Roles Of Two-Component System RscSR And Capsular Polysaccharide(CPS) In The Pathogenicity Of Streptococcus Agalactiae And Regulatory Analysis Of CrRNA On CPS

Streptococcus agalactiae,or group B streptococcus(GBS),can infect a variety of hosts including humans,mammal and fish.Recently,GBS infections has caused huge economic losses to the aquaculture,especially the tilapia industry.However,the pathogenic mechanism of this bacteria is unclear.CRISPR-Cas system consists of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated(Cas)protein in bacteria and archaea.It can not only provide acquired resistance against exogenous nucleic acid,but also regulate the bacterial virulencevia cleavage of endogenous nucleic acid.crRNAs are mature RNAs formed after cleave the CRISPR precursor transcript via further processing.It can be combined with Cas9 protein to form a Cas9-crRNA complex,and guide DNA cleavage by base pairing.Our previous study found that crRNA mutant strain showed significantly decrease in bacterial virulence.The results of RNA-seq and q RT-PCR proved the significantly increased transcription level of two-component system RscSR and part gene of capsular polysaccharide(CPS)synthesis gene cluster(cps B,cps C and cps J)in ΔcrRNA.In this study,the rscSR and cpsE deletion mutant strains and their corresponding genecomplemented strains were constructed using homologous recombination,furthermore,biological characteristics,cell-based assays and bacterial pathogenicity test were carried out.Then regulation effect of crRNA on CPS was analyzed by promoter activity and m RNA degradation assays.Moreover,the regulation of crRNA on CPS was investigated by allelic replacement of cpsE deletion in crRNA mutant.1 Identification of the two-component system RscSR and its influence on the S.agalactiae stress response and virulenceThe function of a putative two-component system(TCS)RscSR was investigated in piscine S.agalactiae GD201008-001.The 3-D structure and conserved domains of its constituent components Rsc S and Rsc R were analyzed using bioinformatics methods.The rscSR deletion mutant strain ΔrscSR was constructed using homologous recombination.The corresponding gene-complemented strain CΔrscSR was also constructed.The growth curve of bacteria was measured in GD201008-001,ΔrscSR and CΔrscSR strain.Acid tolerance ability,oxidative stress tolerance ability,anti-phagocytosis ability and intracellular survival ability were detected,and cytotoxicity to macrophages and pathogenicity in mice were investigated as well.Rsc S and Rsc R are predicted to be a typical histidine kinase and response regulator,respectively.The inactivation of rscSR caused a substantial decrease in all of the abovementioned indexes in S.agalactiae,and it was restored after complementing the gene.The mice pathogenicity testing showed that mice infected with the wild strain all died within 19 hours,while mice infected the rscSR deletion strain all died within 48 hours.This study suggests RscSR plays an important role in stress response and virulence of S.agalactiae,which will provide useful information for further exploration of pathogenic mechanism of this bacterium.2 Roles of capsular polysaccharide in the pathogenic of S.agalactiae The cpsE deletion mutant stain ΔcpsE was constructed using homologous recombination.The corresponding gene-complemented strain CΔcpsE was also constructed.Inactivation of cpsE resulted in an acapsular phenotype shown by transmission electron micrographs and capsule staining as anticipated.The growth curve of bacteria was measured in GD201008-001,ΔcpsE and CΔcpsE strain.Bacterial surface hydrophobicity,auto-agglutination,surface charge,biofilm formation ability,anti-phagocytosis ability,adhesion and invasion ability were detected,and Dot-Blot and pathogenicity in mice were investigated as well.The inactivation of cpsE caused a substantial increase in auto-agglutination,surface charge,biofilm formation,cell adhesion and invasion ability in S.agalactiae,while bacterial surface charge and anti-phagocytosis ability were decreased,and it was restored after complementing the gene.Dot-Blot assay showed that the signal intensity was siginficantly increased in CPS mutant strain.It suggested that impaired CPS exposed the microbial surface immune-related factors.The mice pathogenicity test showed that mice infected with the wild strain all died within 30 hours,while none of mice infected the cpsE deletion strain died in 48 hours.It indicated that CPS is an essential virulence factor for GBS.The results of bacterial burden analysis indicated that at 20 h postinfection,none colonization by the ΔcpsE strain in the brain,blood and spleen,while lots of bacteria colonization by the wild type strain.It demonstrated that CPS mutation led to a significant reduction in the proliferation ability of GBS in host.This study suggests that CPS inhibites the colonization and invasion ability of GBS to host,but it plays a vital role in the proliferation ability of GBS in host,enhancing the bacterial virulence,and it is an essential virulence factor for GBS.3 Regulation of crRNA on the synthesis of capsular polysaccharide in S.agalactiaeIn order to investigate the regulation mechanism of crRNA on CPS synthesis in Streptococcus agalactiae GD201008-001,the transcription levels of 7 genes(cps A,cps B,cps C,cpsE,cps J,cps K and neu A)from the CPS synthesis gene cluster were detected,and the level of CPS production was quantized using sialic acid assay kit.The results showed that crRNA can negatively regulate CPS synthesis genes and inhibite the level of CPS production on the surface of GBS.In order to further explore the regulatory mechanism of crRNA inhibiting the level of CPS production,the m RNA degradation rates of cps A and cpsE were determined via treatment with rifampicin.The results show that crRNA does not act as guides for targeting and degradation of m RNA,thereby regulating the level of CPS production.The results of β-galactosidase activity proved that crRNA can inhibit the level of CPS production by indirectly suppressing the activity of the promoter Pcps A.In order to explore the role of CPS in the pathogenic mechanism in ΔcrRNA,we generated ΔcrRNA-ΔcpsE by allelic replacement of cpsE deletion in ΔcrRNA and complemented the mutant with the cpsE gene on a plasmid vector to constructe ΔcrRNA-CΔcpsE,then the biofilm formation ability and adhesion and invasion ability on b End.3 cells were detected.The results showed that crRNA can inhibit the synthesis of CPS and enhance its biofilm formation ability and the adhesion and invasion ability to b End.3.This study suggested that crRNA can indirectly negatively regulate the level of CPS production,thereby enhancing the colonization,invasion and biofilm formation ability of Streptococcus agalactiae GD201008-001.In summary,this study investigated the roles of two-component system RscSR and capsular polysaccharide in the pathogenicity of Streptococcus agalactiae and the regulation of crRNA on CPS.It provided useful information and evidences for further exploration of pathogenic mechanism of this bacterium.