Streptococcus Suis Endopeptidase O Enhances Pathogenicity To Mice By Cleaving Cathelicindins

Streptococcus suis(S.suis)belongs to the second class of animal disease pathogens in China,caused meningitis,sepsis,and even death in human and animals.The virulence factors and pathogenic mechanisms are the hot spots and difficult points in the research on S.suis.Cathelicidins are mammalian antimicrobial peptides,which have potent antimicrobial activity and immunomodulatory functions,being considered hope in the post-antibiotic era.The Cathelicidins family antimicrobial peptides have been reported in many organisms,including LL-37 in human and m CRAMP in mice.Previous studies in our laboratory found that S.suis tolerates several common antiresistant drugs,while Cathelicidins have good antimicrobial activity against S.suis.S.suis PepO protease is a member of M13 protein family with potential cleavage capacity though have not reported yet.In this research,the cleavage characteristics of PepO protease towards Cathelicidins is confirmed,and made it as an main line,explored the immune escape mechanisms of S.suis.Firstly,PepO was dentified as an exocrine protein by Western bolt.Then,the restriction properties of PepO on LL-37 and m CRAMP were verified with the help of protein gel electrophoresis.It shows that PepO protease cleaves LL-37 and m CRAMP efficiently,at a molar ratio of 1:33.To S.suis and E.coil,LL-37’s bactericidal concentration was 4 μmol/L and m CRAMP was 8 μmol/L.However,the bactericidal activity of both peptides was lost after PepO protease cleavaged them.Nextly,FR-7,IG-7 and LR-11 were synthesised,instead of digestion of LL-37 and m CRAMP for subsequent experiments.After that,a bacterial viability detection kit was used to compare the bactericidal functions of Cathelicidins LL-37,m CRAMP and the truncated peptide FR-7,IG-7,and LR-11,using a laser confocal microscope.It was found that after S.suis still survived in truncated peptide,means the bactericidal activities of Cathelicidins were impaired after truncation.In consider to the immunoregulatory function of Cathelicidins,chemotactic index(CI)of neutrophils isolated from peripheral human and mice bone marrow in response to LL-37,m CRAMP,FR-7,IG-7,LR-11 were detected.It was found that LL-37 has good chemotaxis for human neutrophils,different from FR-7 and IG-7.The results of m CRAMP are consistent with LL-37.Neutrophil’s death stimulated by TNF-α in present or absence of LL-37,m CRAMP,FR-7,IG-7 or LR-11 was quantified to analyze the effect of the different peptides on neutrophils lifespan.The results found that LL-37 had good apoptotic protection to human and mouse neutrophils,better than m CRAMP,while their truncated peptide FR-7,IG-7 and LR-11 were completely lost such capacity.All results above indicated that PepO protease cleaves host Cathelicidins and impairs its antimicrobial and immune regulatory functions.Finally,the research constructed the S.suis infection model in C57BL/6 mice,confirmed the pathogenic ability of PepO protease.Compared with WT,Δpep O showed less pathological damage,viscera load count,and inflammatory factors in mice,confirmed that PepO protease is an important virulence factor that attenuates host immune defense against pathogens.In conclusion,the research concluded that S.suis PepO protease cleaves Cathelicidins,hindering the antimicrobial and immune regulatory functions of Cathelicidins,which mediates immune escape of the S.suis.This study is innovative and rigorous,which provides new ideas and scientific experimental basis for subsequent bacterial immune escape research.