Identification And Characterization Of The Regulatory Roles Of SrtR In Oxidative Stress Response And Virulence In Streptococcus Suis

Streptococcus suis is an important zoonotic pathogen,which can infect human causing meningitis and toxic shock syndrom with public healthy concerns,and can invade swine cansing meningitis and anthritis,among others,leading to huge econmic losses in swine industry.Evasion of clearance by host immune system is crucial for S.suis infection and pathogenesis.Previously,an S.suis serotype 9 strain DN13 was serially pasaged in mice and hypervirulent mouse-adapted strains were successfully obtained in our laboratory.The hypervirulent strains were used to reveal the pathogenic mechanisms of S.suis.Single colonies were rondomly picked from passage 10,20 and 30,respectively,and were designated as SS9-P10,SS9-P20 and SS9-P30,respectively.The LD₅₀0 value of SS9-P10,SS9-P20 and SS9-P30?50%Lethal Dose?was determined in female ICR mice and the results showed that LD₅₀0 value of SS9-P10,SS9-P20 and SS9-P30 decreased 140-fold or more compared to DN13.To elucidate the molecular mechanisms of increased virulence in mouse-adapted strains,phenotypes between the parent strain and mouse-adapted strains were compared.The results showed that the mouse-adapted strains were more resistant to H2O2 challenge and high temperature stress?P<0.05?,and the biofilm formation was also increased?P<0.05?.Whole genome reseqencing identified 150,153 and 155 mutation sites in SS9-P10,SS9-P20 and SS9-P30 compared to DN13,respectively.A total of 138 sites were commonly existed among SS9-P10,SS9-P20 and SS9-P30.The phenotypes and the mutation profiles of SS9-P10,SS9-P20 and SS9-P30 were highly similar,suggesting that one or more mutations in the 138 common sites might be responsible for change in phenotypes.After indivadual analysis,58 non-silence mutations in coding-regions and 1 non-silence mutation in non-coding region were identified among the 138 commonly existed mutation sites.A 12-animo acid deletion in DNA ligase in the mouse-adapted strains did not affect functions of DNA liagase,as indicated by Methyl methane sulfonate assay showing that sensitivities to Methyl methane sulfonate were not significantly differed between DN13 and SS9-P10.For the mutation located in promoter core sequences,CAT assay suggests that activity of the mutated promoter was strengthened as predicted.Mutation in XRE family transcriptional regulator?srtR?coding sequence leads to polypeptide extension and the resultant peptide contains a helix-turn-helix domain for DNA binding in the amino end and a uncharacterized DUF3955 domain in the carboxyl terminus,in contrast to only a helix-turn-helix domain in SrtR in DN13.To test whether srtR has a role in stress response,plasmid-born srtR was introduced into DN13 and H2O2 challenge assay was performed.The results showed that after introduction of srtR,sensitivity of DN13 was decreased.To further demonstrate the role of srtR,DN13-and SS9-P10-background srtR deletion mutants and complementary strains were constructed and characterized.The results showed that strains with functional srtR were more tolerant to H2O2challenge and high temperature stress compared to SrtR deficient strains.In the mouse infection model,srtR was confirmed as virulence-associated gene since srtR positive strains were more virulent compared to their counterpart srtR negative strains.These results demonstrated that srtR participate in anti-oxidative and thermo stress as well as pathogenesis in S.suis.To investigate the mechanisms of SrtR sensing oxidative stress,the only cysteine in SrtR?located at DUF3955 domain?was replaced with alanine(SrtR^C105A)and serine(SrtR^C105S),respectively.The H₂O₂ challenge results showed that SrtR^C105A105A completely abolished the function of SrtR whereas SrtR^C105S105S retained its function as the wild type Srt R,indicating SrtR senses oxidant is thiol-independent.Additionally,a putative motif with multiple conserved amino acids was located at 120-132 in SrtR.Eight of them were individually replaced with alanine to investigate their role in response to oxidative stress.Finnaly,SrtR^N125A125A and SrtR^P130A130A were non-functional while SrtR^G132Aenhanced the activity of SrtR.Further investigations are needed to elucidate the role of these key amino acids.After analysis of transcripts by qRT-PCR,we found that dpr,sodA,and tpx were down-regulatied in SS9-P10-backgroud srt R-deficient strain but partially or completely restored in complementary strain compared to SS9-P10,indicating that dpr,sodA,and tpx were regulated by SrtR.In summary,we identified the first member in XRE family transcriptional regulator,SrtR,involving in oxidative and high temperature stresses,as well as pathogenesis in mice.Sense of oxidants by SrtR is cystenine-dependent but not by thiol oxidation.SrtR was found to regulate dpr,sodA,and tpx in response to oxidative stress.However,the exact mechanisms of sensing oxidants and genes directly regulated by SrtR remain unclear.SrtR also existed in other human pathogens,such as Clostridium difficile and Enterococcus faecium,and SrtR might have similar role in these pathogens.