Elucidation Of The Regulatory Mechanism Of PSMs And Screening Of Virulence Factors In Staphylococcus Aureus

Staphylococcus aureus is a human and animal pathogen that can cause various bacterial infections including relatively benign and fatal systemic infections,and various virulence factors are involved in the pathogenic processes of S.aureus.S.aureus can develop biofilms on host tissues and medical devices,and cause biofilm-associated infections when S.aureus accumulates to form a biofilm at the infection sites.Biofilms constitute a protected environment that is essential for bacteria to resist host immune response and chemotherapies,making therapeutic intervention extremely difficult.Moreover,biofilm dissemination enables bacteria to spread by blood or other body fluids to lead to infections in the body.Phenol soluble modulins(PSMs)play important roles in the biofilm development and the pathogenesis of S.aureus.Although the functions of PSMs have been studied extensively,the regulatory mechanisms controlling the expression of psm genes remain elusive.In this dissertation,the major work is to elucidate the regulatory mechanism of psm operons.We here identified a transcriptional regulator MgrA by using DNA pulldown assay,and further demonstrated that MgrA could specifically bind to the promoter region of psm operons by electrophoretic mobility shift assay and DNase I footprinting assay in vitro.We then constructed the mgrA deletion strain in S.aureus.Real-time quantitative reverse transcription-PCR indicated that MgrA repressed the transcription of psm operons in cultures and biofilms,suggesting that MgrA is a negative regulator of psm expression.We performed RP-UPLC to determine the protein levels of PSMs,and found that the production of PSMs was increased in the mgrA mutant strain,indicating that MgrA decreased the mRNA and protein levels of psm operons.We then detected biofilm formation and detachment in the wild type and the isogenic mgrA deletion strains,and our results indicated that biofilm formation and detachment were significantly increased in the mgrA mutant strain.These findings reveal that MgrA negatively regulates biofilm formation and detachment by repressing the expression of psm genes through directly binding to the promoter region of psm operons.S.aureus can secrete many kinds of virulence factors that play key roles in the pathogenesis of S.aureus.However,previous studies have not in detail revealed the pathogenesis of S.aureus,and there are some unknown virulence factors or virulence-associated regulators whose physiological functions and virulence should be elucidated.Part of this dissertation is to screen for virulence factors and to further investigate their physiological functions and pathogenesis in S.aureus.We constructed a himarl transposon library in MW2,a high virulence community-associated methicillin-resistant S.aureus(CA-MRSA),and use a high-throughput Caenorhabditis elegans-killing model for the determination of S.aureus pathogenicity to identify attenuated strains harboring a transposon inserted mutation gene.We further performed arbitrary primed PCR to identify the mutation gene,and then determined the virulence of these factors by cell infection assays.We also constructed gene knock-out strains by DNA recombination in MW2,respectively.We will perform in vitro and in vivo experiments to investigate their functions in pathogenesis of S.aureus.This research should not only add our knowledge of S.aureus pathogenesis but also provide novel strategy for treatment of S.aureus infection.