| ObjectiveIn this study,the two-component signal transduction system VraSR involved in multiple biological processes was further explored in regulating the biofilm formation and susceptibility of Staphylococcus epidermidis,aiming to provide theoretical basis for clinical control of infection caused by S.epidermidis and to find potential drug targets.Methods1.Construction of S.epidermidis vraSR complementary strain:Using genomic DNA of Staphylococcus epidermidis strain 1457（SE1457）as template,the vraSR gene with the Shine-Dalgarno sequence was amplified by PCR,and ligated into plasmid p CN51 after digestion.The ligation product was transformed into E.coli DH5a.The recombinant plasmid p CN51-vraSR was verified by PCR,enzyme digestion and sequencing,and then transformed into Staphylococcus aureus RN4220,and then transferred into SE1457 vraSR deletion mutant strain（?vraSR）to obtain the vraSR complementary strain?vraSR（p CN51-vraSR）.At the same time,the empty plasmid p CN51 was introduced into?vraSR as the control,which was named?vraSR（p CN51）.The vraSR deletion mutant strain（?vraSR）was derived from SE1457 by allelic replacement using plasmid p KORI.2.Detection of the phenotype of the SE1457 isogenic vraSR mutants:biofilm formation of SE1457 isogenic vraSR mutants were detected by microplate semi-quantitative method,and the susceptibility was detected by the broth dilution method.The viability and tolerance of SE1457 isogenic vraSR mutants to the environmental stressor（vancomycin,SDS,H2O2,Triton X-100）were performed by the colony forming unit（CFU）.The release of extracellular DNA（e DNA）in the biofilm matrix of S.epidermidis strains were quantitated by Real-time PCR（q-PCR）using the gyr B（gyrase B）,serp0306（ferrichrome transport ATP-binding protein A）,leu A（2-isopropylmalate synthase）,and lys A（diaminopimelate decarboxylase A）.The relative quantitation of e DNA in each sample was calculated as total the e DNA（in ng）divided by the biofilm biomass(OD600).The autolysis assay induced by Triton X-100was carried out.The Congo red assays of SE1457 isogenic vraSR mutants were performed for the exploration of the cell wall integrity at the different timepoints（24h,48h,72h,96h）and the morphology of bacterial cell wall was observed by transmission electron microscope.3.The staphylococcal bacteria in logarithmic growth phase were treated with environmental stressor（vancomycin,ampicillin,chloramphenicol,H2O2,SDS）for30min,and RNA samples were extracted,then the transcriptional levels of vra S/vra R were analyzed by qRT-PCR.Transcriptome sequencing（RNA-seq）was used to analyze the differentially expressed genes between SE1457 and?vraSR mutant,and the genes probably regulated by VraSR（especially for the genes related to biofilm formation and susceptibility）were verified by qRT-PCR.To determine the interaction between VraR and the promoter regions of putative target genes,the recombinant protein VraR（His6-VraR）of S.epidermidis was purified using Ni-NTA column chromatography,and electrophoresis mobility shift assay（EMSA）was carried out using DNA probes labeled with biotin.Results1.PCR and sequencing results showed that the recombinant plasmid p CN51-vraSR was successfully constructed.qRT-PCR indicated that the transcriptional level of vraSR in?vraSR（p CN51-vraSR）complementary strain was1.8 folds compared with that of SE1457 parent strain.To investigate the effect of vraSR deletion on biofilm matrix production,the amount of extracellular DNA（e DNA）was quantified by Real-time PCR（q-PCR）(ng/OD600)with SYBR.The relative concentration of e DNA in 24-h biofilm of?vraSR and?vraSR（p CN51）strains was 2 folds higher than that of SE1457 and?vraSR（p CN51-vraSR）.2.Effect of vraSR deletion on the biological phenotypes of Staphylococcus epidermidis:（1）The biofilm formation of Staphylococcus epidermidis was determined by the semi-quantitative microplate assay.Staphylococcal biofilms on 96-well microtiter plates at 6,12,24,and 48 h were stained with crystal violet and the biomass was quantified by measuring the optical density at 570 nm.The biofilm formation amount(OD570)of the parent strain SE1457 at the above time points were 1.74±0.10,2.45±0.20,2.74±0.11 and 2.82±0.15,respectively.Compared to SE1457 parent strain,?vraSR produced less biofilm（P<0.01）with lower values of OD570at the above time points（0.81±0.07,1.22±0.1,1.71±0.05 and 1.76±0.10,respectively）.The biofilm formation of?vraSR（p CN51-vraSR）complementary strain was restored to the level of SE1457 parent strain.The biofilm formation amount(OD570)of?vraSR（p CN51-vraSR）complementary strain at the four observation points were1.36±0.06,1.80±0.04,2.31±0.17 and 2.79±0.09,respectively.The biofilm formation of?vraSR（p CN51）empty control strain was similar to the level of?vraSR mutant.（2）The susceptibility of SE1457 isogenic vraSR mutants were determined by the broth dilution method.The MICs of the parent strain SE1457 to vancomycin,ampicillin,kanamycin and chloramphenicol were 4μg/m L,0.5μg/m L,8μg/m L and8μg/m L,respectively.The MICs of?vraSR were 1μg/m L,0.25μg/m L,8μg/m L and8μg/m L,respectively.?vraSR increased the susceptibility to the cell wall antibiotics（vancomycin and ampicillin）compared with that of SE1457 parent strain,whereas there was no significant difference in the susceptibility to kanamycin and chloramphenicol.The susceptibility to vancomycin and ampicillin was restored when the complementary plasmid p CN51-vraSR was introduced into?vraSR.The MICs of?vraSR（p CN51-vraSR）complementary strain were 4μg/m L,0.5μg/m L,8μg/m L and8μg/m L,respectively.However,there had no effect on the susceptibility of?vraSR when empty control plasmid p CN51 was electroporated into it,while the MICs of?vraSR（p CN51）vector control strain were 1μg/m L,0.25μg/m L,8μg/m L and 8μg/m L,respectively.（3）The growth curve of Staphylococcus epidermidis was determined by spectrophotometer(OD600).The vraSR deletion had no effect on the growth of SE1457,we further studied the viability and tolerance of SE1457 isogenic vraSR mutants to the environmental stressor（vancomycin,SDS,H2O2,Triton X-100）using colony forming unit（CFU）.Staphylococcal strains were diluted into TSB（1:200）and incubated at 37℃with shaking at 180 rpm,and an aliquot of 100mL was taken for CFU counting at the different stage of the growth curve.The viable cell（CFU/m L）of SE1457 incubated at 8h,12h,24h and 48h were 1.52×109,2.19×109,3.23×109and3.18×109,respectively.The viable cell of?vraSR was decreased compared with that of parent strain SE1457（P<0.05）,while the values of?vraSR CFU counting were1.19×109,1.02×109,5.6×108,and 1.21×108.CFU was used to evaluate the tolerance of staphylococcal cells to the environmental stressor.The log-phase bacteria of SE1457 isogenic vraSR mutants were exposure to the chemical agents（vancomycin,SDS,H2O2,Triton X-100）,and aliquots were taken out for CFU counting every 12h until to 120h.Compared to SE1457,the tolerance of?vraSR was significantly reduced after 72-h exposure to vancomycin,48-h exposure to SDS and Triton X-100,and no viable bacteria were detected.There was no significant difference in tolerance to oxidative stress H2O2between SE1457 and?vraSR.The difference in the autolysis between SE1457 and?vraSR was detected using Triton X-100 induced autolysis assay.The mid-log phase cultures of SE1457 isogenic vraSR mutants were adjusted to an OD600value of 1.0,incubated with 0.2%Triton X-100 for 6 h and measuring using a spectrophotometer at 600 nm.The results showed that the autolysis rate of?vraSR was 74%after 3-h incubation,which was significantly high than that of parent strain（34%）.The cell wall integrity of SE1457 isogenic vraSR mutants was assessed by Congo red absorption assay.Compared with SE1457 parent strain,?vraSR absorbed more amount of Congo red,and the central part of the colony in?vraSR mutant was redder.The cell wall morphology of SE1457,?vraSR and?vraSR（p CN51-vraSR）.strain were observed by transmission electron microscopy.The results showed that compared with SE1457 wild strain,the cell wall surface of?vraSR was rougher and the cell wall was discontinuous.The cell wall thickness of SE1457 was 37.93±7.2μm、?vraSR was 29.79±2.8μm（P<0.01）、ΔvraSR（p CN51-vraSR）was 35.04±4.52μm;The phenotype ofΔvraSR（p CN51-vraSR）complementary strains returned to the wild level.The phenotype of?vraSR（p CN51）empty control strain was similar to?vraSR.3.A proposed mechanism of VraSR in regulating Staphylococcus epidermidis biofilm formation and susceptibility:（1）The transcription level of vraSR in SE1457 upon the environmental stressor was analyzed by qRT-PCR.The transcription level of vraSR in SE1457 treated with10-fold MIC concentration of vancomycin and ampicillin were up-regulated 4.8 and7.2 folds,respectively.Upon the SDS stress,the vraSR expression of SE1457 was also upregulated about 7 folds.However,the transcription levels of vraSR in SE1457treated with chloramphenicol,oxidative pressure（H2O2）,heat（42℃,45℃）and hyperosmosis（1.5%Na Cl）did not change significantly.These results suggest that VraSR selectively responds to cell wall antibiotics（vancomycin,ampicillin）and SDS,and that S.epidermidis VraSR may play a role in adaptation to environmental stress.（2）The differentially expressed genes of SE1457 and?vraSR were analyz ed by transcriptome sequencing（RNA-seq）.A total of 314 differentially expres sed genes were found,among which 249 genes were up-regulated and 65 gene s were down-regulated.The results were further verified using qRT-PCR,and t he transcription level of ica A（N-acetylglucosaminyltransferase）,the gene related to biofilm formation,was decreased 2 folds in?vraSR mutant,and the ica R expression was increased 4 folds.The transcription level of lrg A（antiholin like protein）was down-regulated about 10 folds,and cid A（holin like protein）was up-regulated about 2 folds.The transcription level of gnt K related to glucose metabolism was down-regulated about 10 folds,and glp D expression was down-regulated about 2 folds.The gene of sdh A related to pentose phosphate pathw ay was upregulated about 2 folds.The transcriptional level of genes directly re lated to staphylococcal drug resistance,such as sgt B（glycotransferase）,mur AA（UDP-N-acetylglucosamine-1-carboxyvinyltransferase）,mur E（UDP-N-acetylmuramo yl-L-alanyl-D-glutamate-L-lysine ligase）,and pbp2（penicillin binding protein）,d id not change significantly（P<0.05）,suggesting that S.epidermidis VraSR doe s not directly regulate the drug resistance.（3）Electrophoretic mobility shift assay（EMSA）was used to detect the binding of recombinant VraR（His-tagged VraR）to the promoter regions labeled with Biotin.Electrophoretic mobility shift assay（EMSA）further demonstrated that phosphorylated VraR（VraR-P）could bind to the promoter regions of vraSR,ica,lrg AB and cid A genes,while VraR-P could not bind to the promoter regions of pbp2,sgt B,mur E and mur AA genes.ConclusionStaphylococcus epidermidis VraSR is autoregulated upon the environmental stress targeting cell wall synthesis,and it promotes biofilm formation by regulating the ica operon transcription.Furthermore,S.epidermidis VraSR regulates bacterial cell death through modulating Cid A/Lrg A system,and indirectly affect drug resistance in association with the alterations to multiple metabolism pathways.This study provides a probable regulatory network for S.epidermidis biofilm formation and cell death. |
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