Role And Mechanism Of WalK(S221P) Mutation In The Virulence Of Vancomycin-intermediate Staphylococcus Aureus

Staphylococcus aureus is a widespread Gram-positive pathogen that can cause diseases such as mild skin and soft-tissue infections,pneumonia,endocarditis,and sepsis.Rapidly acquired resistance to antibiotics in S.aureus has raised a great concern.Vancomycin has been proven effective in treating severe S.aureus infections,however,the treatment failure has increased since the first vancomycin-intermediate S.aureus(VISA)strain Mu50 was reported in 1997.The mechanisms underlying VISA formation are complex.Hundreds of mutations in certain genes discovered in VISA strains after compared with their vancomycin-susceptible counterparts(VSSA)have been associated with VISA formation,and the most mutations observed within genes encoding two-component systems(TCSs),such as walKR,vraSR and gra RS.Importantly,VISA is often presented a decline in its virulence,while the mechanisms underlying this phenomenon remain unclear.In our previous studies,we isolated a clinical ST239 type VISA strain termed XN108(vancomycin MIC=12?g/ml).A new WalK(S221P)mutation has proven to be associated with vancomycin resistance of XN108 after bacterial genome sequencing and comparison and the allelic replacement experiments.However,whether Wal K(S221P)mutation affects bacterial virulence is not clear.In this study,the virulence between XN108 and its Wal K(S221P)-cured strain K65 was compared with mouse models.RNA sequencing(RNA-seq)was performed to analyze the differentially expressed virulence genes in XN108and K65.Then,the mechanism underlying WalK(S221P)effects on the virulence of S.aureus was explored.Finally,the WalK(S221P)mutation was separately introduced into S.aureus strains of different genetic backgrounds,including methicillin-resistant(MRSA)strain USA300 and methicillin-susceptible(MSSA)strain Newman,to detect the variation of vancomycin resistance and the expression alteration of bacterial virulence of the mutants.The main results are as following:1.Reversion of WalK(S221P)in XN108 results in enhanced virulence of K65 in mouse modelsXN108 is a VISA with vancomycin MIC of 12?g/ml,and its Wal K(S221P)-reverted strain K65 exhibited 4?g/ml of vancomycin MIC.The mouse infection model was established by subcutaneously injecting flanks of mice with 5×10~8 CFU of XN108 and K65,respectively.Results showed that the abscesses caused by K65 between 1 and 7 days were significantly larger than those caused by XN108.Histological examination indicated that the K65-infected mouse skin presented extensive leukocyte infiltration,a damaged structure of skin,and a reduced dermal fat layer compared with that of XN108-infected mice.With murine systemic infection model,we observed that the bacterial loads in the spleen and kidney of the K65-infected mice were significantly higher than those in the XN108-challenged mice after 7 days of infection.These data suggest that reversion of Wal K(S221P)can enhance the bacterial virulence of VISA.2.Expression levels of virulence-associated genes are upregulated after reversion of Wal K(S221P)mutation in XN108RNA-seq was performed for XN108 and K65,and 13 differentially expressed genes(DEGs)were selected and confirmed by RT-q PCR.RNA-seq revealed that a total of 193DEGs of fold changes?2 were present in K65 compared with XN108,including 118unregulated and 75 downregulated genes.Among them,the expression levels of virulence genes,such as hla,lip,agr A and RNAIII were significantly unregulated in K65 compared with those in XN108.Hemolytic activity of K65 increased in comparison with that of XN108.3.The effect of WalK(S221P)on the virulence of VISA is Agr-dependentGiven the downregulation of agr genes in Wal K(S221P)-carried VISA strain XN108,we hypothesized that the effect Wal K(S221P)on VISA virulence is Agr-dependent.To test this hypothesis,a reporter vector(pOS1-agr)was constructed and the?-galactosidase assay demonstrated that?-galactosidase activity was significantly lower in Wal K(S221P)-carried XN108 than that in K65,suggesting an effect of WalK(S221P)mutation on the agr activity in S.aureus.We then searched the possible binding site of Wal R response regulator in the promoter regions of agr,and a typical Wal R-binding site was predicted.The electrophoretic mobility shift assay(EMSA)showed that Wal K-phosphorylated Wal R could directly bind to the agr promoter region,indicating that Wal KR may regulate the virulence of VISA through directly controlling Agr activity.Together,WalK(S221P)mutation impairs the virulence of VISA through an Agr-dependent pathway.4.Introduction of WalK(S221P)enhances vancomycin resistance in S.aureus of different genetic lineages but impairs the virulenceTo further investigate whether Wal K(S221P)mutation can affect vancomycin resistance and bacterial virulence in S.aureus of different genetic lineages,the Wal K(S221P)mutation carried MSSA strain(K-Newman)and MRSA strain(K-USA300)were constructed by homologous recombination technologies.E-test revealed the MIC of K-Newman increased up to 3.0?g/ml compared with wild-type(WT)Newman(MIC=1.0?g/ml),and K-USA300 also presented an increasing vancomycin-resistant phenotype(MIC=1.5?g/ml)compared with WT USA300(MIC=0.75?g/ml).RT-q PCR determination demonstrated that the expression levels of most regulators and virulence factors,including those encoding?-toxins(hla)and virulence regulators(agr A,RNAIII,gra R,and rot),were decreased in K-Newman and K-USA300 compared with those in their WT strains.The hemolytic activity of K-Newman as well as K-USA300 was also reduced compared with their progenitors.Taken together,these results indicate that bringing in a single Wal K(S221P)mutation into S.aureus of different genetic lineages can not only enhance their vancomycin resistance but also reduce the virulence of those mutant strains.In conclusion,our data indicate that a single WalK(S221P)mutation in staphylococcal TCS contributes vancomycin resistance,and reversion of Wal K(S221P)in VISA results in enhanced virulence presented as increased expression levels of virulence-associated genes.The?-Galactosidase assay and EMSA confirmed that the effect of Wal K(S221P)on the virulence of VISA is Agr-dependent.Introduction of Wal K(S221P)into S.aureus strains of different genetic backgrounds enhances the vancomycin resistance but impairs the virulence of the mutants.Our data provide new evidence to facilitate the understanding of mechanism underlying regulation of bacterial virulence in VISA.