Effect And Mechanism Of Collagenase-Like Protease In Promoting Meningitis Induced By Streptococcus Suis Type 2

Streptococcus suis serotype 2(SS2)is an important zoonotic pathogen that causes tremendous economic losses to the pig industry and seriously threatens public health security.SS2 infection can cause several symptoms such as meningitis,endocarditis,arthritis,and sepsis,among which meningitis is the most severe disease,however,the mechanism of pathogen-host interaction to induce meningitis is unclear.In our previous work,through the interaction between the SS2 genome-wide phage display library and the Blood-Brain Barrier(BBB)model in vitro,14 virulence factors that may play a role in SS2 destruction of BBB were screened,one of which was Collagenase-like protease(Clp).This protein was identified as the putative collagenase of the U32 peptidase family,and the roles of Clp in SS2 breakthrough BBB to induce meningitis have not been reported,so the current study was carried out on the effect and mechanism of Clp in the occurrence of SS2 meningitis:1.Through prokaryotic expression,the high-purity and soluble SS2 recombinant protein r Clp was successfully induced to be purified.In addition,the Clp gene deletion strain ΔClp and its supplementary strain CΔClp of SS2 SC19 were constructed by homologous recombination technology.The growth rate measurement and bacterial morphology observation showed that compared with the wild strains,the deletion strain had shorter chain length and there was no change in growth rate.2.To elucidate the role of Clp in the invasion of BBB by SS2 infection,the differences in virulence and tissue colonization between wild and mutation strains were compared using mouse infection models.It was found that the deletion of the Clp gene significantly increased the survival rate of mice,reduced the colonization ability of bacteria in the blood and various tissues and pathological damage to the brains and lungs.The h CMEC/D3 monolayer BBB model was constructed in vitro,and the determination of cell Trans epithelial electric resistance(TEER)showed that the recombinant protein r Clp could significantly increase the permeability of the monolayer barrier.After the deletion of the Clp gene,the destructive effect of SS2 on the monolayer barrier decreased significantly.The Evans blue(EB)content and the bacterial amount of the crossing model also showed the similar results.In addition,experiments in mice found that after the deletion of the Clp gene,the EB content of SS2-infected mouse brain decreased significantly,while the pretreatment of r Clp increased the content of EB in the brain after SS2 infection.3.To explore the mechanism of Clp destroying BBB,this study found that Clp could significantly promote the adhesion of SS2 to h CMEC/D3 through bacterial adhesion experiments.WB detected the expression of intercellular tight junction on h CMEC/D3 after treated with recombinant protein r Clp and ΔClp strain,and found that Clp could significantly inhibit the expression of intercellular tight junction proteins ZO-1 and Occludin.In addition,the apoptosis of on h CMEC/D3 stimulated by recombinant protein r Clp and ΔClp strains was analyzed by RT-PCR,flow cytometry,and WB,and it was found that Clp may induce apoptosis of h CMEC/D3 through mitochondrial apoptosis pathway and cell receptor ligand apoptosis pathway,and these two pathways were carried out independently.4.Through pull down,mass spectrometry,flow cytometry,and other techniques,the protein of h CMEC/D3 interacting with Clp was identified,and found that PABPC1 and G3BP1 may be their interacting proteins.In summary,our results indicate that Clp is a new candidate virulence protein,which participates in the pathogenic process of SS2,induces apoptosis of cerebral microvascular endothelial cells by promoting bacterial adhesion,and inhibits the expression of tight junctions between cells,thereby destroying BBB and increasing its permeability.Its interacting proteins may be PABPC1 and G3BP1.This study further reveals the pathogenic mechanism of SS2 breakthrough in BBB and induces meningitis,provideing a theoretical basis for better treatment and prevention of SS2-induced meningitis.