Suilysin-mediated Complement Evasion Of Streptococcus Suis

The complement system consists of series of small proteins which were located in the serum,tissue fluid and cell surface.Over 30 proteins and protein fragments are included in the complement system,namely serum proteins,serosal proteins and cell membrane receptors.The complement system,which represents as a chief component of innate immunity,not only participates in inflammation,but also enhances the adaptive immune response.The complement system could identify pathogenic microorganisms quickly and remove the invading pathogens by effector cells,however there are increasing immune escape mechanisms of pathogenic microorganisms.Streptococcus suis(S.suis)is an important zoonotic pathogen which has 33 serotypes and the serotype 2(SS2)is the most virulent serotype.S.suis can survive in animal blood or tissues for a long time through escaping the complement system and the mechanism is unclear.Complement factors C3a and C5a are important chemokines and are involving in inflammation and phagocytosis.In this research,we found that several C3aR-and C5aR-binding proteins,scuh as suilysin(SLY).In addition,SLY could inhibite C3a and C5a through competitivly binding with their receptors,thus SLY could inhibit the chemotaxis caused by C3a and C5a.1.Identification of C3aR-and C5aR-binding proteins from SS2 secreted and membrane proteins:We have screened more than one C3a and C5a receptor-binding proteins with the bacterial two-hybrid system.SLY is a virulence factor of SS2 which has interactions with both C3aR and C5aR.2.Validation of the interactions of SLY to C3aR and C5aR:The interactions between SLY and C3aR/C5aR have been validated again by the far western blot.3.Confirming the binding site of C3aR/C5aR:The results of ELISA tests showed that the second extracellular loop of C3aR(C3aR_e2)binds to SLY.4.The influence of recombinant protein SLY on chemotaxis:The human monocyte leukemia cells(THP-1)has been selected as the objects of C3a and C5a chemokines tests.Firstly,the expression of C3aR and C5aR on the THP-1 cell surface was detected using flow cytometry;then the THP-1 cells were incubated with SLY at different concentrations,and chemotactic responses were carried out with chemokines C3a and C5a,respectively.In addition,a parallel control group was set to observate the influence of SLY for cell number.When the SLY concentration was less than 1?m,the cell number has unobvious changes,which can ensure the validity of the chemotactic result.With increasing concentrations of SLY,the chemotaxis capacity of C3a and C5a has been suppressed.In a word,SLY can inhibit the chemotaxis capacity of C3a and C5a.5.The influence of the SLY mutant strain for chemotaxis:Comparing the growth curves between the wild strain and the SLY mutant strain,we found that lack of SLY had no obvious effect on the growth of the strains.The wild strain and the mutant strains was cultured in the same inoculation quantity simultaneously,and the supernatants were obtained respectively.The THP-1 cells were incubated with the supernatants,and chemotactic responses were carried out with chemokines C3a and C5a.In addition,a parallel control group was set to observate the influence of the mutant strain and the wild strain.We found that the cell number has no obvious changes when incubated with the supernatants,proving the validity of the chemotactic results.Compared with the mutant strain,the inhibitory effect for C3a and C5a chemokines of the wild strain are more apparent.