Molecular Mechanisms Of Vancomycin-intermediate Resistance Conferred By A Novel Mutation Of WalK And Identification Of Toxin-antitoxin Like Element In Staphylococcus Aureus

Staphylococcus aureus is an important human pathogen that can lead to various infections,ranging from mild skin and soft tissue infections to life-threatening endocarditis,osteomyelitis,necrotizing pneumonia,toxic shock syndrome and sepsis.By horizontal gene transfer of mobile genetic elements and mutations in chromosomal genes,S.aureus is notorious for its extraordinary ability to develop resistance after the introduction of new antibiotics for the treatment of staphylococcal infections.Antibiotics such as penicillin and methicillin were introduced to effectively treat penicillin-susceptible S.aureus（PSSA）and penicillin-resistant S.aureus（PRSA）infections in the mid-20th century.However,the methicillin-resistant S.aureus（MRSA）strains were quickly generated and then spread globally,making vancomycin as the last line in the clinical treatment of serious staphylococcal infections.With the treatment failure by vancomycin and poor clinical outcomes,the emergence and spread of vancomycin intermediate-resistant Staphylococcus aureus（VISA）has raised more concerns in recent years.While the majority of VISA were reported to evolve from hospital associated MRSA,which display a similar genetic background of clonal complex 5 and 8,the methicillin-susceptible S.aureus（MSSA）-derived VISA from clinical or laboratory isolates are reported rarely and the mechanism underlying the generation is still largely unknown.In this study,we identified a total of 10 mutations composed of nine point mutations and one frameshift mutation in 9 genes（walK,NWMN₀306,lpl4nm,vraA,NWMN₁213,lexA,vraS,putP,and NWMN₂281）through comparative genome analysis from laboratory-derived VISA strain.We focused the role of a novel point mutation of WalK（I237T）as it is located in the sensor protein of WaIKR,the only necessary two-component signal transduction system of S.aureus,which is vitally important in the regulation of cell wall metabolism,autolysis,and cell death.Our results indicated that the introduction of WalK（1237T）by allelic replacement can confer vancomycin resistance in MSSA with common VISA characteristics,including thickened cell walls,reduced autolysis,and changed virulence.Consistent with these phenotypes,real-time quantitative reverse transcription-PCR revealed the altered expression of several genes associated with cell wall metabolism and virulence control.In addition,fluorescence-based promoter activity and β-galactosidase assays revealed WalK（I237T）can decrease promoter activities of slel and oatA,and increase promoter activity of mgt.Electrophoretic mobility shift assay indicated that WaIR can directly bind to the promoter regions of slel,oatA,and mgt,suggesting that the WalK（I237T）can regulate expression of target genes by WalR.These findings broaden our understanding of the regulatory network by WalKR system and decipher the molecular mechanisms of developmental VISA resistance in MSSA with point mutations.In addition,we found a Toxin-antitoxin-like element which consists of SA1833 and SA1832 in the genome of N315.It shares some commen features with the type II TA system,as the components are expressed in a co-transcriptional manner,and the proteins they encoded can form a stable complex and bind to its own promoter sequence to achieve self-regulation.However,overexpression of SA1833 and SA1833-SA1832 by plasmid pRMC2 can inhibit cell growth equally which emplying the deficiency of antitoxin component to neutralize the toxicity of the toxin,so it is difficult to classify it as a classic type Ⅱ TA system and identified as a novel TA-like element instead.Through a variety of environmental stress stimuli,we found this system can be upregulated significantly under SOS,oxidative and stringent stress.Among them,the response of SA1833-SA1832 to SOS and oxidative pressure is dependent on the LexA-RecA pathway.For the further research,we will conduct a more in-depth study on its molecular mechanisms of the inhibition to cell growth and the physiological processes that it may participate in.