Regulation Of Streptococcus Suis Biofilm Formation By GGDEF

Streptococcus suis(Streptococcussuis,S.suis)is an important zoonotic pathogen,which can cause meningitis,septicemia,pneumonia,endocarditis and arthritis,causing serious harm to the health of humans and pigs.Biofilm is a self-protection mechanism for bacteria to adapt to external environmental pressure.the formation of biofilm can greatly enhance the tolerance of bacteria to antibiotics and make infection uncured for a long time.Therefore,the research on the regulation mechanism of biofilm has become one of the focuses of researchers at home and abroad,but the specific regulation mechanism of Streptococcus suis biofilm is still not very clear.C-di-GMP is an important second messenger molecule regulating the formation of bacterial biofilm.Previous studies have found that the genome of Streptococcus suis 86-5192 contains a gene GGDEF encoding c-di-GMP cyclase,but its regulatory effect on biofilm formation is not clear.In this study,Streptococcus suis was taken as the research object.Firstly,the protein function of GGDEF was predicted by bioinformatics analysis,and then its effect on the biofilm formation and virulence of Streptococcus suis was confirmed.Finally,the molecular mechanism of Streptococcus suis biofilm formation and virulence was preliminarily explored.In this study,the biofilm forming ability of Streptococcus suis 86-5192 was confirmed by Congo red plate test and crystal violet test.The results showed that strain86-5192 showed typical red,dry and rough rdar colony morphology on Congo red plate,and formed obvious biofilm in liquid medium,indicating that strain 86-5192 had strong biofilm forming ability.Bioinformatics analysis of GGDEF protein of Streptococcus suis showed that it contained a conserved GGDEF domain,suggesting that it is a potential cdi-GMP synthetase.In order to explore the effect of GGDEF on biofilm formation of Streptococcus suis,a gene knockout strain of GGDEF was constructed by homologous recombination.Congo red plate test,crystal violet test and scanning electron microscope observation showed that compared with wild strains,GGDEF deletion down-regulated 86-5192 biofilm formation.Secondly,the overexpression recombinant plasmid of GGDEF gene was constructed.The results of Congo red plate test showed that GGDEF overexpression upregulated the biofilm formation of Salmonella typhimurium UMR1 compared with wild strains.In addition,the results of bacterial motility experiment showed that the overexpression of GGDEF significantly reduced the motility of Salmonella typhimurium UMR1 and Escherichia coli MG1655 compared with wild strains,and the number of flagella on bacterial surface after GGDEF overexpression was significantly lower than that of wild strains.The results of in vivo experiments in mice showed that compared with wild strains,GGDEF deletion significantly reduced the virulence of Streptococcus suis 86-5192 to mice.Finally,the molecular mechanism of GGDEF regulating the formation and virulence of 86-5192 biofilm was explored by q PCR.The results showed that compared with wild strains,GGDEF deletion significantly down-regulated the transcription level of biofilm regulatory genes lux S and virulence factors ef,fbps and sly,and up-regulated the transcription level of gdh.In summary,this study explored the regulatory effect of GGDEF on biofilm formation and virulence of Streptococcus suis 86-5192.It was found that GGDEF inhibited the biofilm formation of Streptococcus suis by down-regulating the transcriptional level of lux S,reduced the virulence of Streptococcus suis by downregulating the transcriptional levels of ef,fbps and sly genes,and inhibited bacterial motility by inhibiting flagella synthesis.This study provides reference and reference for further exploring the regulatory mechanism of GGDEF on the formation of Streptococcus suis biofilm,and also provides a theoretical basis for the effective prevention of Streptococcus suis related diseases.