Study On The Streptolysin O Of Streptococcus Pyogenes

Streptococcus pyogenes is a kind of pathogen which take human as the host strictly,and it exists in human feces and nose pharynx ministry widely. S.pyogenes can cause relatively mild infections including pharyngitis, scarlet fever, impetigo and life-threatening invasive infections, such as Rheumatic fever. Streptolysin O is one of the important toxin streptococcus produced.lt can not only cause hemolysis and aslo be eukaryotic cell membrane damage toxins.SLO plays an important role in inflammation of streptococcus infection.The bioinformatics analysis of Streptococcus pyogenes Streptolysin O showing that SLO contains a Thiol_cytolysin domain which is composed of 461 amino acid, and a transmembrane domain in the amino terminus.In this research, we used pET101-GENE protein expression system to construct the recombinant bacteria which can express Streptolysin O fusion protein. The fusion protein was purified by using Ni-NTA Resins affinity chromatography. The purified recombinant proteins have hemolytic activity. Using the purified recombinant protein as immunogen to immune rats for four times, the antiserum was harvested by centrifugal. The antiserum titer was tested though Elisa and reached 12800. The hemolysins proteins of the culture supernatants in Streptococcus pyogenes, Streptococcus equi and Streptococcus suis were detected by Western Bloting with antiserum. The results showed that the polyclonal antibody can only react with Streptococcus pyogenes Streptolysin O.We construct two isogenic SLO mutants (ATM and AThiol_cytolysin) to investigate the contributions of the SLO different functional structure to the biogenesis of established virulence factors such as SpeB, GAPDH and NADase in S.pyogenes. ATM mutant has a deletion of the N-terminus segment containing a non-cleavable Sec-dependent signal sequence and a transmembrane domain (MT) of SLO. AThiol_cytolysin mutant was an in-frame SLO deletion mutant, in which the Thiol_cytolysin of SLO was knock out. Our results indicated that both of mutant strains losed the ability of hemolysis, and the SLO proteins were not detected by Western blot analysis with with anti-SLO serum in the culture supernatants of two SLO mutants. Analysis of NADase activities showed there is no difference between mutants and wild type. Comparing with the wild strains, the GAPDH activity of ATM and AThiol_cytolysin is 17.3% and 90.5%, respectively. Comparing with the wild strains, analysis of SpeB by SDS-PAGE electrophoresis show that the maturation of SpeB in ATM mutant is more slowly, but faster in AThiol_cytolysin mutant. Cell adhesion experiment shows that less numbers of ATM bacteria bind to cell than that of the wild strain (58%), however more AThiol_cytolysin bacteria wthich adhere to the cell than that of the wild strain (129%). Moreover, the ability of cell permeability is decline in two mutant strains.In conclusion, that SLO transmembrane domain deletion and Thiol_cytolysin deletion could result in losing the ability of hemolysis, reducing the cell damage and decreasing the toxicity in S. pyogenes. The transmembrane domain deletion of SLO delayed the maturation of SpeB and reduced the adhesion to the cell in S. pyogenes.