The ArlR-MgrA Regulatory Cascade Regulates PIA-dependent And Protein-mediated Biofilm Formation In Rbf-dependent And Rbf-independent Pathways

Staphylococcus aureus is one of the most common pathogens that can cause a broad range of infections.And biofilms lead to significant problems during the treatment of Staphylococcus aureus infections.Biofilms consist of exopolysaccharides,extracellular proteins and extracellular DNA,and they provide the bacteria a protection against antibiotics and host immune system.Rbf has been reported to play a role in the regulation of the ica operon and the production of PIA,then promote the biofilm formation.Our previous work found that the LuxS/AI-2 system had an inhibitory effect on Rbf and Rbf could participate in quorum sensing as the potential receptor of AI-2.So we further aimed to improve the understanding of the whole Rbf-centered regulatory network of biofilm formation.Our data indicated that two-component system response regulator ArlR and global gene regulator MgrA both directly inhibited the expression of Rbf as a regulatory cascade.The ArlR-MgrA regulatory cascade indirectly inhibited the expression of the ica operon and PIA production via Rbf,but they also directly bound the promoter region of the ica operon to enhance the expression of ica operon and PIA production.Combining the results of RT-PCR and PIA detection assay,we determined that the upregulation of ArlR-MgrA regulatory cascade on ica operon played a major role.Extracellular metalloproteinase Aur could cleave and mature serine protease SspA,then mature SspA cleaved and activated cysteine protease SspB.All of these proteases had inhibitory effects on biofilm formation.We found Rbf directly inhibited the expression of aur,proving the upregulation of Rbf on biofilm formation didn’t rely only on the control of PIA production.In addition,we also confirmed the ArlR-MgrA regulatory cascade indirectly enhanced the expression of aurr by repressing the expression of rbf and directly enhanced the expression of aur by binding the promoter region of aur.All these data suggested that the inhibitory effect of ArlR and MgrA on biofilm formation was fulfilled by their upregulation on extracellular protease Aur.σ~B is an important sigma factor in S.aureus,which has an icaR-independent inhibitory effect on ica operon and also an inhibitory effect on extracellular proteases including Aur.Our results showed that whether in σ~B-inactivated strain NCTC8325 or in σ~B activity restored strains,the regulatory pathways identified in this study remained the same,suggesting that these regulatory pathways are σ~B-independent.Meanwhile,we studied the influence of the LuxS/AI-2 system on Aur and found the LuxS/AI-2 system didn’t have a significant impact on aur in spite of its inhibitory effect on Rbf.At last we investigated a novel two-component system response regulator SAOUHSC 01314,and proved it could enhance biofilm formation by repressing the expression of Aur.