Virulence Regulation By The Toxin-antitoxin System SavRS In Staphylococcus Aureus

Staphylococcus aureus is a ubiquitous human pathogen responsible for increasing numbers of infectious morbidity and mortality in both communal and hospital settings.These infections range from superficial skin lesions to life-threatening inflammation,sepsis,endocarditis,and toxic shock.These virulence factors are cumulative and modulated by diverse regulatory factors,such as two-component system,quorum sensing,and sigma factors family,which give them a vast ability to survive,to induce infection,and to evade host immune defense.Toxin-antitoxin?TA?systems have been found in diverse bacteria and archaea and play broad biological roles.A typical TA system consists of a stable toxin that can cause cell death by disrupting an essential cellular process and a labile antitoxin that can prevent the toxin from exerting its toxicity by forming a complex with toxin.TA systems have a general role in stabilization of genomic parasites,growth control,programmed cell death,and persister cell formation under stress stimuli.More and more studies have demonstrated a regulatory role among TA systems.The majority of their regulatory roles are implemented by toxins containing site-specific endoribonuclease activity.Here,we have characterized a regulatory role is implemented by antitoxin in Staphylococcus aureus NCTC8325.A novel type ? toxin-antitoxin system,SavRS encoding by SAOUHSC₀2756-02757,can regulate more than its own transcription.SavRS can bind directly to the promoters of virulence genes and regulate the transcription of these loci.The toxicity of SavS is lethal to bacterial cells,and this toxicity can be encounted by the expression of the antitoxin SavR.Electrophoretic mobility shift assay and isothermal titration calorimetry data indicated that the antitoxin SavR acted as the primary repressor bound to its own promoter,while the toxin SavS formed a complex with SavR to enhance the ability to bind to the operator site.DNase I footprinting assay identified the SavRS-binding site containing a short palindrome?5'-ACT**AGT-3'?and long palindrome?5'-TACGCTA****TAGCGTA-3'?in the promoter region.Further,mutation and DNase I footprinting assay demonstrated that the two palindromes were crucial for DNA binding and transcriptional repression.More interestingly,genetic deletion of the savRS system led to the increased hemolytic activity and pathogenicity in a mouse subcutaneous abscess model.We further identified two virulence genes,hla and efb,by real-time quantitative reverse transcription-PCR and demonstrated that SavR and SavRS could directly bind to their promoter regions to repress virulence gene expression.We also found that the persister cell formation of in the savS mutant is significantly decreased with ampicillin treatment.We chose seveval genes,of which the transcription levels were changed through RNA-seq and futher assessed by real-time quantitative reverse transcription-PCR.In addition,we overexpressed the rmpH and checked the levels of persister cell formation.We have also characterized a putative TA system that is encoded by SAOUHSC 02691-02692.It is a type ? TA system that can autoregulate its own transcription and the exertion of its toxicity.The toxin inhibits cell growth only when the TA element is deleted.Further,we analyzed the structure of toxin-antitoxin complex and checked the phenotypes of the muant strains.