Carbonic Anhydrase,Small RNA Rss03 And Its Target Fe-S Cluster Assembly Proteins Affect The Oxidative Stress Response And Virulence Of Streptococcus Suis

Streptococcus suis is not only an important bacterial pathogen of pigs,causing important economic losses to the pig industry worldwide,but also a zoonotic pathogen for those who have close contact with pigs or pig-products.The host’s immunocompetent cells,such as macrophages,neutrophils,eliminate pathogenic bacteria through the production of reactive oxygen species,including superoxide,hydrogen peroxide,hydroxyl free radicals,damaging bacterial proteins,lipids,and DNA.Therefore,pathogenic bacteria must have corresponding strategies combating oxidative stress to resist the elimination by immunocompetent cells.At present,how S.suis combats oxidative stress is still very limited.Based on our previous studies,we focused on S.suis carbonic anhydrase（SCA）,small RNA rss03 and its target Fe-S cluster synthesis protein（Suf CDSUB）for further study,and clarified their roles in oxidative stress response and virulence of S.suis,which contributes to the understanding of oxidative stress response and pathogenic mechanism of S.suis.1 The effect of SCA on the oxidative stress response and virulence of S.suisIn our previous study,compared with control group,the protein level of S.suis SCA was significantly increased in the H₂O₂treatment group.In this study,the deletion mutant?sca was constructed using S.suis virulent strain SC070731,and the effect of SCA on the growth of S.suis under different conditions including H₂O₂treatment was evaluated;the assay of mouse macrophage cells RAW264.7 phagocytosis and survival,pig blood survival and zebrafish infection experiment were performed.The analysis of growth curves under different conditions showed that compared with the wild-type strain,the growth of?sca in the absence of CO₂was significantly inhibited,and its OD₆₀₀value of 12 h growth was lower than 0.5.Under the condition of 5%CO₂,although the lag phase of?sca was longer than that of the wild-type strain,the OD₆₀₀value of both wild-type strain and?sca was close to 1.6 at stationary period;in the absence of CO₂,with the gradient increase of Na HCO₃concentration in the medium,the growth ability of?sca was significantly improved.Compared with?sca,adding unsaturated fatty acid（UFA）oleic acid and UFA derivatives such as Tween-80 and Tween-20 significantly promoted the growth of?sca.The survival rate of the wild-type strains was 99.48%after H₂O₂treatment for 20 min,while the survival rate of?sca was significantly reduced to 74.04%（P<0.01）.There was no significant difference in the phagocytosis rate of RAW264.7 to the wild-type strain and?sca;the survival assay showed that after 3 hours of antibiotic treatment,the survival rate of the wild-type strain was 77.98%,while the survival rate of?sca in RAW264.7 was significantly reduced to 49.23%（P<0.01）.Pig blood survival assay showed,the survival rate of the wild-type strain was 110.02%,but the survival rate of?sca in pig blood was only 51.39%（P<0.01）.The zebrafish infection experiment showed that the survival rate in the group infected with SC070731 was 13.3%,while the survival rate in the group infected with?sca was 53.3%（P<0.0001）.The above results indicate that the SCA,necessary for S.suis to utilize CO₂and HCO₃^-,is involved in the synthesis of UFAs,and it contributes to the oxidative stress response of S.suis.Moreover,the enzyme promotes the survival of S.suis in pig blood and macrophages,and the deletion of sca attenuates the virulence of S.suis in zebrafish infection model,suggesting that it plays an important role in S.suis pathogenesis.2 The effect of sRNA rss03 and its target Suf CDSUB on the oxidative stress response and virulence of S.suisIn our previous,sRNA rss03 promotes the survival of S.suis in the blood,the protein level of Suf CDSUB in the rss03 deletion strain was significantly reduced,and Suf CDSUB was predicted to be a target candidate of rss03.In this study,theβ-galactosidase translation fusion assay confirmed that rss03 binds to the 5’UTR region of suf C,thereby positively regulating the translation level of suf CDSUB m RNA.Under 1 mmol/L H₂O₂treatment,compared with the wild-type strain,the growth of?suf CDSUB and?rss03 were significantly inhibited,while the growth level of the rss03 complementary strain C-rss03was similar with wild-type strain.Under iron limit condition（200μmol/L BIP）,compared with the wild-type strain,the growth of?suf CDSUB and?rss03 under were significantly inhibited,while the growth level of C-rss03 was similar with wild-type strain.There was no significant difference in the phagocytosis rate of RAW264.7 to the wild-type strain,?suf CDSUB,and?rss03;the survival assay showed that after 3 hours of antibiotic treatment,compared with the wild-type strain,the survival rate ofΔsuf CDSUB andΔrss03in RAW264.7 were significantly reduced（P<0.0001）,while the survival rate of C-rss03 in RAW264.7 was equivalent to that of the wild-type strain.The?suf CDSUB（survival rate80%）andΔrss03（survival rate 73.3%）were significantly less virulent than the wild-type strain（survival rate 13.3%）in zebrafish infection model,and the virulence of C-rss03（survival rate 13.3%）was restored to wild-type level.The above results indicate that rss03regulates Fe-S cluster binding protein by binding to the suf C 5’UTR region,thereby affecting the oxidative stress response and virulence of S.suis.