Identification of &beta;-haemolysin-encoding genes in <em>Streptococcus anginosus.</em>

Streptococcus anginosus is an emerging pathogen, but little is known about its virulence factors. To detect the genes responsible for &beta;-haemolysis we performed genomic mutagenesis of the &beta;-haemolytic S.&nbsp;anginosus type strain ATCC 12395 using the vector pGhost9:ISS1. Integration site analysis of 15 non-haemolytic mutants identified a gene cluster with high homology to the genes of the streptolysin S (SLS) encoding sag gene cluster of S.&nbsp;pyogenes. The gene cluster harbours 10 open reading frames displaying significant similarities to the S.&nbsp;pyogenes genes sagA-sagI, with the identities on protein level ranging from 38 to 87%. Complementation assays of S.&nbsp;anginosus sagB and sagD integration mutants with the respective genes confirmed their importance for &beta;-haemolysin production and suggest the presence of post-translational modifications in S.&nbsp;anginosus SLS similar to SLS of S.&nbsp;pyogenes. Characterization of the S.&nbsp;anginosus haemolysin in comparison to the S.&nbsp;pyogenes SLS showed that the haemolysin is surface bound, but in contrast to S.&nbsp;pyogenes neither fetal calf serum nor RNA was able to stabilize the haemolysin of S.&nbsp;anginosus in culture supernatants. Inhibition of &beta;-haemolysis by polyethylene glycol of different sizes was carried out, giving no evidence of a pore-forming haemolytic mechanism. Analysis of a whole genome shotgun sequence of Streptococcus constellatus, a closely related streptococcal species that belongs to the S.&nbsp;anginosus group, revealed a similar sag gene cluster. Employing a genomic mutagenesis strategy we were able to determine an SLS encoding gene cluster in S.&nbsp;anginosus and demonstrate its importance for &beta;-haemolysin production in S.&nbsp;anginosus