Regulatory Mechanism Of Virulence Gene Expression By Rbf In Staphylococcus Aureus

Staphylococcus aureus is a gram-positive pathogenic bacterium and leading cause of morbidity and mortality from community-and hospital-acquired infectious diseases.These infectious diseases range from minor skin lesion to life-threatening sepsis pneumonia and toxic shock.The virulence feature of S.aureus as a pathogen is mainly due to the fact that S.aureus can produce a large arsenal of virulence factors,including toxins,proteases,and secreted exoproteins.Alpha-toxin and PSMa are categorized as membrane-damaging toxins,which are typical virulence factors of S.aureus.These virulence factors can attach to the cytoplasmic membrane in receptor-mediated or non-receptor mediated manners and lead to membrane distintegration,thus causing acute infections.Meanwhile,biofilm formation provides a protecting environment enabling the bacteria cells to proliferate by limiting antibiotic access and shielding the bacterial pathogen from host immune defense,which often causes chronic infections.The production of virulence factors is under control of diverse regulatory factors,of note,two-component systems,SarA family proteins,agr quorum sensing system,and ?factor.Rbf was originally identified as a transcriptional regulator,and contained a consensus region signature of AraC/XylS family.The AraC/XylS family protein have a highly conserved region containing two helix-turn-helix(HTH)DNA binding motifs at the C terminal.The mechanisms of this modulation revealed that Rbf can inhibit the transcriptional regulator IcaR,a negative regulator of icaADBC,then can lead to increased production of polymeric N-acetylglucosamine(PNAG)to promote the biofilm formation.Recently,studies have demonstrated that Rbf can directly upregulate sarX,a member of SarA family,which then can activate icaADBC by downregulating IcaR.Biofilm formation is often involved in chronic infections and pronounced toxin production is characterized to be responsible for acute infections.These are the two main virulence traits that can affect the S.aureus infection.Although Rbf has been recognized to play a major role in biofilm formation,its involvement in the regulation of acute infections has been unknown.In this study,we were interested in the virulence control by Rbf.We first performed hemolytic activity assays in the WT and rbf mutant strains and observed the extremely increased relative hemolytic activity in the rbf mutant strain.We investigated the transcript levels of genes encoding important toxins regulated by Rbf.Our data indicated that the transcript levels of hla and psma,encoding alpha-toxin and PSMa,respectively,were significantly increased in the rbf mutant strain compared with the WT strain.Results of ?-galactosidase activity assay and EMSA revealed that Rbf can directly bind to the hla and psma promoter regions to repress their expression in S.aureus.Furthermore,the increased production levels of alpha-toxin and PSMa explain well the difference in hemolytic capacity observed in the rbf mutant strains,as these are the most important hemolytic toxins of S.aureus.Meanwhile,the decreased expression level of hla and psma further confirmed the toxins control by Rbf in the rbf-overexpressed strain compared with the WT strains NCTC8325 and MW2.Mouse subcutaneous abscess model can be used to investigate a number of virulence factors related to the pathogenesis of skin and soft tissue infections.In the model of mouse subcutaneous abscess,the rbf mutant strain exhibited significantly increased pathogenicity than that in the WT strain.In conclusion,we have revealed that Rbf can negatively regulate the hemolysis activity and directly repress the expression of hla and psma.Furthermore,the rbf mutant strain exhibited significantly increased pathogenicity compared to the WT strain in the model of mouse subcutaneous abscess accordingly.Our findings provide a novel insight into the virulence regulation and acute infections mediated by Rbf in S.aureus.