Control Of Virulence By The AraC/XylS Family Transcriptional Regulator Rsp In Staphylococcus Aureus

BackgroundThe Gram-positive bacteria Staphylococcus aureus can cause a variety of human diseases, ranging from superficial abscesses and wound infections to deep systemic infections such as endocarditis, osteomyelitis, toxic shock syndrome （TSS） and septicaemia. A considerable increase in the prevalence of MRS A has been observed globally during the last decade after the introduction of methicillin in clinical settingsInfections due to MRS A are difficult to treat because of the restricted spectrum of antimicrobials of proven efficacy. Pandemic CA-MRSA strains represent a serious problem for the public health system. The recently emerging CA-MRSA strains combine antibiotic resistance with high virulence and transmissibility, enabling them to spread and cause severe infections in healthy people. Remarkably, in the US, the estimated number of deaths due to MRSA infections exceeds that due to HIV/AIDS. However, the virulence factors and pathogenesis of MRSA are still far from being understood, a situation calling for enhanced research efforts in that area.The expression of these virulence factors is under the control of a network of regulatory systems, the most important global regulator is agr, controlling the expression of most of the exoprotein genes. Further support for the concept of global regulation of virulence was obtained by the subsequent isolation of other pleiotropic mutants including sarA, sae, sarS and rot etc. The virulence regulatory network in Staphylococcus aureus is multifactorial, and much remains to be explored.Part1Transposon mutagenesis of S. aureus USA500 and screening of a transposon libraryObjective To build a transposon library through the mariner-based transposon system and screen the gene that regulated the hemolytic capacity of Staphylococcus aureus.Methods Mariner-based transposon mutagenesis was performed in the clinical isolate S. aureus strain BD02-25 （USA500） and a transposon library was built. Then screen the transposon library by detection of hemolytic capacity on sheep blood agar plate.Arbitrary primed （inverse） PCR and nucleotide sequence were used to detect the gene that regulated the hemolytic capacity of Staphylococcus aureus.Results A large number of clones （about 10,000） were used for screening. In our study,4/25 clones which were significantly deficient in hemolytic activity had a single pBTn insertion at 4 different locations in the gene （SAUSA300₂326）. This gene codes for a 701-amino-acid protein with homology to the AraC/XylS family of transcriptional activators, shares 99% identity and 100% similarity in amino acid sequence with the biofilm regulator gene rsp of S. aureus.Conclusions AraC/XylS family transcriptional regulator Rsp can regulate hemolytic capacity in S. aureus.Part 2The function of AraC/XylS family transcriptional regulator Rsp in S. aureus.Objective To investigate the regulation of virulence by the AraC/XylS family transcriptional regulator Rsp in S. aureus.Methods Plasmid PKOR1 was used to delete the rsp gene from the genome of S. aureus strain BD02-25. For genetic complementation, the rsp gene was amplified by PCR and cloned in plasmid pRB473. The growth curves of wild type, mutant strain and complement strain were drawn. Semiquantitative biofilm assays were performed. Mice bacteremia and skin abscess models were built to study the function of Rsp protein on virulence. Then we analyzed different expression profiles in widetype, mutant, and complement strain by RNA-sequence and using quantitative RT-PCR method to verify the results of RNA-sequence.Results Growth curves showed that the growth of the rsp mutant strain grew slower than complement strain and wildtype, but no difference was observed after 24 hours of growth. The semiquantitative biofilm assays suggested that rsp downregulated biofilm formation. The mouse bacteremia experiment showed that the survival rate of rsp mutant was significantly higher than the wild-type strain. The mouse skin abscess model experiments indicated that the abscess area of the wild-type strain was significantly larger than that of rsp mutant. The RNA-sequence suggested that rsp can upregulate many proteins （such as hemolysin, lysin leukocyte protease aur, ssp and regulatory proteins agrD, sarR etc）, a variety of proteins can be reduced （such as autolysin atl, surface protein sasG, isaB and regulatory proteins bccT, opuCa, opuCb etc）. Quantitative RT-PCR validated the results of RNA-sequence that rsp can upregulate the expression of virulence factors （hla, RNA â…¢ and PSM）, downregulate the expression of the bacterial biofilm formation associated factor （icaA） and metabolic -related factors.Conclusion AraC/Xyls transcription regulatory protein Rsp can upregulate the virulence of S. aureus.Part 3The regulatory mechanisms of Rsp protein in S. aureus virulenceObjective To analyze the regulatory mechanisms of the AraC/XylS family transcriptional protein Rsp on S. aureus virulence.Methods Inducing, expression and purification of Rsp protein with DNA binding domain, observing the interaction between Rsp protein and the target gene promoter by electrophoretic mobility shift assay （EMSA）.Results Electrophoretic mobility shift assay showed that Rsp protein played a positive regulatory role in virulence of S. aureus through binding the agrD gene promoter.Conclusion The AraC/XylS family transcriptional protein Rsp regulates its target genes by direct DNA binding at the promoter region, thus affecting the virulence of S. aureus.