Molecular Mechanism Of Virulence Gene Expression Modulated By Rsp In Staphylococcus Aureus

Staphylococcus aureus is a detrimental and versatile human pathogen responsible for a wide diversity of community-and hospital-acquired infections,ranging from innocuous skin infections to life-threatening conditions like pneumonia,osteomyelitis and infective endocarditis.The pathogenicity of the bacterium is a sophisticated process involving multiple virulence factors,such as exotoxins,enzymes,and surface protein adhesins.The expression of virulence factors is coordinately controlled by three global regulators,including Agr system,SarA family proteins,and SaeRS two-component system,which are believed to enable S.aureus to survive and to elicit subsequent infection in different environments.The AraC/XylS family is a group of transcriptional regulators with a highly conserved 99 amino acid region containing two helix-turn-helix（HTH）DNA binding motifs at the C terminal.Members of this family are widely distributed in prokaryotes and mainly involved in metabolism of carbon sources,responses to environmental stress and pathogenesis.In S.aureus,6 AraC/XylS-like proteins are predicted and one of which,Rsp,has been reported to play a crucial role in the regulation of virulence genes via an agr-dependent pathway.However,whether the target genes are under the direct control of Rsp remains unknown.So in this dissertation,we aim to further investigate the regulatory mechanism of Rsp in S.aureus NCTC8325.β-Galactosidase assay and electrophoretic mobility shift assay demonstrated that Rsp could bind to the rsp promoter to positively regulate its own expression.Deletion of rsp in S.aureus NCTC8325 resulted in decreased haemolytic activity.Real-time quantitative reverse transcription-PCR indicated that the transcript levels of virulence genes,hla and psm,were significantly decreased in the rsp mutant strain.The agr locus is the most thoroughly investigated virulence regulatory element in S.aureus,which has a pronounced effect on the expression of hla and psm.Our results indicated that Rsp can modulate the haemolytic activity of S.aureus and the expression of hla and psm independent of agr.Furthermore,electrophoretic mobility shift assay indicated that Rsp can directly bind to the promoter regions of hla and psm.The mouse model of subcutaneous abscess showed that the rsp mutant strain displayed a significant defect in virulence compared to the wild-type strain.Collectively,these findings reveal that Rsp positively regulates the virulence of S.aureus by promoting the expression of hla and psm through direct binding to their promoter regions.We have also found that an AraC/XylS-like protein encoded by SAXN108₂546,is involved in antibiotic resistance in vancomycin-intermediate resistance S.aureus（VISA）strain XN108.Deletion of SAXN108₂546 in strain XN108 caused a significant decrease in glycopeptide and daptomycin resistance and an increase in Triton X-100-induced autolysis.