The Two-component Signaling System VraSR_ss Regulatory Network Modulates Multidrug Resistance And Full Virulence In Streptococcus Suis Serotype 2

Streptococcus suis is an important zoonotic pathogen responsible for various swine diseases and human cases with streptococcal toxic shock syndrome.Among the described serotypes based on capsular antigens,S.suis serotype 2(SS2)is considered the most prevalent in most countries.With the massive use of multiple antimicrobial agents for S.suis prophylaxis and therapy,the emergence of resistance to multidrug has been frequently reported in S.suis worldwide.Two-component signaling systems(TCSs)are ubiquitous in prokaryotes and represent a major mechanism through which bacterial cells sense and respond to external stimuli.As an important post-transcriptional regulator,RNA-binding proteins(RBPs)can modulate different genes expression by modulating RNA translation,processing,and stability.To survive in diverse environments,bacteria can employ a variety of regulatory systems to build a complex regulatory network.At present,the mechanism through which SS2 regulates antimicrobial resistance and coordinates gene expression during the infection process remains unclear and requires further exploration.1 Identification of the two-component signaling system VraSRss signal molecules and phosphory lation sitesA typical TCS consists of a membrane-bound sensor histidine kinase(HK)and a cytoplasmic response regulator(RR),while the phosphorylation between them is critical for the TCS functions.VraSR,a well-known TCS,was identified to confer resistance to diverse cell wall-targeting antibiotics in Staphylococcus aureus.In this study,we found that VraSRss is a homolog carried by S.suis that shares～50%sequence identity with the VraSR of S.aureus.Domains and three-dimensional structures of VraSRss were predicted and confirmed that VraSRss is a typical TCS.According to the phylogenetic tree based on amino acid sequences,VraSRss are conserved among different serotypes of S.suis,which further suggested that VraSRss have important biological roles.Our data demonstrated that both peptides,β-lactams,and aminoglycosides antimicrobials could activate vraSRss genes.In addition,bioinformatics analysis identified four potential phosphorylation sites in VraSRss,while the site-specific mutation further confirmed that VraSss H138 and VraRss D56 were the real phosphorylation active sites of this TCS.These results provide valuable reference for the further study of VraSRss molecular functions.2 VraSRss contributes to the multidrug resistance of Streptococcus suis serotype 2S.suis has received increasing attention for its involvement in severe human infections worldwide as well as in multidrug resistance.In this study,we identified that VraSRss is involved in susceptibility to a variety of antimicrobials,which are classified based on differing chemical structures and compositions,including aminoglycosides,β-lactams,and peptides.Further analyses demonstrated that the yvqFss gene,located upstream of vraSRss,shared the same promoter with the TCS genes,which was directly regulated by VraSRss,as shown in electrophoretic mobility shift assays.Notably,YvqFss and VraSRss constitute a novel multidrug resistance module of SS2 that participates in resistance to certain groups of antimicrobials.Given both the high conservation of YvqFss,VraSss,and VraRss among different serotypes of S.suis,this multidrug resistance module may provide valuable insights to other multidrug-resistant S.suis strains.Subsequent qRT-PCR assays showed that VraSRss regulates the expression of penicillin-binding protein PBP2A,which involves the synthesis of peptidoglycan and participates in the resistance to cell wall-targeting antibiotics.In addition,our data confirmed that the phosphorylation is critical for the VraSRss to regulate the multidrug resistance of SS2.3 VraSRss regulates the full virulence of Streptococcus suis serotype 2When invading the host,SS2 strains encounter diverse stimuli.TCSs are usually employed to sense and respond to extracellular signals during the infection process.In this study,we found that VraSRss inactivation significantly attenuated bacterial virulence in animal models,which,coupled with the significant activation of VraSRss expression observed in host blood,strongly suggested that VraSRss is an important regulator of SS2 pathogenicity.Furthermore,RNA-sequencing analyses identified 106 genes that were differentially expressed between the wild-type(WT)and △vraSRss strains,including genes involved in capsular polysaccharide(CPS)biosynthesis.Subsequent studies confirmed that VraSRss indirectly regulated the transcription of CPS gene clusters and,thus,controlled the CPS thickness shown by transmission electron microscopy.Decreased CPS biosynthesis caused by vraSRss deletion subsequently attenuated antiphagocytosis against macrophages,which partially clarified the pathogenic mechanism mediated by VraSRss.In addition,we found that VraSRss regulates the expression of maltose utilization gene cluster(malQXCDARP),which also involves in the CPS biosynthesis and contributes to the SS2 virulence.Taken together,our data suggest that VraSRss constructs a complex regulatory network to control CPS biosynthesis of SS2,thereby facilitating adaption to harsh host stimuli.4 The RNA-binding protein RBPS1 plays as a global regulator to the pathogenicity of Streptococcus suis serotype 2RBPs can modulate different genes expression by post-transcriptional regulation.In this study,we identified an RBP harboring S1 domain,named RBPS1,which facilitated SS2 adhesion to host epithelial cells and contributed to bacterial pathogenicity.Comparative proteomic analysis identified 145 proteins that were differentially expressed between the WT and ΔRBPS1 strains,which included several virulence-associated factors,such as extracellular protein factor(EF),SrtF pilus,LgAl protease,SBP2 pilus,and peptidoglycan-binding LysM’.In addition,the most downregulated protein,GlnQ,was investigated and showed that GlnQ could facilitate SS2 binding with host fibronectin and adhesion to host cells.Further analyses found that RBPS1 and the TCS vraSRss form a single operon and share one promoter.βgalactosidase assays indicated that RBPS1 can interact with the 5’ untranslated region(5’UTR)of vraRss,which combined with the proteomic and transcriptional data indicated that RBPS1 participates in the VraSRss regulatory network.These results can partly explain the pathogenesis mechanism mediated by RBPS1.In addition,the regulatory mechanisms by RBPS1 to EF,srtF,IgAl,sbp2,lysM’,and glnQ were explored and indicated that RBPS1 regulates genes expression in diverse pathways including post-transcriptional regulation and thus plays as a global regulator.These findings enrich the regulatory systems of S.suis and provide valuable insights into the bacterial pathogenicity.