Molecular Mechanism Of Biofilm Formation Modulated By AI-2/SaaR In Staphylococcus Aureus

Quorum sensing(QS)is a widespread mechanism used by bacteria for cell-to-cell communication.QS coordinates social activities,such as bioluminescence,bacterial competence,virulence,antibiotic production,and biofilm formation.The QS bacteria produce and secrete chemical signal molecules called autoinducers to communicate.In general,Gram-negative bacteria use acylated homoserine lactones as autoinducers,and Gram-positive bacteria use processed oligopeptide to communicate.Studies have indicated that a QS system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer 2(AI-2),which is synthesized by the LuxS enzyme in the activated methyl cycle.LuxS is widely present in bacteria,leading to the suggestion that AI-2 is a universal language for interspecies communication.To date,the detailed mechanism by which AI-2 functions has been characterized in only a few species.In Vibrio harveyi,AI-2 is detected and transduced by a two-component system.Borate-AI-2 is recognized by a periplasmic binding protein LuxP that interacts with cytoplasmic response regulator proteins,initiating a phosphorylation cascade to subsequently regulate the expression of the luciferase structural operon.In Escherichia coli and Salmonella typhimurium,AI-2 is first taken up by the Isr transporter,and then the expression of genes controlled by the QS system is initiated.However,it is not clear how AI-2 is detected or what functions are regulated by this signaling molecule in many luxS-containing bacteria,especially in Gram-positive bacteria.Staphylococcus aureus is a major opportunistic pathogen that could cause a variety of infectious diseases ranging from minor skin infections to life-threatening infections.Many S.aureus infections are involved in biofilm formation,including endocarditis,septic arthritis,osteomyelitis,and infections associated with implanted medical devices.The luxS gene has been identified in S.aureus and has been shown to function in AI-2 production.Our previous work showed that the LuxS/AI-2 system could control virulence-related gene expression and decrease biofilm formation via an icaR-activation pathway.However,the detailed mechanism by which AI-2 functions remains unclear in S.aureus.Our data indicated that inactivation of luxS in S.aureus NCTC8325 resulted in increased biofilm formation and higher PIA production compared with the wild-type strain.The transcript level of saaR,a positive regulator of biofilm formation,was significantly increased in the luxS mutant.All of the parental phenotypes could be restored by genetic complementation and chemically synthesized AI-2,which suggested that AI-2 has a signaling function to regulate biofilm formation and saaR transcription in S.aureus.Moreover,we showed that the luxS/saaR double mutant produced approximately the same amount of biofilms and PIA as the saaR mutant.In the saaR mutant,deletion of luxS couldn’t enhance biofilm formation and icaA transcription.These results indicated that the LuxS/AI-2 system regulates PIA-dependent biofilm formation via repression of saaR in S.aureus.Electrophoretic mobility shift assay and β-galactosidase assay showed that SaaR could specifically bind to its promoter and upregulate its own expression.Furthermore,we determined the three-dimensional structures of SaaR and its complex with AI-2 by cyro-EM.We found that SaaR underwent a significant conformational change upon AI-2 binding,suggesting that SaaR is an AI-2 receptor in S.aureus.