Molecular Mechanism Of Vancomycin Resistance In Staphylococcus Aureus Regulated By Nitric Oxide Synthase NOS

Staphylococcus aureus is a major human pathogen.The emergence of multi-drug resistance has caused global concern about S.aureus infection,especially resistance to vancomycin,a drug of last resort.A growing body of research focuses on the mechanisms of vancomycin resistance.Nitric oxide synthase(NOS)of S.aureus has been demonstrated to generate NO endogenously,which plays an important role in vancomycin resistance.However,the underlying mechanism remains unclear.In this study,we defined the role of NOS in vancomycin resistance in a clinical VISA isolate XN108.The NOS deletion strain had decreased resistance to vancomycin and other cell wall-associated antibiotics,a thinner cell wall,and increased autolysis activity.Furthermore,treatment with NOS inhibitor dramatically decreased vancomycin resistance of VISA isolate XN108 and Mu50.S-nitrosylation,a posttranslational modification on cysteine thiol mediated by NO,has been found to regulate protein function.Therefore,a proteomic analysis was used to identify proteins that can be S-nitrosylated by NO generated endogenously in XN108.S-nitrosylation was found to be a prominent growth feature of S.aureus.MgrA,a global regulatory protein involved in autolytic activity,multidrug resistance and virulence,was found to be S-nitrosylated at the unique cysteine residue Cys12.Allelic replacement was performed to generate the mgrAC12S mutant strain by replacing the cystine residue with a serine,which resulted in removal the S-nitrosylation.The mgrAC12S mutant strain showed decreased resistance to vancomycin,decreased cell wall thickness and increased autolysis activity.The growth defeciency caused by vancomycin in the Δnos strain could be compensated by the addition of low concentration of NO donor.However,the mgrAC12S strain,in which the MgrA cannot be S-nitrosylated,lost the ability to recover its growth rate in vancomycin containing medium when NO donor was added.This result suggested that loss of S-nitrosylation on MgrA severely impairs the ability of S.aureus to sense NO.The RT-qPCR results showed that the transcriptional levels of genes involved in autolysis,such as lytN and sarV,were significantly up-regulated in the mgrAC12S mutant strain,while no significant changes were observed in the transcription of genes involved in cell wall synthesis.This result suggested that the changes occurred in cell wall thickness and autolysis activity may be primarily mediated by the alteration of cell wall degradation process.Electrophoretic mobility shift assay(EMSA)and Chromatin immunoprecipitation assay(ChIP)were performed to further elucidate the mechanism of vancomycin resistance mediated by NOS-derived NO.MgrA S-nitrosylation mediated by NOS-derived NO can facilitate the promoter binding ability of MgrA and increase the repression of genes involved in cell wall metabolism,which results in decreased autolysis activity and mediates the increase of cell wall thickness,and finally contributes to S.aureus vancomycin resistance.In addition to MgrA,at least one additonal regulator,WalR,also employs S-nitrosylation for NO signal transduction to confer resistance,indicating that the transcription mechanism tuned by S-nitrosylation may be ubiquitous in bacteria.In conclusion,our data defined the critical function of S.aureus NOS in vancomycin resistance,and revealed that transcription regulated by S-nitrosylation underlies a mechanism for NO-mediated bacterial antibiotic resistance.Our study will provide new insights for the clinical treatment of VISA or other pathogens.