Prevalence Of S. Aureus In Goats And NLRP3 Inflammasome Activation Induced By TSST-1

Staphylococcus aureus is a gram-positive extracellular bacterium mainly colonized in the skin surface and nasopharynx of human and animals.After infection,it will cause a variety of diseases of human and animals,such as pyopymia,sepsis,mastitis,arthritis,etc.,which seriously threatens public health safety.TSST-1,an important virulence factor of S.aureus,belongs to the family of suprantigen toxins and plays an important role in the pathogenesis of S.aureus.Like other superantigen toxins,TSST-1 has two domains:A and B.It directly crosslink between MHC class II molecules on antigen-presenting cells and T cell receptors?TCRs?bearing specific V?elements and induce high levels of pro-inflammatory cytokine production.A lot of studies have shown that S.aureus can promote the production of preinflammatory cytokines,anti-inflammatory cytokines and chemokines by activating Toll-like receptors in innate immune model receptors and NOD-like receptors,thus triggering the inflammatory response of the body.However,there is no research on the correlation between TSST-1 and host cell inflammatory response.However,there are currently no studies on the correlation between TSST-1 and host cell inflammatory response.In this study,genotyping of S.aureus isolated from the nasal cavity of goats in a certain area of Chongqing showed that the tst1 gene had a higher carrying rate.Based on this,we purified the recombinant TSST-1 toxin by prokaryotic expression.After infecting mouse peritoneal macrophages with TSST-1,we used fluorescence quantitative RT-PCR,ELISA and Western Blot to study the maturation and secretion mechanism of IL-1?in the model of TSST-1 infected macrophages.The experimental results are as follows:1.To investigate the epidemic characteristics and drug resistance of S.aureus from goats in ChongqingIn this study,167 goat nassal swabs from farms in Dazu,Yongchuan and Shizhu areas of Chongqing were taken to S.aureus isolating and identifying,suprantigen toxin gene testing,genotyping and drug resistance analysis.Fourty-five strains of S.aureus were detected,with a total detection rate of 26.9%?45/167?.Dazu has the highest detection rate of 29.8%?26/45?.Twenty-seven different MT types were obtained from MLVA molecular typing,with the maximum Simpson index of 0.8707.A cladogram drawn from MLVA results revealed that the isolates in the same region were highly similar.The results of supra-antigen toxin gene testing showed that sel and tst1 had the highest detection rate?20%,9/45?,followed by sec?17.8%,8/45?.Eight different gene types were observed in the genotyping results of superantigenic toxin,of which the sec-sel-tst1 type was the most,accounting for 8.9%?4/45?.The drug resistance rate of S.aureus to penicillin in this study was the highest,up to 71.11%?32/45?,followed by cotrimoxazole31.11%?14/45?.One strain was resistant to more than three antibiotics,and the multiple resistance rate was 2.2%.2.The inflammatory effect of the TSST-1 on macrophagesThe recombinant expression plasmid,pGEX-6P-1,of TSST-1 was transformed into E.coli BL21,the recombinant TSST-1 protein was expressed and purified successfully.The TSST-1 was used to infect macrophages in the abdominal cavity of WT mice,and the secretion level of IL-1??L-1??TNF?and IL-6 in the infection system was detected by ELISA.The results showed that TSST-1 could significantly induce the secretion of inflammatory cytokines under the action of ATP.It was known that the 135 amino acid of TSST-1 toxin will lose its superantigenic activities when mutated from histidine to alanine.The mutated superantigenic toxin,mTSST-1,was obtained by the same method.The mTSST-1 was used to infect macrophages in the abdominal cavity of WT mice,and the secretion level of inflammatory cytokines in the infection system was detected by ELISA.The results showed that the secretion level of inflammatory cytokines could also be induced under the action of ATP,with no significant difference from that of TSST-1.Above results showed that under the action of ATP,TSST-1 could induce mouse peritoneal macrophages to produce inflammatory cytokines,independent of its superantigen activity.3.The TSST-1 induces activation of NLRP3 inflamemomes in macrophages through the TLR4 pathwayIn order to study the effect of inflammasomes on IL-1?maturation and secretion in TSST-1-infected macrophages,TSST-1+ATP was used to infect peritoneal macrophages in wild type mice and mice with the absence of inflammasome components(Nlrp3^-/-,Caspase-1^-/-,Asc^-/-).The secretion of IL-1?and TNF?inflammatory cytokines were detected by ELISA,and the activation of Caspase-1 after infection was detected by Western Blot.The results showed that the secretion of IL-1?decreased significantly in the peritoneal macrophages from the three kinds of knockout mice after infection.To explore which cell surface receptors are involved in the recognition of TSST-1,TSST-1+ATP was used to infect peritoneal macrophages of WT mice and of mice lacking TLR2and TLR4 components.The results showed that the secretion of IL-1?decreased significantly after TLR4 was knocked out.The results of RT-PCR on the secretion of IL-1?after TSST-1+ATP infection of WT and Tlr4^-/-mouse macrophages also showed that TLR4-mediated signaling pathway was involved in the production of IL-1?.Together,the above results suggest that after TSST-1+ATP infection,TLR4 receptor recognizes the signal and leads to activation of downstream inflammation-related signaling pathway,inducing the activation of NLRP3 inflammome,thereby activating Caspase-1 and cutting pro-IL-1?,making it mature and secreted.