Functional Analysis Of LuxS In Infection And Pathogenicity Of Piscine Streptococcus Agalactiae

Streptococcus agalactiae,also known as group B streptococcus(GBS),is an opportunistic Gram-positive pathogen.It has a broad host range of infection and is associated with neonatal meningitis and bacteremia in humans,dairy cattle and fish.S. agalactiae-induced diseases have affected many species of cultivated fish including Nile tilapia.This pathogen causes typical symptoms of streptococcosis in tilapia,including abnormal behavior,meningitis and septicemia,usually is highly contagious and lethal.Quorum sensing(QS)is an intercellular cell-cell communication mechanism that controls the expression of variety genes involved in the adaption and survival in environment and encode virulence factors.The LuxS/AI-2 QS system mediated by autoinducer-2(AI-2)has been identified in both Gram-negative and Gram-positive bacteria.LuxS/AI-2 QS system acts as one of the global regulatory networks in bacteria which response to the number and species of bacteria present in a community and regulate the expression of a number of phenotypes even the behaviors in bacteria.In this study,we investigated the role of the lux S/AI-2 QS system in the pathogenicity and immune evasion of S.agalactiae.Transcriptome analysis,followed by the lux S gene deletion and subsequent functional studies,confirmed that impaired bacterial immune evasion was due to the inactivation of lux S.EMSA and point mutation were used to reveal the regulatory mechanism of lux S during intracellular survival of S.agalactiae,which may give a more thorough understanding of role for lux S in S.agalactiae.1.Detection of LuxS/AI-2 system in S.agalactiae S-ribosylhomocysteine(SRH)lyase(LuxS)mediated QS system employs AI-2 as an intracellular molecule controls many behaviors of bacteria.Comparison and analysis of the S.agalactiae GD201008-001 genome reveals the presence of LuxS encoding gene in the strain.To determine whether the culture supernatants from S. agalactiae possessed AI-2-like activity,Vibrio harveyi BB170 was used as a reporter strain.A strong fluorescence signal produced by V.harveyi BB170 could be detected induced by the culture supernatants of S.agalactiae indicated that S.agalactiae GD201008-001 can produce and secrete AI-2 molecules.To evaluate the function of AI-2,LuxS and Pfs were expressed and purified to synthesize AI-2 in vitro.The Ellman’s assay was used to calculate the concentration of AI-2 from culture supernatants which showed an increased AI-2 level in exponential phased and reached a peak concentration at early stationary phase.These results and productions pave the way for further research on the LuxS/AI-2 QS system in S.agalactiae.2.The impact of lux S/AI-2 system on bacterial virulence in S.agalactiae After confirmed that S.agalactiae GD201008-001 possesses a functional lux S gene and produces AI-2,we constructed a lux S gene mutant(Δlux S)and the corresponding complementary strain to further evaluate the function of LuxS/AI-2 QS system in S. agalactiae.We found that inactivation of lux S caused a marked decrease in biofilm formation,hemolytic activity without affecting bacterial growth in THB medium.Except for hemolytic activity,the altered phenotypes due to the lux S deletion were AI-2-independent.Moreover,the deletion of lux S depressed the anti-phagocytosis and intracellular survival ability but promotes the cytotoxic of S.agalactiae in an AI-2 independent manner.Further investigation indicated that high levels of the proinflammatory cytokines IL-1β and IL-6 could be induced in macrophages co-incubated with the lux S deletion mutant and synthetic AI-2,single or combined.Also, the results of tilapia and mice infection showed that inactivation of lux S significantly decreased the virulence of S.agalactiae.We obtained brain tissues from mice had developed the neurological symptoms after infection to perform pathologic section.The pathologic anatomic observation showed a massive meninges haemorrhage occurred in the WT-infected group but congestion of blood vessel found in the Δlux S-infected group.Up-regulated the expression of cytokines induced by lux S deficiency were found in brains of both tilapia and mice but only in spleen from mice.Increased proinflammatory effects of the lux S mutant were restored in the lux S complemented strain but could not be restored by AI-2 addition.All the findings suggest that lux S is involved in virulence-associated phenotypes and immunological evasion of S.agalactiae,and furthermore,this involvement is mostly AI-2-independent.This study will provide valuable insights into our understanding of the role of the LuxS/AI-2 QS system in the pathogenesis of S.agalactiae.3.The RNA-seq analysis of lux S deletion mutantsTo figure out the specific regulatory mechanism of lux S impact the bacteria virulence,RNA-seq analysis was performed to compare the gene transcription of Δlux S and WT.Genes with over 2-fold change were considered differentially expressed.A total of 264 genes were identified in the Δlux S strain,including 155 upregulated and 109 downregulated genes.The expression patterns of 10 up-regulated genes and 10 downregulated genes randomly selected to measure the expression levels in order to validate the reliability of RNA-Seq results.The GO enrichment analysis of down-regulated genes showed the function of most genes involved in nucleotide biosynthetic process and carbohydrate transport or metabolic process,including the PTS sugar transporter system,the sugar ABC transporter system and the putative fructose metabolic operon(fru RKI).However,there were compensatory increase in riched nucleotide salvage and fatty acid biosynthetic process.Considering the nucleotide salvage process was found to be closely associated with methionine cycle,we speculate the compensatory increase of genes involved in nucleotide salvage process was due to lux S deficiency caused methionine cycle disorders.To repair deficiency in methionine cycle of Δlux S,sah H gene from Pseudomonas aeruginosa PAO1 was heterologous expressed in Δlux S.However,there was no significant effect on the decreased biofilm formation,anti-phagocytosis,intracellular survival ability of Δlux S by heterologous expression of sah H with high transcription level.These results demonstrated that methionine cycle disorders even changes in nucleotide biosynthetic process had no responsibility for decreased virulence,especially the immune evasion of Δlux S.These results indicated that loss of lux S resulted in down-regulated genes involved in carbohydrate transport or metabolic process,which might be responsible for decreased virulence of Δlux S.4.lux S contributes to immune evasion of S.agalactiae via regulating the transcription of fru RKI operon The previously RNA-seq data showed that,lux S deficiency caused 109 genes more than2-fold down-regulated.Most of these genes involved in the PTS sugar transporter system and embden meyerhof pathway.Different carbohydrates defined medium(CDM)used to evaluate ability of Δlux S to utilize carbon source.The results show that the growth kinetics of Δlux S exhibited significant weakening in fructose-or glucose-defined medium with a low concentration of fructose or glucose.Among those 2-fold down-regulated genes,there’s a putative fructose metabolic operon(fru RKI)which is composed of fru I,fru K and fru R.The fru R encodes an Deo R family regulator which has been reported to act as a repressor of the whole operon.The fru K encodes a 1-phosphofructokinase,which is the key kinase of the glycolytic flux involved in both fructose and glucose metabolism in Streptococcus spp.The fru I encodes a transporter of fructose.RT-PCR confirmed that fru I,fru K and fru R genes comprised an operon in S.agalactiae.Meanwhile,we also demonstrated that the fru RKI operon was important for S.agalactiae intracellular survival via constructing the fru RKI operon knockout strain and overexpressed fru R,fru K,fru I single gene in Δlux S.However,all fru RKI related editing strains had no significant change in ability of anti-phagocytosis.fru RKI operon also helped the S.agalactiae survive in H2O2,but each of fru R,fru K,fru I overexpression can not restore the decreased H2O2 resistance in Δlux S.High-level expression of cytokines can be detected in Δfru RKI infected macrophages.These observations indicated that loss of lux S resulted in a lowlevel expression of fru RKI,which might be responsible for decreased immune evasion ofΔlux S.5.The mechanism of lux S in regulating the fru RKI operonThe lux S gene has been demonstrated to play a crucial role in immune evasion of piscine Streptococcus agalactiae in AI-2 independent way,but the regulatory pathways remain largely unknown.In this study,transcriptome analysis,followed by the lux S gene deletion and subsequent functional studies,confirmed that impaired bacterial survival inside macrophages due to the inactivation of lux S was associated with reduced transcription of the fru RKI operon.Further studies focus on the underlying mechanism of regulation ship between lux S and fru RKI operon.Amino acid sequence analysis and ability on DNA binding showed that lux S could not to influence the transcript of fru RKI operon by binding its promoter.An unusually low level of β-galactosidase activity was detected in the Δlux S,which showed an inhibitory action on fru RKI promoter and was consistent with the downregulated transcription level of fru RKI.We identified a cis-acting sequence,which was a catabolite responsive element(cre)recognized by catabolite control protein A(Ccp A),in the fru RKI promoter.Mutations on cre site can completely eliminating the effects of lux S on fru RKI operon,which showed the recovered intracellular survival ability even with lux S deficiency.We performed EMSA using purified LuxS protein to interfere the combination of Ccp A to the promoter of fru RKI operon.Unexpectedly,Ccp A showed similar binding capacity to fru RKI promoter when incubated with or without increasing amounts of purified LuxS.Competitive EMSA performed to confirm that lux S indirectly regulated the expression of fru RKI operon by competitively binding with the catabolite control protein A(Ccp A)via catabolite responsive elements(cre).However,the same mechanism had no effects on transcription of lux S.Collectively,our study identifies a novel and previously unappreciated role for lux S in bacterial intracellular survival,which may give a more thorough understanding of the immune evasion mechanism in S.agalactiae.