| ObjectiveTo analyze the bioinformatical characteristics of two component regulatory system VraSR of Staphylococcus epidermidis(SE1457),and express response regulator VraR protein.To explore the biological function of SE1457 VraSR,susceptibility and biofilm formation of SE1457 and its isogenic vraSR mutant were conducted combining with RNA sequencing,which can provide theoretical basis for the prevention of staphylococcal infection.Methods1.Based on the genome of the S.epidermidis SERP62A(ATCC35984),the primers were designed and the vraSR fragment of SE1457 was amplified and sequenced(3independent repeat).The nucleotides,amino acid,and domain of SE1457 VraS/VraR were analyzed using bioinformatical software.VraS/VraR analogs of S.aureus USA300,S.aureus Col,S.aureus Newman,S.aureus Mu50,S.aureus MW,Bacillus subtilis substr 168,Streptococcus mutans substr UA159,and Lactococcus lactis substr MG1363were downloaded from Genbank database,then sequence alignment and phylogenetic analysis were performed using Vector NTI and DNAMAN software.2.Expression of SE1457 VraR:the fragment of vraR was PCR-amplified from SE1457 genome,and cloned into vector pET-28a,then the resulting recombinant plasmid pET28a-vraR was transferred into E.coli BL21.Expression conditions(e.g.temperature,IPTG concentration,inducing time,etc.)were explored for suitable expression of recombinant protein 6His-VraR.3.The disc diffusion test and tube dilution method were used for susceptibility detection of SE1457 and its isogenic vraSR mutant against common antibiotics(e.g.vancomycin,ampicillin,penicillin G,kanamycin,chloramphenicol,tetracyline,and levofloxacin),respectively.Microtiter plate assay was used for the detection of biofilm formation of SE1457 and its isogenic vraSR mutant incubated for 6h,12h,24h and 48h.4.The RNA samples of SE1457 and its isogenic vraSR mutant incubated for 6h were extracted for RNA sequencing performed by Shanghai Biotechnology Corporation(3 independent experiments).Results1.The gene of vraS(locus serp1423)contains 1047 bp,encoding 348 amino acids,vraR(locus serp1422)contains 630 bp,encoding 209 amino acids,and the fragment of11 bp are overlapped between vraS and vraR.There are two open reading frames(ORFs,serp1425 and serp1426)in the upstream fragment of vraSR,and 4 bp of vraS is overlapped by the serp1424,and there is a reverse repeating sequence structure in the downstream of vraS.Moreover,Staphylococcus epidermidis VraSR has high homology with other bacterial analogues,especially with that of S.aureus up to 90%,sharing homology of 50-70%with that of Streptococcus mutans and Lactococcus lactis,to 50%with that of Bacillus subtilis.the protein of VraS is a transmembrane protein with a hydrophobic value of up to 4.0 in the transmembrane region;VraR is a cytosolic protein(containing HTH motif)that accepts a phosphate group,changes its conformation,exposes a DNA binding site,and binds to the promoter region of target gene,then regulate the transcription of downstream genes.2.The recombinant plasmid pET-28a-vraR was successfully constructed after verification by PCR,digestion reaction and sequencing.The recombinant protein6His-VraR was expressed with a concentration of 0.5mM IPTG at 22℃for overnight inducing.3.The drug susceptibility of SE1457 wild strain andΔvraSR mutant to vancomycin,ampicillin,penicillin G,kanamycin,chloramphenicol,tetracycline and levofloxacin was determined by disc diffusion method.The results showed that the sensitivity(Inhibition Zone)of SE1457 to the above seven drugs were 15±0.5 mm,40.2±2.1 mm,45.3±2.6mm,32.4±0.82 mm,27.6±0.66 mm,34±1.32 mm,34.5±1.0 mm,respectively.The sensitivity of theΔvraSR mutant to the above seven drugs(inhibition zone)were19±0.54 mm,43.5±1.8 mm,49.6±1.9 mm,33.5±0.79 mm,28.5±0.79 mm,34.9±2.18mm,34.8±0.99 mm,respectively.It is suggested that theΔvraSR mutant is more sensitive to the drug acting on the bacterial cell wall(such as vancomycin,ampicillin,penicillin G)than the wild strain SE1457(?~2=213,p=0.0001),and there was no significant difference against the drugs that inhibits bacterial protein synthesis(such as kanamycin,chloramphenicol,tetracycline)or drugs that inhibit DNA synthesis(such as levofloxacin)(?~2=189,p=0.267).The drug sensitivity ofΔvraSR mutan and its parental strain SE1457 to vancomycin,ampicillin,penicillin G,kanamycin,chloramphenicol,and levofloxacin were detected by tube dilution test,and showed that the MIC value of parental strain SE1457 against the above drugs were 4μg/mL,0.5μg/mL,0.5μg/mL,16μg/mL,8μg/mL,and 0.5μg/mL,respectively;the MIC value ofΔvraSR mutan were 1.0μg/mL,0.25μg/mL,0.25μg/mL,16μg/mL,8μg/mL,and 0.5μg/mL,respectively.It demonstrated that theΔvraSR mutan were more sensitive to vancomycin,ampicillin and penicillin G than that of SE1457 parental strain(?~2=9.44,p=0.037).And there was no significant difference against kanamycin,chloramphenicol,levofloxacin betweenΔvraSR mutan and its parental strain(?~2=0.001,p=1.000).4.The amount of biofilm formation(OD570)at different stages of biofilm formation(6h,12h,24h,48h)in SE1457 wild strain andΔvraSR mutant was determined by microplate quantitative method.The results showed that the biofilm formation amounts in SE1457 wild strain were 1.52±0.09,2.13±0.27,2.55±0.05 and 2.67±0.19 at the above four time points respectively;the biofilm formation amounts ofΔvraSR mutants were 0.72±0.02,0.95±0.06,1.23±0.23 and 1.41±0.21,respectively.It is suggested that the biofilm formation ability ofΔvraSR mutant is significantly lower than that of wild strain(t=3.787,p=0.009).5.In order to compare the transcriptional profiles of the mutant strainΔvraSR and the wild strain SE1457,RNA was extracted from the log phase(6 hours)bacteria and subjected to transcriptome sequencing for 3 times.It was found that 73 genes were differentially expressed,of which 58 genes were down-regulated and 15 genes were up-regulated.These genes are mainly involved in glycometabolism,phosphate metabolism,cell wall synthesis,global regulon,etc.,and there is no significance on the transcriptation levels of genes involved in drug resistance(pbp2,murA,sgtB).Conclusions1.Among the two component regulatory system VraS/VraR,sensor is VraS,and response regulator is VraR,the genes of vraS/vraR may form an operon with upstream two genes of serp1424 and serp1425.2.The suitable expression conditions for recombinant protein 6His-VraR are a concentration of 0.5mM IPTG,a temperature of 22℃,and inducing time of overnight incubation.3.TheΔvraSR mutant increased susceptibility to the antibiotics targeting on the bacterial cell wall than the parental strain SE1457,and there was no significant difference against the drugs that inhibits bacterial protein synthesis,meantime,ΔvraSR mutant decreased the ability of biofilm formation comparing with SE1457.RNA sequencing indicated that S.epidermidis VraSR is not directly involved in drug resistance,it modulates biofilm formation and susceptibility by influencing the integrity of bacterial cell wall,glucose mechanism and protein synthesis. |
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