Study On The Molecular Mechanism Of Vancomycin Intermediate Resistance In Staphylococcus Aureus

Staphylococcus aureus is a major human pathogen that can cause a varity of diseases.Antibiotic resistance of S.aureus is serious along with the widespread use of antibiotics in clinic.The emergence and prevalence of methicillin-resistant S.aureus（MRSA）have brought new challenges to the treatment of clinical S.aureus infections.Vancomycin is considered as the last line of drug in the treatment of severe MRSA infections.With its increasing used from 1980 s,S.aureus isolates with reduced vancomycin-susceptibility were emerged.Vancomycin-intermediate S.aureus（VISA）was first reported in Japan in 1997 and subsequently reported in many countries.In 2017,WHO listed VISA as one of the 12 "superbugs" needed to be controlled.Therefore,it is of great importance to explore the mechanisms underlying VISA formation,which can facilitate the development of new strategies to control VISA infections.The mechanism of vancomycin performing bactericidal effect is based on its binding activity to the D-alanyl-D-alanine（D-Ala-D-Ala）of the precursors for peptidoglycan biosynthesis.The mechanism of VISA is very complicated and no horizontal-transferred gene clusters were identified in VISA isolates.Genome sequencing and comparative analysis of VSSA/VISA strain pairs or series revealed that dozens of mutated genes are closely related to the formation of VISA.The mutations in certain genes have cumulative effects.The mutations mostly occurred in the two-component system（TCS）genes,such as walKR,vraSR,and graSR.The WalKR two-component system is essential for the viability of Staphylococcus aureus,playing a central role in controlling cell wall metabolism.VraSR system plays a central role in maintaining the integrity of the cell wall peptidoglycan and coordinating the S.aureus response to cell wall damage,much like an antibiotic resistance mechanism.GraSR is a glycopeptide resistance associated TCS,which controls stress reponse and cell wall metabolism signal transduction pathways.In addition to the vancomycin-intermediate resistance,VISA strains exhibit some common phenotypes,such as thickened cell wall and reduced autolysis.How diverse mutations in certain genes lead to the formation of common phenotype in VISA is not known.We isolated and characterized a VISA strain XN108 that exhibits a vancomycin MIC of 12 μg/ml.In this study,the comparative genomic analysis was performed to screen potential mutations that may contribute to the vancomycin resistance in XN108.Gene knockout,complementation,and replacement were used to test the effect of these mutated genes on vancomycin resistance.Transcriptomics and metabolomics revealed that a new metabolism regulatory cascade CcpA-GlmS plays important roles in mediation of vancomycin resistance in VISA.The main research contents and results are summarized as following:1.Effect and mechanism of WalK（S221P）mutation in mediating vancomycin resistance in XN108Comparative genomics analysis was performed based on the genomes of XN108（GenBank accession number: CP007447）and VSSA TW20（NC₀17331）,and the results showed that three mutations,WalK（S221P）,GraS（T136I）,and RpoB（H481N）,might be responsible for the vancomycin resistance in XN108.WalK（S221P）is a novel mutation site discovered in XN108.We first mutated the WalK coding gene of VSSA strain N315 to WalK（S221P）,and the vancomycin MIC of the mutant increased from 1.5 μg/ml to 8 μg/ml;while replacing WalK（S221P）in XN108 with WalK（P221S）from N315,the MIC of vancomycin decreased from 12 μg/ml to 4 μg/ml.Transmission electron microscopy revealed that N315-WalK（S221P）strain had thickened cell wall,while the cell wall thickness of XN108-WalK（P221S）was decreased.Autolysis experiments also confirmed that the autolysis ability of the mutants changed accordingly.These results indicate that the novel WalK（S221P）mutation plays an important role in the vancomycin resistance of XN108.Protein phosphorylation analysis showed that the autophosphorylation level of WalK（S221P）was significantly decreased compared with that of wild-type WalK,which led to a decrease in the ability to activate WalR,and then decreased binding ability of WalK（S221P）phosphorylated WalR to the promoter DNA fragment of the WalR target gene lytM.These results indicate that WalK（S221P）can cause a decrease in the ability of WalR to regulate target genes and plays an important role in mediating vancomycin resistance in XN108.2.WalK（S221P）and GraS（T136I）synergistically mediate the vancomycin resistance in VISA XN108To further investigate the roles of mutations in XN108 resistance,the WalK（S221P）,GraS（T136I）,and RpoB（H481N）mutations were stepwise cured with the relative genes from a VSSA strain DP65.The mutants XN108-WalK（P221S）,XN108-GraS（I136T）,XN108-RpoB（N481H）,XN108-WalK（P221S）/GraS（I136T）,and XN108-WalK（P221S）/ GraS（I136T）/RpoB（N481H）were termed as K65,S65,B65,KS65,and KSB65,respectively.Antibiotic susceptibility test showed that the vancomycin resistance of K65 and S65 were decreased,while that of B65 was consistent with XN108.Recovery of both WalK（S221P）and GraS（T136I）completely converted XN108 into a VSSA phenotype（KS65）.The cell wall thickness and autolysis ability of certain mutants were changed accordingly to the vancomycin resistance levels.These findings indicate that a VSSA phenotype can be completely reconstituted by gradually recovering the certain mutations in XN108.Recovery of WalK（S221P）and GraS（T136I）is sufficient to concert XN108 into a VSSA phenotype,while RpoB（H481N）mutation has marginal effect on the vancomycin resistance in XN108.3.CcpA-GlmS metabolism regulatory cascade mediates vancomycin resistance in VISA strains of diverse genetic lineagesA variety of mutations in certain genes are associated with VISA formation,whereas VISA strains show several common phenotypes such as thickened cell wall and decreased autolysis.There is a "black box" between the upstream mutated genes and the downstream common phenotypes in VISA.Elucidation of this black box will facilitate the understanding of VISA formation.We found that the increase in VISA resistance level was accompanied by upregulation of CcpA,a global regulator of carbon metabolism.To investigate the role of CcpA in VISA,we knocked out ccpA gene in XN108 and found that the vancomycin MIC of XN108ΔccpA was reduced to 4 μg/ml.After complement of ccpA,the vancomycin resistance level of the complement strain was restored.Subsequently,knock-out of ccpA in K65 and Mu50 strains also resulted in a significant decrease in the vancomycin resistance level,indicating that CcpA is involved in the regulation of vancomycin resistance in VISA strains of diverse genetic lineages.EMSA experiments confirmed that the two-component system WalKR can directly regulate ccpA,while GraSR may regualte ccpA expression indirectly.Metabolomic analysis of 39 metabolites in XN108ΔccpA and XN108 revealed that the accumulation of glutamine（an important precursor for cell wall synthesis）in the XN108ΔccpA,accompanying with the decreased expression of glutamine fructose-6-phosphate transaminase GlmS.We speculated that CcpA may play a key role in the regulation of VISA resistance by controlling the expression of GlmS.EMSA showed that CcpA can directly bind to the promoter of glmS,indicating that CcpA can directly regulate GlmS to form a CcpA-GlmS regulatory cascade.Deletion of glmS gene in XN108 resulted in decreased vancomycin resistance（the vancomycin MIC of XN108ΔglmS is 3 μg/ml）.Overall,this work reveals an unknown mechanism underlying the promotion of common phenotypes in VISA by major mutant genes.