Study On The Regulation Mechanism Of Two-component System PhoBR To The Pathogenicity Of Piscine Streptococcus Agalactiae

Streptococcus agalactice,which is one of the most important pathogenic bacteria in fish culture,is responsible for significant economic losses in the fish farm industry worldwide.Fish with streptococcosis often exhibit typical symptoms and clinical signs,including swimming abnormalities,septicaemia,meningoencephalitis,subcutaneous abscesses in the caudal peduncle region and exophthalmia(“pop-eye”).In the process of the infection,the pathogen could sense and response to extracellular signal through the two component system(TCS),and the regulate the expression of the various genes to complete its pathogenic process.They can sense the environmental phosphate concentration and response to phosphate starvation.The PhoB is a response regulator of PhoR-PhoB two-component regulatory systems and transcriptional activator that binds to DNA and regulates transcription of genes.It has been known that the most genes in the Pho regulon are not only involved in the phosphate uptake and metabolism,but also associated with bacterial biofilm formation and virulence related genes expression.And phoB mutantscan lead to the decrease of bacterial adhesion,colonization and virulence.However,there is no information about the phoB of S.agalactiae and its role in S.agalactiae virulence.In this study,Streptococcus agalactiae strain TOS01,which was isolated from the golden pompano(Trachinotus ovatus Linnaeus),was selected as the parent strain and one phoB deleted strain,named as SA?phoB,was obtained by homologous recombination.In addition,the virulent-related genes regulation of phoB was analyzed through the transcriptome and proteomics,and its regulation function on hemolysin was verifed by lacZ reporter gene and bacterial one hybrid system,which will clarify the role of PhoB in regulation of virulence genes.The main results and conclusions are as follows:1.Characterization and identification of streptococci from Golden pompano in ChinaA total of 9 streptococcal strains,TOS01-TOS09,were isolated from cage-cultured diseased golden pompano in Beihai,Zhanjing and Shenzhen,China between 2012 and 2014.Both physiological and biochemical methods and molecular biological techniques were used to determine that isolates TOS01-05 were S.agalactiae,TOS06-08 werw S.iniae and TOS09 was S.dysgalactiae subsp.dysgalactiae.Pathogenicity assays revealed that S.agalactiae(LD50 was 6.38×104 CFU)was more virulent for golden pompano than S.iniae(LD50 was 1.47×107 CFU)and S.dysgalactiae subsp.dysgalactiae(LD50 was 2.57×106 CFU)when they were challenged by i.p.injection.The results of antibiotic susceptibility showed that all strains were extremely susceptible to cefradine,erythromycin,cefotaxime,which will contribute to the diagnosis and prevention of the streptococcal.2.Construction of the mutant strain SA?phoB and its basic biological characteristicsStreptococcus agalactiae strain TOS01 was selected as the parent strain with high virulent.The upstream and downstream fragments of phoB genes are amplied from TOS01,the erm genes was amplied from plasmid pAT18.Then the PCR amplicons were cloned into the temperature-sensitive Streptococcus-E.coli shuttle vector pSET4 s one by one.One phoB deleted strain(SA?phoB)was obtained by homologous recombination.In addition,the complementation strain(C?phoB)was constructed through cloning the phoB gene into the E.coli/Gram-positive shuttle plasmid pDL276.The research results of biological characteristics of mutant strain show that SA?pho B could propagate stability in vitro with dozens of long chains and smoother surface,but a lower growth rate than the parent strain.The biofilm thickness of SA?phoB was significantly higher than that of TOS01 and C?phoB,but lower hemolysis activity,cell invasion rate,cell adhesion rate and anti-phagocytic rate;The challenge experimentwas used to compare virulence of S.agalactiae TOS01 and S.agalactiae SA?phoB,the results show that SA?phoB was less virulent than TOS01 in golden pompano.The low concentration of the mutant SA?phoB as a live attenuated vaccine could yielded RPS values of 93.1%,which indicating that SA?phoB may be an effective S.agalactiae attenuated live vaccine candidate.3.Regulation of biofilm formation and virulence genes by PhoBFive hundred and twenty-one genes and sixty proteins were separated by RNA-seq and 2-DE.The metabolic pathways participated by the differently expressing genes and their fuctions were analyzed using bioinformatics analysis,revealing that the mutant SA?phoB enhanced the expression of glycolysis-associated genes expression to impove the carbohydrate catabolism and energy supply;reduced synthesis of free amino acid and enhanced metabolism of fatty acid and expression of capsular polysaccharide-related genes to increased biofilm thickness.Among of them,33 virulence related genes and proteinswere regulated by PhoB in Pi-limited condition,which involved in adherence,colonization and invasion of S.agalactiae to host cells,and 12 genes and proteins were related to biofilm formation and the expression levels of the genes encoding capsular polysaccharide were down regulated,indicating that the PhoB of Streptococcus agalactiae was involved in the regulation of bacterial biofilm formation and virulence gene expression.4.Functional identification of PhoB regulation of hemolysin genesThe interaction between PhoB and promoter region of cyl,hemolysin III,hemolysin A and ciaR by construcinglacZ reporter gene vector and bacterial one-hybrid system,revealing that PhoB can be directly combined with the promoter region of hemolysin A and ciaR,but not with the promoter region of cyl and hemolysin III,suggesting that phosphate control of these genes is mediated indirectly by PhoB.Binding of PhoB to the conserved sequence in S.agalactiae was TTGGAGAA(T/G)by constructing 18 bp DNA random fragment library and using bacterial one-hybrid system.