Study On The Inhibition Of Streptococcus Suis Biofilm Formation By Tylosin Targeting CysM

Streptococcus suis(S.suis)is an important zoonotic pathogen,which is a serious hazard to the health and safety of humans and pigs.S.suis can form biofilm(BF).The bacteria forming biofilm have a high degree of drug resistance and can evade the attack of the immune system,making the infection unhealed for a long time.Hence,the study on the regulatory mechanism and the search for new drug targets of S.suis biofilm have become one of the focus of scientific researchers at home and abroad.Cysteine synthase(CysM),as one of the key enzymes in the cysteine biosynthesis pathway,does not exist in mammalian cells,so it is considered as a potential drug target.Tylosin,as one of the macrolide drugs,is widely used in veterinary clinics.Early studies have shown that tylosin can interfere with the biofilm formation of S.suis,but whether CysM is the target has not been reported yet.Therefore,this study reveals the intervention effect of tylosin on the biofilm of S.suis by exploring CysM as the target,and clarifies the mechanism of tylosin interfering with the biofilm formation of S.suis.The specific research results are as follows:(1)Using crystal violet staining and scanning electron microscopy to study the biofilm formation ability of S.suis,the results showed that compared with tylosin-resistant S.suis,the biofilm formation and formation ability of the cysM deletion strain was significantly reduced.However,although the amount and ability of biofilm formation of the cysM complement strain was partially restored,it did not return to the level before knockout.There are still significant differences,indicating that the cysM gene plays a key part in the biofilm formation of S.suis;Tylosin with 1/4 MIC(80 ?g/m L)can significantly interfere with the biofilm formation of tylosin-resistant S.suis,cysM deletion strain and cysM complement strain,and destroy the three-dimensional structure of the biofilm.Tylosin has the ability to interfere with the biofilm formation of S.suis.(2)The extracellular polysaccharides,extracellular DNA and protein in the biofilm matrix were quantitated by using the Congo red binding method,phenol-chloroform method and Bradford method.The results showed that compared with tylosin-resistant S.suis,the content of EPS,e DNA and protein in the cysM deletion strain were significantly reduced.The content of EPS and protein in the cysM complement strain recovered to the level before knockout,while the e DNA of the cysM complement strain recovered partially,but did not return to the level before knockout,there is still a significant difference;1/4 MIC(80 ?g/m L)of tylosin can significantly reduce the content of EPS,e DNA and protein in tylosin-resistant S.suis and cysM complement strain.It can significantly reduce the content of EPS and e DNA in the cysM deletion strain,but has no significant effect on the protein content of the cysM deletion strain.(3)Real-time fluorescent quantitative PCR and Westren blot were used to reveal the regulation of tylosin on CysM.The results showed that tylosin can significantly down-regulate the transcription level of cysM gene and the expression of CysM protein.At the same time,although the biofilm capacity of the cysM deletion strain was significantly reduced,tylosin still interferes with the formation of the biofilm of the cysM deletion strain.The results showed that although tylosin regulates CysM and interferes with the biofilm formation,there may also be other mechanisms.(4)Tylosin-resistant S.suis,cysM deletion strain and cysM complement strain were used to determine the content of intermediate metabolites in the cysteine biosynthetic pathway,related gene expression and enzyme activities.The results showed that compared with tylosin-resistant S.suis,the content of cysteine,homocysteine and S-adenosylmethionine in the cysM deletion strain was significantly reduced,and the cysM gene was supplemented.The content of cysteine,homocysteine and S-adenosylmethionine in the strains all recovered to the level before knockout;Tylosin with 1/4 MIC(80 ?g/m L)can be significantly reduced.The content of cysteine,homocysteine and S-adenosylmethionine in tylosin-resistant S.suis,cysM deletion strain and cysM complement strain.Compared with tylosin-resistant S.suis,the expression of cys E,met I,met K,met E,mtn N and lux S genes in the cysM deletion strain was significantly reduced,while in the complement strain,there is no significant difference in the expression of met I and met E genes.Although the gene expression levels of cys E,met K,mtn N and lux S have been partially restored,they are still significantly different from the gene expression levels before knockout;Tylosin with1/4 MIC(80 ?g/m L)can significantly reduce the expression of cys E,cysM,met I,met K,met E,mtn N and lux S genes in tylosin-resistant S.suis.At the same time,tylosin with 1/4 MIC(80 ?g/m L)can also significantly reduce the enzyme activities of Cys E,CysM and Mcc B of Tylosin-resistant S.suis.The results indicate that tylosin has a regulatory effect on CysM protein and cysteine synthesis pathway.(5)Based on BLI technology,enzyme activity analysis and FT-IR analysis,it is clarified whether tylosin can bind to CysM protein to interfere with the biofilm formation of S.suis.The results showed that tylosin can directly bind to CysM protein,inhibit the activity of CysM,affect the composition and content of the secondary structure of CysM protein,and change its conformation.In summary,from the perspective of the CysM target,this project reveals the mechanism of tylosin's intervention in the biofilm formation of S.suis from the two levels of regulation and direct binding.The problem lays the foundation and provides new ideas for the development of CysM inhibitors to control bacterial biofilm infections.