Research On Function Of Two-Component Regulatory System VicRK In Streptococcus Suis Serotype 2

Streptococcus suis?S.suis?is an important zoonotic pathogen.It can cause huge economic losses,and seriously affect human public safety.Based on capsular polysaccharides,29 serotypes have been proposed for S.suis.Among them,serotype2 is regarded as the most virulent and frequently isolated serotype.Denmark is the first country been reported infected with S.suis.There are many countries been reported many cases of human S.suis infection in Europe,North America,South America,Oceania and Asia.The two-component regulatory systems?TCSs?play an important role in bacterial growth metabolism and virulence.Bacteria can sense external changes and make important responses by TCSs.The YycG/YycF two-component system was first discovered in Bacillus subtilis and later named WalK/WalR due to its important role in cell wall metabolism.In Streptococcus pneumoniae it was named VicK/VicR.It has been reported that VicRK plays an important role in the chain length,morphology,growth,metabolism,cell wall homeostasis,biofilm,and cytotoxicity of some bacteria,but there is no relevant report in Streptococcus suis.The aim of this study is evaluate the importance of VicRK to S.suis2.The details are as follows:1.Construction and biological characterization of the vicK gene deletion mutantThe vicK gene was successfully deleted in S.suis 2,and the complementation strain C?vicK was constructed by electroporation on the basis of the?vicK mutant.Gram staining was used to study the differences in chain length and morphology among wild strain,mutant strain?vicK and complement strain C?vicK.The results showed that there is no different in the chain length and morphology,indicating that the vicK gene has no effect on the chain length and shape to SS2.When strains grew in fresh medium?TSB with 10%newborn bovine seru?at a static condition there is no different with them.When they grew under the following oxidative stress conditions:vigorous shaking?high oxygen tension?,medium containing 0.5 mmol/L H₂O₂,0.5mmol/L diamide,or 2 mmol/L paraquat,there is also no different among them.When they grew in a condition?only have TSB?,difference had been seen,and the growth rate of mutant?vicK was lower than the wild-type and complemented strain.The result show that vicK gene had no effect on SS2 growth except under low nutrient conditions.2.The effect of the vicK gene on pathogenicity to SS2Through the hBMEC?human brain microvascular epithelial cells?adhesion and Hep-2 cells?human laryngeal carcinoma epithelial cells?adhesion and invasion experiment,we find that the adhesion and invasion ability of?vicK was significantly lower than the other two strains.When the three strains incubated the human blood,we find the viability of the?vicK was significantly lower than the wild strain,indicating that vicK plays a role in S.suis evasion of innate immune.In the competition experiment of piglets,we found that the colonization ability of?vicK in various tissues was lower than the wild strain SC19.The result suggest that virulence of?vicK was significantly attenuated.To investigate the role of VicRK in regulating the virulence of SS2,we performed RNA-seq experiments and verified the accuracy of the data.Through the RNA-seq data we found nine potential virulence genes.The EMSA?gel electrophoresis experiments?demonstrated that these virulence genes are regulated by the VicRK TCS.