Identification Of Oxidative Stress Response Proteins In Streptococcus Suis And Screening Of Small RNA Rss03 Targets

Streptococcus suis(S.suis)is an important zoonotic pathogen that can cause meningitis,endocarditis,pneumonia,arthritis,sepsis and even death in pigs.The host immunocompetent cells,such as macrophages and neutrophils,can produce reactive oxygen species,such as superoxide,hydrogen peroxide(H2O2),and hydroxyl free radicals.These reactive oxygen species cause damage to bacterial large molecules such as proteins,lipids,and DNA,which facilitates the killing of bacteria.However,little is known about the mechanism of oxidative stress response of S.suis.Our previous research found that small RNA(sRNA)rss03 is involved in the oxidative stress response of S.suis and promotes the survival of S.suis in the blood,but its regulation mechanism is unknown.In this study,we identified the oxidative stress response proteins and rss03 targets,which contribute to better understanding of the mechanism of the oxidative stress response in S.suis and the role of rss03 in pathogenesis.1.Identification of oxidative stress response proteins in S.suis by a proteomic approachIn order to reveal the mechanism of oxidative stress response in S.suis,a comparative proteomic method was used to identify involved proteins.After H2O2 treatment,54 differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry(LC-MS/MS)from whole-cell extract,of which 28 were up-regulated and 26 were down-regulated in the treatment group compared to the control grown in THB medium.These differentially expressed proteins are mainly involved in processes such as metabolism,biosynthesis,and stress response.Two proteins,YbaB/EbfC family nucleoside-related protein(YED)and 2-amino-4-hydroxy-6-hydroxymethyl dihydropteridine diphosphate kinase(AHH),which were significantly up-regulated in the H2O2 treatment group,were selected for verification.Their deletion mutants(?YED and?AHH)were constructed.Growth curve analysis showed that the two mutants grow similarly to the wild-type strain in THB medium.The H2O2 stress experiment showed that after H2O2 treatment the survival rate of the wild-type strain is significantly higher than that of two mutants,which was 97.12%(wild-type strain),72.69%(?YED),and 79.49%(?AHH).Phagocytosis and survival assay showed that after 1 h of phagocytosis,uptake of the wild-type strain by RAW264.7 macrophages was not significantly different to any of the mutant strains.However,the survival rate of two mutants was significantly lower than that of wild-type strain.In this study,the differentially expressed proteins identified in H2O2 stress condition are helpful to reveal the mechanism of oxidative stress response in S.suis,of which YED and AHH contribute to the survival of S.suis in macrophages,suggesting that they play a role in S.suis pathogenesis.2.Screening of sRNA rss03 TargetsIdentification of sRNAs targets is important to reveal the functions and regulatory mechanisms of sRNA,which is little known in Gram-positive bacteria.Our previous research found that S.suis sRNA rss03 participates in the oxidative stress response of S.suis and promotes the survival of S.suis in the blood,but its targets are unknown.In this study,the rss03 targets were screened by using MAPS(MS2 affinity purification coupled with RNA sequencing),LC-MS/MS,and bioinformatics analyses.The plasmid pSET2-MS2-rss03 expressing MS2-rss03 and the control plasmid pSET2-MS2 were constructed.MAPS results showed that compared with the control group,there were 155 differentially expressed genes,of which 116 genes were significantly up-regulated and 39 genes were significantly down-regulated in MS2-rss03 group.LC-MS/MS analysis results showed that there were 53 differentially expressed proteins,of which 34 proteins were up-regulated and 19 proteins were down-regulated in ?rss03.There are 8 candidates jointly screened by MAPS and LC-MS/MS analses,and it is worth noting that the operon NJAUSS_RS04010-NJAUSS_RS04015 involved in the metabolism of glycerol and sugar metabolism is significantly enriched in the MS2-rss03 group by MAPS analysis.These expression of two proteins was up-regulates in ?rss03 and it is predicted that rss03 can bind to the ribosomal binding site(RBS)region of these two genes.Through LacZ translation fusion,it is clear that rss03 negatively regulates these two direct targets.In addition,LC-MS/MS analysis results showed that the expressions of SufD,SufS,SufU,and SufB is significantly down-regulated in ?rss03.These four proteins are encoded by the SufCDSUB gene cluster,which is involved in the synthesis of iron and sulfur clusters and plays an important role in oxidant stress response and iron metabolism.Furthermore,it is predicted that rss03 can bind to the 5'UTR of SufC which is far from the RBS region.It is speculated that the long 5'UTR of mRNA structurally sequesters the RBS and binding of the rss03 to the 5'UTR of SufC mRNA liberates the RBS,thus promoting protein translation.It is suggested that in addition to participating in anti-oxidative stress and promoting the survival of S.suis in the blood,rss03 may also have other important biological functions,such as participating in glycerol and sugar metabolism,which may help S.suis to proliferate in the host.