Research On The Molecular Mechanism Of Toxic-shock-like Syndrome Induced By Streptococcus Suis Serotype 2-activated Inflammasome

The two large-scale of human S.suis epidemics in China(the first was 25 cases with 14 deaths in Jiangsu in 1998,and the second was 204 cases with 38 deaths in Sichuan in 2005)have raised serious concerns for global public health and have challenged the conventional conception of S.suis infections as sporadic in humans.Elucidation the mechanism of the pathogenicity of S.suis and the interaction between the pathogen and the host made a great significance for prevention from S.suis infection and providing therapy for the disease.Based on a mouse model,this paper reveals the relationship of Streptococcus suis type 2(SS2)-induced STSLS with inflammasome and inflammatory “storms”.1.Activation of inflammasome triggering cytokines storm played a role in STSLS in SS2 infected mice.In the murine model,infection of epidemic Chinese strain SC-19 could induce STSLS,typical characteristic with an acute exaggerated high level of inflammatory cytokines,multiple tissues damage and high mortality.A specific inhibitor of Caspase-1,YVAD-CHO,inhibiting the activation of inflammasomes significantly relieved symptoms associated with the STSLS and improved the mice survival.Further,we blocked IL-1? and IL-18 signal which are the substrate of active caspase-1 and the quintessential pro-inflammatory cytokines released by the pyroptosis cells to verify the role of the inflammasome in SS2 induced-STSLS.The results showed blocking IL-1? did not improve the survival of mice,but the use of IL-18 inhibitory antibody could alleviate the STSLS caused by SC-19 infection.In general,activation of inflammasome had been shown to play a role in the STSLS.2.NLRP3 inflammasome played a role in STSLS in SS2 infected mice.In order to explore whether Streptococcus suis could activate inflammasome and what kind of inflammasome it activated,we reconstructed four major type inflammasomes of NLRP1 b,NLRP3,NLRC4,and AIM2 in 293 T cells.After infection with MOI= 10 of SC-19,it was found that SC-19 mainly activated NLRP3 inflammasome.Then we use CRISPR for NLRP3 Gene knockout.The wild type cells and the NLRP3 deficiency cells were infected with SC-19 under the same conditions.And the results showed SC-19 only activated the wild type cells while ds DNA could activate both.And we found that K+ efflux was the important process of the NLRP3 inflammasome activation.To further elucidate the role of inflammasome activation in STSLS,we used the specific inhibitor of the NLRP3 inflammasome,MCC950,to observe the role of NLRP3 inflammasome activation in the STSLS induced by SC-19.And the results showed that the use of MCC950 could alleviate the symptoms associated with the STSLS including the reduction of inflammatory factors,the alleviation of tissue damage,and the decrease in mortality.It suggested that infection of SC-19 induced the activation of NLRP3 inflammasome promoting the development of STSLS.3.Suilysin secreted by SC-19 activated the NLRP3 inflammasome.In order to investigate the components of SC-19 activating inflammasome,we collected different deletion mutants preserved in our laboratory and constructed a suilysin hemolytic mutants named msly(P353L).Specifically,suilysin of this strain was expressed without hemolysis.Subsequently,PMA-differentiated THP1 were infected with live or heat-killed SC-19,and we found that only live bacteria could activate the inflammasome.Then the cells were infected with different live mutants,only the suilysin deletion mutant and msly(P353L)could not activate the inflammasome.Further to explore the relationship between suilysin and inflammasome activation,we expressed recombinant suilysin and found that the purified recombinant suilysin could activate the inflammasome in a dose-dependent manner.Finally,we added different concentrations of cholesterol,which is the inhibitor of the hemolytic activity of suilysin,and the results showed that cholesterol could dose-dependently inhibit the activation of inflammasome caused by S.suis infection.In conclusion,our results declared that the main component of SC-19 for activating inflammasome was suilysin dependent on the hemolysis.4.The ability of inflammasome activation of SS2 played an important role in STSLS.To further investigate the role of inflammasome activation of SC-19 in STSLS in vivo,we used msly(P353L)and SC-19 to infect mice,respectively.And we found a significantly alleviated STSLS associated syndromes in the mutant-infected group,confirming that inflammasome activation was a key factor in the progress of STSLS.It suggested msly(P353L)induced a reduced risk of STSLS.Due to the reduced virulence of this mutant,we tried to use this strain as an attenuated vaccine and found that one dose immunization could provide complete protection,the mutant msly(P353L)had the potential to become an attenuated vaccine.5.The occurrence of STSLS was markedly related to the higher expression of suilysin.We compared the suilysin expression of the epidemic strain SC-19 and the European virulent strain P1/7 and we found that the expression of suilysin was significantly higher than the epidemic strain,and the degree of inflammasome activation of the different strains was closely related to the expression of suilysin.To explore the impact of suilysin in Streptococcus suis induced STSLS,we constructed a hemolysin expression plasmid and replenished the plasmid into the suilysin-deficient strain and P1/7 strain with lower-expressed suilysin.Our results show that deta-sly replenishing SLY of could express suilysin and regain the virulence,meanwhile over-expressed suilysin could increase the mortality rate of P1/7.These results suggest that the high expression of suilysin in SC-19 played an important role in STSLS.In conclusion,we illustrated that an NLRP3 inflammasome-triggered cytokines storm contributed to STSLS.