| Streptococcus suis is not only a major bacterial pathogen for pigs worldwide,but also an emerging zoonotic agent.In humans and pigs,meningitis is a major manifestations of S.suis infections.At present,the mechanism of meningitis is still unclear.Recent studies indicate that bacterial RNA-binding proteins not only play a key role in the regulation of bacterial small RNAs,but also participate in important biological functions,including bacterial movements,biofilms formation.In this study,intracranial subarachnoidal route of infection was established for investigating S.suis meningitis in a mouse model and MS2 purification technique was used for enriching S.suis RNA binding protein.These findings contribute to better understanding S.suis pathogenesis and provide a new perspective for the prevention and control of the disease caused by S.suis.1 Influence of biofilm on Streptococcus suis meningitis in a mouse modelA suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens.Several routes of infection in mouse have been developed to study the pathogenesis of S.suis infection.However,the intraperitoneal,intranasal,and intravenous routes of infection are not suitable for studying the roles of S.suis surface components in meningitis directly in brain,like extracellular matrix from biofilms.Although the intracisternal inoculation was used for S.suis infection,the precise injection site has not been described.Here,the intracranial subarachnoidal route of infection was described in mouse to investigate the roles of biofilms in S.suis meningitis.S.suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mouse through the injection site located 3.5 mm rostral from the bregma.The histopathological analysis and the increased mRNA expression of TLR2 and cytokines of brain from mice injected with biofilm state cells have clearly indicated that S.suis biofilm plays definitive roles in S.suis meningitis.This route of infection has obvious advantages over other routes of infection,allowing to study the host-bacterium interaction and the effect of bacterial components on host immune responses directly in the brain,and mimic bacterial entrance into the central nervous system.Thus,this route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria.In addition,it can also be used to test the efficacy of drugs against bacterial meningitis.2 MS2 purification technique for enriching Streptococcus suis RNA-binding proteinsBacterial RNA-binding proteins not only play a key role in the regulation of small RNAs,but also participate in the regulation of bacterial virulence.However,the reports on the identification and function of bacterial RNA-binding proteins are rare in gram-positive bacteria.In order to establish an ideal method for screening S.suis RNA-binding protein,S.suis small RNAs rss04 and rss06 were selected.The MS2-MBP that binds to MS2 sequence was purified;the plasmids pSET2-MS2-rss04 and pSET2-MS2-rss06,expressing the fusion RNAs MS2-rss04 and MS2-rss06 respectively,as well as the negative control plasmid pSET2-MS2-negative,were constructed and then electroporated into the S.suis small RNA deletion mutants △rss04,△rss06,and wild strain P1/7,respectively.Using MS2 recognition sequence tag purification technology,RNA and proteins were extracted from eluent.Quantitative real-time PCR results showed that rss04 and rss06 were significantly enriched 16-fold and 110-fold,respectively,in the samples expressing fusion RNAs,whereas no enrichment of rss04 and rss06 was detected in the negative control group.The above results indicate that the MS2 purification system has been successfully established and can be used for screening S.suis RNA-binding proteins.The collected proteins were subjected to high performance liquid chromatography-mass spectrometry analysis.It was found that 50 proteins existed only in the samples expressing the fusion RNA but not in the control samples.The candidate proteins were further analyzed,and 9 proteins contain RNA-binding domains.One of them,DNA binding response regulator,not only has an RNA-binding domain,but also has an OmpR domain associated with bacterial virulence.These results lay the foundation for furthering the function of S.suis small RNA and elucidating the mechanism of small RNA regulation of virulence. |
