[11]  J. Timoney,et al.  Serum bactericidal responses to Streptococcus equi of horses following infection or vaccination. , 1985, Equine veterinary journal.

[12]  G. Dunny,et al.  Development of a system for genetic and molecular analysis of Streptococcus agalactiae. , 1985, Research in veterinary science.

[13]  K. Timmis,et al.  Cloning and expression in Escherichia coli of the streptolysin O determinant from Streptococcus pyogenes: characterization of the cloned streptolysin O determinant and demonstration of the absence of substantial homology with determinants of other thiol-activated toxins , 1984, Infection and immunity.

[14]  P. Cleary,et al.  Insertional inactivation of streptolysin S expression in Streptococcus pyogenes , 1983, Journal of bacteriology.

[15]  C. Lai,et al.  The role of protease in streptolysin S formation. , 1983, Archives of biochemistry and biophysics.

[16]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[17]  C. Lai,et al.  Streptolysin S: improved purification and characterization. , 1978, Archives of biochemistry and biophysics.

[18]  P. Bazeley.  STUDIES WITH EQUINE STBEPTOCOCCI. 5.: SOME RELATIONS BETWEEN VIRULENCE OF Streptococcus Equi AND IMMUNE BESPONSE IN THE HOST , 1943 .

[19]  P. Bazeley.  STUDIES WITH EQUINE STREPTOCOCCI, 3.: VACCINATION AGAINST STRANGLES. , 1942 .

[20]  P. Bazeley.  Studies with Equine Streptococci. 2. Experimental Immunity to Str. equi. , 1940 .

[21]  T. Tanaka,et al.  Cloning and sequencing the streptolysin O genes of group C and group G streptococci. , 1994, DNA sequence : the journal of DNA sequencing and mapping.

[22]  J. Alouf,et al.  Streptococcal toxins (streptolysin O, streptolysin S, erythrogenic toxin). , 1980, Pharmacology & therapeutics.