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2013 ARCHIVES OF CLINICAL MICROBIOLOGY
Vol. 4 No. 5:3 doi: 10.3823/274
Our Site: http://www.imedpub.com/
Transcriptomic study reveals new pathways and genes involved in Enterococcus faecalis V583 response to a therapeutic dose of Vancomycin
Tânia Ribeiro1, Neuza Teixeira1, Ryoji Yokohata2, Jiro Nakayama2, Michael S. Gilmore3, Maria de Fátima S. Lopes1,4*
1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal. 2 Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka, Japan. 3 Harvard Medical School, Massachusetts Eye and Ear Inﬁrmary, Boston, Massachusetts, USA. 4 IBET, Oeiras, Portugal.
Maria de Fátima Silva Lopes. Address: IBET, Apartado 12, 2781-901 Oeiras, Portugal; Phone: +351214469559; Fax: +351214421161
Background: An enterococcal strain carrying the VanB resistance type can become susceptible if impaired in other genes unrelated to the vanB operon. This fact alone illustrates the lack of knowledge on the vancomycin mode of action. This antibiotic is still usable to treat serious infections caused by multiresistant enterococcal strains, but may not be so for long. This work was thus set up to gather a body of knowledge that can be used in the future to increase efﬁcacy against both vancomycin resistant (VRE) and susceptible enterococci (VSE).
Methods and Findings: Microarrays were used to detect the genetic response
of the VanB carrying strain Enterococcus faecalis V583 to a therapeutic dose (10 mg/ml) of vancomycin. Besides the vanRS genes, two other two-component systems were induced. The therapeutic dose of vancomycin was found to act as an anti-virulence agent, by turning-off the Fsr quorum-sensing system. Key regulators and metabolic enzymes, involved in trafﬁcking carbon sources into glycolysis and isoprenoid synthesis and utilization, were also affected in order to support cell-wall synthesis. Also, cell-wall modiﬁcation involving lipotheicoic acid synthesis, DNA repair and protein folding were highly responsive functions to the vancomycin dose tested.
Conclusions: Overall, our results provide clues on the ability of a VRE strain to This article is available from: www.acmicrob.com
stand vancomycin and on the mode of action of the antibiotic. VRE response to a vancomycin therapeutic dose involves an intricate regulatory network and metabolic adjustment which is worth solving as it can help ﬁnding new targets to ﬁght both VRE and VSE infections.
During the ﬁve decades of vancomycin usage, eight genotypes of resistance to this antibiotic have been described in only one genus, Enterococcus. Vancomycin, a cell-wall active glycopeptide antibiotic, was ﬁrst introduced to clinical practice in the 60’s and in 1986 the ﬁrst resistant enterococcal strain was isolated [�]���������������������������������������� ���������������������������������������� . Since then, vancomycin resistant enterococci (VRE) have been isolated from endocarditis, bacterae© Copyright iMedPub
mia, urinary tract infections and wound infections, and have emerged as one of the major nosocomial agents in hospitals . It is currently unknown why so many different resistance genotypes have converged in the genus Enterococcus. S. aureus (2002) recently acquired vancomycin resistance from an enterococcal strain . Among the eight resistance types, VanA and VanB constitute the two most widely disseminated, both conferring resist-
are responsible for survival and growth maintenance in the presence of the antibiotic. including all of the factors that contribute to its resistance. Cultures were then diluted 20 fold and grown in BHI until an optical density of 0. Although the genetic basis for vancomycin resistance is known.com/ ance by the same mechanism and encoding related enzymes ��������������������������������������������������������� . and that Fsr quorum-sensing system. GelE-. Cultures were split in two. V583. 12] has been studied in a resistant strain. vanB encodes a ligase that synthetizes the depsipeptide D-Ala-D-Lac. 2002)  are induced by vancomycin in both VRE and in vancomycin susceptible enterococci (VSE). vanB and vanX are essential for resistance phenotype. 7]. to vancomycin. To achieve this goal. The regulatory and resistance genes are transcribed from distinct promoters that appear to be co-ordinately regulated . clinical isolate derived strain. we subjected E. We found that CroRS (ef3289-ef3290). the ﬁrst VRE genome to be sequenced. and TCS06 (ef1260-ef1261. Expression of the resistance genes is regulated by the vanRBSB two-component system. besides those involved in resistance to the antibiotic. which is composed of a membrane-associated sensor kinase (VanSB). faecalis V583. with VanB type of resistance FusR RifR. faecalis V583 ∆fsrB. vanHB encodes a dehydrogenase that reduces pyruvate to D-Lac. Strains and primers used in this study. VaR. and VanXB hydrolyses the D-Ala-D-Ala dipeptide synthetized by the native Enterococcus Ddl ligase. Knowledge of the factors that contribute to the “resistome” of Enterococcus for vancomycin will be important for the design of new drugs to treat resistant infections.imedpub. and a cytoplasmic response regulator (VanRB) that acts as a transcriptional activator [5. Relevant characteristics Strains V583 JH2-2 JH22∆croR JH22∆err06 SAVE 9 SAVE 11 VI13 Plasmids pAT80 Primers Gene Sequence (5´-3´) PRvanRSPHHAXcat  Clinical isolate. The molecular basis for the range of MIC values conferred by each genotype is also not clear. Moreover. 5:3 doi: 10. revealing an intricate array of genes involved in response of E. For the microarray experiments E. responding to the antibiotic. one that is increasingly compromised by resistance [13. 4 No. Pellets were held at 4ºC prior to RNA extraction. studying erythromycin response in a resistant strain showed that other gens. Among genes differentially regulated by vancomycin during the time of the experiment are three two-component systems. faecalis to chloramphenicol and erythromycin [11. besides the VanRS. resistance is due to synthesis of peptidoglycan precursors ending in the depsipeptide D-alanyl–Dlactate (D-Ala–D-Lac) that binds glycopeptides with reduced afﬁnity . and 5 ml samples of each were collected 10 min (t10) and 30 min (t30) following vancomycin addition.3823/274 Our Site: http://www.iMedPub Journals 2013 ARCHIVES OF CLINICAL MICROBIOLOGY Vol.45 at 600 nm was reached. faecalis V583 was grown overnight in Brain Heart Infusion (BHI) at 37°C. faecalis typical intrinsic resistance to antibiotics. GBAP    This study This study  Reference 2 © Copyright iMedPub . Methods Bacterial strains and growth conditions Strains used in this study are described in Table 1. The vanB operon contains the vanYBWHBBXBV resistance genes . As a key last line drug for treating multidrug resistant enteroccoccal infections. faecalis V583 to two distinct antibiotics. and vanH. Control and experimental cultures (BHI and BHI plus vancomycin) were then further incubated. there are additional details that remain to be elucidated. The cellular response of E. named as is by Hancock and Perego. plasmid free croR deletion mutant of JH2-2 err06 deletion mutant of JH2-2 JH2-2 with pAT80 JH2-2 ∆croR with pAT80 E.4-0. which is part of the E. and centrifuged for 10 min at 4°C. Moreover. It is currently not understood why a vancomycin resistant strain becomes susceptible if impaired in the two-component system (TCS) CroRS (for “ceftriaxone resistance”) . and vancomycin (Sigma) was added to one of the cultures to a ﬁnal concentration of 10 µg/ml. Table 1. 14] it is important to understand how the organism responds to vancomycin. faecalis core genome . In both cases. A therapeutic dose of vancomycin (10 µg/ml) was chosen to represent the range between peak levels of 20-40 µg/ml and trough serum levels of 5-10 µg/ml achieved in therapy . are repressed by the cell-wall active antibiotic. and examined the transcriptional response by microarray to identify the cellular pathways affected. and the genes directly regulated by it. These studies showed valuable in identifying genes putatively responsible for the E. Samples were immediately suspended in RNA Protect solution (QIAGEN). we observed additional genes that are highly induced suggesting possible contributions to the resistome of Enterococcus for vancomycin.
com/technology/index. For hypridization experiments. faecalis V583 to sense the quorum regulator of the fsr system. faecalis gene.com/ croR CCAATCATGGATGGAATGGAAG (forward) CCAACTCCCCACACTGTTTG (reverse) GCTGTTCTCTATTGGCGCTTA (forward) GCCAGACCTAAACCAGTACC (reverse) GCAAAGTTTCAGCCAATGTTCC (forward) GGATGACTTGAGGACCCACTT (reverse) GCAGGCGTTTCAGATCGAG (forward) GGCATCCGCCATAAATTGACG (reverse) TCATTCATTGACCAG (forward) AACGGATAACACAGGGG (reverse) CATTCTTAAAACTTTCAGCCAC (forward) TAACTTTGATCGCCGG (reverse) CCTACCCTGTCTTTGTGAAGC (forward) ATTGTCCTGCTGCTTCTATCG (reverse) This study croS This study err06 This study ehk06 This study For each condition three batches of RNA were puriﬁed from three separate biological replicates and analysed by hybridization to the microarray. average relative fold change = 2SLR. coding for CroRS and ef1260-ef1261 proteins. Additionally. according to the manufacturer’s instructions. additional pathogenicity island genes of strain MMH594. tetM. known to be absent in V583.affx). for an SLR of <0. Primers used in PCR reactions are described in Table 1. in strain JH2-2 and JH2-2∆croR were performed using semi-quantitative RT-PCR. gelE  sprE  Semi-quantitative reverse transcriptase PCR (RT-PCR) Studies on the effect of vancomycin on the expression of croRS and err06-ehk06 genes. on the basis of previous comparisons in which transcript amounts were also determined by Northern blotting or reverse transcription-PCR . A full list of probe sets including genes represented and excluded from the microarray is available in the ArrayExpress public repository at http://www.3823/274 Our Site: http://www. and collected before and 30 minutes after exposure to the antibiotic. Average relative fold changes were calculated from the average of the signal log ratio (SLR) from three separate experiments by using the following equations: for an SLR of ≥0. 10 μg of each RNA sample was used. namely the vanA operon. fluorescent labelling.uk/ under accession number E-MEXP-1090. Briefly. faecalis plasmid and antibiotic resistance genes or clusters from other E. cDNA synthesis. bcr operon. vanB This study Microarray experiments Total RNA extraction was performed using RNeasy Mini columns (QIAGEN). pRE25 plasmid/cat. �������������� the microarray design includes a total of 3582 probes representing 3182 predicted ORFs from the chromosome of strain V583. and pTEF3 were included. vanG operon.iMedPub Journals 2013 ARCHIVES OF CLINICAL MICROBIOLOGY Vol.affymetrix. vanE operon. average relative fold change =-1×2(-1×SLR). E. Semi-quantitative RT-PCR was done using serial cDNA dilutions. GBAP (gelatinase biosynthesis-activating pheromone). blaZ. Details of the algorithm used in construction of custom Affymetrix GeneChips are available at the manufacturer’s website (www. Tennesse). faecalis strains for which nucleotide sequences had been reported previously. RNA was puriﬁed from DNase I using the RNA clean-up kit (Qiagen). (2007) [��]������ﬂ ������ﬂ . Total RNA extraction was performed as before using RNeasy Mini columns (Qiagen).ac. microarrays included 111 Affymetrix designed eukaryotic and prokaryotic negative control probe sets. Results from this experiment can be accessed through GEO platform under the accession number GSE52263. © Copyright iMedPub Susceptibility testing MIC values for vancomycin were determined by E-test (Biodisk). with incubation at room temperature for 30 min. Quorum-sensing experiments The effect of vancomycin on the ability of E. pTEF2. as well as the genes present on the V583 endogenous plasmids pTEF1. and E.imedpub. 4 No. 5:3 doi: 10. For quantitation of mRNA. oligonucleotides array hybridization and preliminary data analysis were performed by the company Genome Explorations (Memphis. DNA was digested using 1 U of RNase-free DNase I (Roche) at 37ºC for 1 hour. 16S rRNA was included as a control. according to manufacturer´s instructions. DNA contamination of the RNA samples was assessed by a standard (30 cycles) PCR reaction with primers targeting a chromosomal E. without reverse transcriptase addition. DNA was eliminated on the columns by adding RNase-free DNase (QIAGEN). reverse transcription was used to generate cDNA using the Transcriptor High Fidelity cDNA Synthesis reagents (Roche). Relative changes of 2-fold in independent experiments were considered indicative of differences in transcript amounts. Prior to PCR reactions. RNA samples were collected from cells grown in presence and absence of vancomycin (10 µg/ml). The custom Afﬁmetrix microarrays used were described previously by McBride et al. ebi. was directly evaluated by 3 . faecalis ATCC29212 was used as a control. and using 1 µg of RNA as template.
Transcriptome of V583 in the presence of vancomycin In total. termed V583DfsrB. respectively. Hypothetical proteins and proteins of unknown function constituted 17% and 23%. which is unable to produce GBAP by itself. were included among those responding to vancomycin. These results highlight the complexity of the E. murM (ef2658) and ef2585. To half. This allowed us to identify genes which respond either to vancomycin. Figure 1 shows the growth curves of V583 cultures. Nine genes were upregulated at one time point and downregulated at the other. or to the changes introduced by the induction of the vanB operon genes in response to the antibiotic. only GBAP (10nM). cells were collected for RNA extraction and expression levels of gelE. Figure 1. as well as other genes involved in cell-wall synthesis. mobile and extrachromosomal element functions.3823/274 Our Site: http://www. At OD 0. which was chemically synthesized . 5:3 doi: 10.imedpub. Several genes involved in cell-wall synthesis and modiﬁcation. faecalis V583 exposed to a therapeutic dose of vancomycin for 10 and 30 min. but is able to sense and activate Fsr upon GBAP addition. vanB (ef2294) and vanH Results The expression proﬁle of E. and transport and binding. namely murB (ef2733). and encode proteins putatively involved in cell envelope (ef0095 and ef2713). 4 © Copyright iMedPub . This strain. protein synthesis and transcription. ef1017. 30 min following exposure to the antibiotic. ef1018. The functional categories most affected by vancomycin were cell envelope. The culture exposed to vancomycin enters stationary phase before the control culture. exposed and not exposed to vancomycin. and signal transduction/transport and binding (ef1012. energy metabolism. hypothetical proteins of unknown function (ef0054). we used an fsrB mutant of V583 [��]������������������������� ������������������������� . Figure 2 shows all functional categories of genes that were differentially expressed under the conditions tested. faecalis cell-wall synthesis and modiﬁcation. To the other portion. 4 No.4. ef1019 and ef2213). Of this total. including genes involved in fatty acid and phospholipid metabolism. faecalis V583 response to vancomycin exposure. sprE and vanB genes were evaluated. and in energy metabolism. was grown in BHI to an OD ~0. both GBAP (10 nM) and vancomycin (10 µg/ml) were added. was compared with the expression proﬁle of V583 cells grown in the absence of the antibiotic. and the vancomycin resistance genes vanX (ef2293). and after 10 or 20 minutes. 41% of genes were affected by changes of more than 5 fold. ef2476 and ef2857 ).4 10 µg/ml vancomycin was added to half of the culture and growth was followed in the presence (square) and absence (asterisk) of the antibiotic. 10 min after exposure to vancomycin no differences in growth were observed between the exposed culture and the control one. Figure 3 highlights those that are directly or indirectly involved in E. faecalis V583 growth curve in the presence and absence of vancomycin. Four genes coding for PBPs were up-regulated (ef0746. was added. were differentially transcribed. Some functional classes of genes were relatively unaffected by vancomycin exposure. nucleotide metabolism (ef1714). of the genes differentially expressed in response to vancomycin. ef0680. To control the timing of the response of this operon. E.com/ measuring changes in expression of fsr regulated genes gelE and sprE. regulatory functions and signal transduction. and the culture was then split. 130 different genes.iMedPub Journals 2013 ARCHIVES OF CLINICAL MICROBIOLOGY Vol. vanY (ef2297 ). corresponding to 4% of the V583 chromosomal genes represented in the array ships. A full list of genes differentially expressed in the presence of vancomycin is given in Table S1.
glucosamine. which is provided by the increased expression of ef 2295. L-lysine. Regulatory functions. D-Glu. EF1364 EF3316 EF2295 Mevalonate IPP EF2495 UPP D-‐Lac EF2294 Pyruvate GLYCOLYSIS D-‐Ala D-‐Ala-‐D-‐Lac EF2585 Pep@doglycan EF2658. B. D-Lac. EF2476 EF2857. Purine/pyrimidine/nucleoside and nucleotides. Signal transduction.6P EF1503 UDP-‐MurNAc-‐L-‐Ala-‐D-‐Glu-‐L-‐Lys-‐D-‐Ala-‐D-‐Lac EF2733 Fru-‐6P EF2151(GlmS) GlcN-‐6P UDP-‐MurNAc Glucose-‐6-‐P Figure 3. DNA metabolism. N-acetylmuramic acid. EF2293 EF2297 Fru-‐1. Citrate Oxaloacetate EF3319 EF3320 EF3321 Acetyl-‐CoA EF1363. Pyruvate is likely fuelled into EF2295 reaction through increased levels of Fru-1.com/ Figure 2. uridine diphosphate. I. 4 No. fructose. Biosynthesis of cofactors/prosthetic groups/carriers. Protein fate. D-Ala. which allows Fructose-6P to be increasingly converted into Fru-1. D-glutamate. L-Lys. Grey bars: genes found to be differentially expressed with fold-change values below 5. isopentenyl pyrophosphate. Transport and binding. L. M. A. Hypothetical proteins. Black bars: genes found to be differentially expressed with fold-change values above or equal to 5. Fru. cell envelope. UPP. Single steps between metabolites are shown as single arrows and pathways involving several steps and loci are shown as multiple arrows on a line. Number of differentially expressed genes by functional category and fold-change value. Down-regulated (light grey) and up-regulated (underlined) loci are shown. D. which converts pyruvate into D-Lac. V583 loci and pathways involved. F. or related to.3823/274 Our Site: http://www. Energy metabolism. D-lactate. D-alanine.imedpub. UDP. the increased expression of ef 2294 requires D-lac. undecaprenyl pyrophosphate. As peptidoglycan synthesis induced by the up-regulation of the vanB operon also requires UPP. EF0746 EF0680. H. this is likely achieved by increasing the input of acetyl-CoA into UPP synthesis. N. Central intermediary metabolism. E. cellular processes. © Copyright iMedPub 5 .6P entering into glycolysis. C. Unknown function. This is possible due to repression of ef1503 and ef 2151. G.6P. Overall. Aminoacid biosynthesis. IPP.iMedPub Journals 2013 ARCHIVES OF CLINICAL MICROBIOLOGY Vol. and affected by vancomycin. J. 5:3 doi: 10. MurNAc. K. GlcN. peptidoglycan biosynthesis.
we further analysed these highly affected genes.1 -11.9 -3.1 15.3 11.1 29. putative transcriptional regulator.5 49. As this huge response to vancomycin can provide some insights into its mode of action.com/ (ef2295 ). the precursor of the D-lactate synthesized by the highly up-regulated vanH gene. biotin carboxyl carrier protein conserved hypothetical protein Role Cell envelope Cell envelope Unknown function DNA metabolism Unknown function Hypothetical proteins Regulatory functions Regulatory functions Transport and binding proteins Energy metabolism Unknown function Transport and binding proteins/Cellular processes Regulatory functions Unknown function Protein fate Protein fate Cell envelope/Cellular processes Cellular processes/Signal transduction Cell envelope/Cellular processes Unknown function Unknown function Unknown function Transport and binding proteins/Energy metabolism Hypothetical proteins Fold change t10 4. 4 No.6 -7.5 129. including four that exhibited decreases ( Table 2).2 10. LysR family. by locus number.7 6 © Copyright iMedPub . were repressed.5 219. at least at one of the assayed times.1 3.1 t30 9. were up-regulated (ef1363.3823/274 Our Site: http://www.7 13.6 14. EmrBQacA family protein transcriptional regulator. as well as its putative function and role. there were also other 18 genes for which mRNA abundance exhibited a change of more than 9-fold.5 19.2 176.5 28.3 9. List of genes. putative hypothetical protein DNA repair exonuclease family protein hypothetical protein conserved hypothetical protein transcriptional regulator.4 7. Fold change values are given for each gene. differentially expressed at both time points (t10 and t30 min) under exposure to 10 µg/ml of vancomycin. Also down-regulated was the citrate gene cluster (ef3316 to ef3326 ). Table 2.2 3. according to NCBI.6 8.iMedPub Journals 2013 ARCHIVES OF CLINICAL MICROBIOLOGY Vol. putative sulfatase domain protein drug resistance transporter. putative hypothetical protein D-alanyl-D-alanine dipeptidase D-alanine-D-lactate ligase D-speciﬁc alpha-keto acid dehydrogenase vancomycin B-type resistance protein VanW D-alanyl-D-alanine carboxypeptidase hypothetical protein hypothetical protein cell-envelope associated acid phosphatase sodium ion-translocating decarboxylase. which is further converted into the carrier lipid UPP (undecaprenyl pyro- phosphate) required for cell-wall peptidoglycan biosynthesis.1 145.8 4.2 14. both involved in fructose metabolism.9 132.8 241.6-bisphosphatase.8 6. Genes highly affected by vancomycin Aside from the van operon (ef2292-ef2297 ). LysR family magnesium-translocating P-type ATPase fructose-1. which converts acetyl-coA into IPP (isopentenyl pyrophosphate). which codes for the enzymes involved in citrate transport and conversion into oxaloacetate and pyruvate .5 13.3 7. ef1364 and ef2495 ).3 14.0 10.8 -12.5 -4.7 -12.2 3.3 10. Locus EF0559 EF0746 EF0802 EF0972 EF1097 EF1231 EF1302 EF1303 EF1304 EF1503 EF1813 EF1814 EF1815 EF2292 EF2293 EF2294 EF2295 EF2296 EF2297 EF2896 EF3244 EF3245 EF3325 EF3326 Gene name vanV vanX vanB vanH vanW vanYB Descriptions polysaccharide biosynthesis family protein penicillin-binding protein.4 15.0 203.1 170.1 13. 5:3 doi: 10.imedpub.9 -5.2 26.3 131. with fold-change values higher than 9. Genes coding for the enzymes involved in the mevalonate pathway.9 217.5 188. glmS (ef2151) and ef1503 genes.9 123.7 -9. Down-regulation of both genes likely directs D-fructose-6P into glycolysis for pyruvate synthesis.
namely flippases and translocases. The vancomycin molecule has been found to chelate ions . ef1231 is a Ser/Thr romoterse with a signal peptide © Copyright iMedPub region and a transmembrane segment. which corresponds to the regulator protein). as shown in Table S1. FsrAC (TCS15. between 100 and 250 ( Table 2). also containing the LytR-CpsA-Psr motif. vanRBvanSB had a discrete increased expression (fold-change values of 2. but low level identity and hydrophobic character suggests that ef2896 is a membrane protein potentially involved in cellenvelope protein folding. aureus . Biotin is required for proper activity of several biotin-dependent carboxylases. speculatively. Although bioinformatics analysis does not allow reliable prediction of their activity ( Table S2). Reichman and Grundling (2011) have proposed that ef1813 and ef1264 work together in LTA (lipotheichoic acid) synthesis in E. ef0559 is annotated as involved in polysaccharide synthesis. suggesting a possible role for reactive oxygen species (ROS) in vancomycin mode of action. CroRS two-component system The croRS romoter has been found previously to respond to vancomycin in E. which was also overall down-regulated by vancomycin. aureus.3823/274 Our Site: http://www. ef3289ef3290) and TCS06 (ef1260. and to control virulence associated genes . respectively.0/3. vanYB.8/3. In the present work we observed that it also responds to vancomycin in a strain carrying the VanB type of resistance.imedpub. ef0972 ( Table 2) and ef1587 ( Table S1). A similar system in S. N���������������������� o known structural domains could be identiﬁed with conﬁdence for ef2896. 4 No. ef1303 and ef1815 ) were highly induced by vancomycin. ef1813 encoded protein has a sulphatase domain (Table S1) and is paralogous to ef1264. 5:3 doi: 10. Although further studies are needed it is possible that ef0559 acts as the E. ef1097 is predicted to be regulated by the Fsr system in OG1-RF . raising a possible connection between induction of these three genes by vancomycin and depletion of free magnesium in the extracellular environment.2. They also predict the existence of a permease. Three were up-regulated. and one. All the putative codY operon genes are induced by vancomycin exposure at similar levels. HslVU chaperones (ef1646-ef1647) were found to be up-regulated by vancomycin ( Table S1). Proteins with the same motifs include those involved in sporulation. leads to susceptibility to vancomycin 7 . The VanRS two-component system VanRS induced all genes from the vancomycin resistance operon (ef2292 to ef2299). This protein has 12 transmembrane domains. is possibly a drug-resistance transporter but its subtrates are currently unknown. Two-component regulatory systems responding to vancomycin Out of 17 TCS predicted in the V583 genome [��]���������� ���������� . Half of the genes listed in Table 2 code for hypothetical proteins of unknown function. is also highly induced by vancomycin. including TCS or proteins involved in cell-division or metabolism . annotated as involved in DNA repair and metabolism. for 10/30 min exposure) ( Table S1). is under the control of ef1302 and ef1303. Recently. ef2298-ef2299). Three orphan LysR family regulators (ef1302. as expected from two-component systems. was down-regulated ( Table S1). the orphan regulator ef1815. Paralogues to ef3245. ef3245�������������������������������������������������� is a LytR-CpsA-Psr protein with a type 2 phosphatidic acid phosphatase superfamily domain (PAP2). vanHB. These proteins are encoded in what appears to be the codY operon of E. faecalis permease LtaA. was highly downregulated by vancomycin. which would act as the LtaA protein of S. Ser/Thr phosphatases are often involved in activation/deactivation of other proteins by dephosphorylation. although to different levels. four responded to vancomycin.6-biphosphatase key metabolic enzyme. ef3244 and ef3245 also appear to be in an operon.0 and 3. annotated as a putative MFS protein. LysR regulators often control the transcription of genes responsive to environmental conditions . and are also up-regulated by vancomycin ( Table S1). it suggests that these genes may be involved in biotin transport. faecalis JH2-2. has been shown to respond to glycopeptides and other cell-wall active agents. which codes for the fructose1. ef1503. cell-envelope and transport and binding. as often found in LysR regulators . which is annotated as an ltaS gene. Similarly. MsrRA. and ef3325 and ef3326 are part of the citrate transport and metabolism. Ef0802 is in operon with ef0803. Two genes. Of those. ef1814. Using several bioinformatics tools we were able to provide some prediction on their putative role ( Table S2 for details). Mutation in croR gene in V583. and likely has a role in cell-wall maintenance ( Table S2). vanW. CroRS (TCS05. carrying the VanB type of resistance. a vancomycin susceptible strain . namely VanRS (TCS11. are ef1212 and ef1569 (Psr). Genes corresponding to increased levels of mRNA are mainly involved in regulatory functions. annotated as magnesium translocating P-type ATPase. faecalis – ef1648ef1647 (hsIV )-ef1646 (hsIU )-codY. ef1820 which corresponds to FsrC. were also higly up-regulated. showing huge fold-change values. which are transcribed in the opposite direction compared to ef1304.iMedPub Journals 2013 ARCHIVES OF CLINICAL MICROBIOLOGY Vol. which may be the positive transcriptional regulator of ef1813 and ef1814. Further studies are needed to deﬁne the substrate of ef1231. or part of operons. signalling or chaperoning. faecalis. vanB and vanXB were the most strongly induced genes. the kinase component of this TCS). vanV . as does the LtaA transporter protein. It is possible that ef1304.com/ Three of these four highly reduced messages are either regulated by.
namely TCS06 and TCS11. 8 © Copyright iMedPub . This result demonstrates that CroRS is essential for expression of the VanA phenotype in E. We added chemically synthesized GBAP to cultures of this strain. As shown in Figure 5B. Recently.imedpub. We wondered if the same was true for the VanA type of resistance. However. Vancomycin MIC values for the constructed transformants. faecalis. We thus introduced pAT80 plasmid. in the absence and presence of vancomycin. 5:3 doi: 10. demonstrating that vancomycin somehow prevents the Fsr system from TCS 06 We detected increased message levels in the presence of vancomycin. were 256 mg/ml and 4 mg/ml. We measured the expression levels of err06 and ehk06 genes in JH2-2DcroR strain. of the TCS06. it was shown that cell-wall ac- C Figure 4. err06 (ef1260). and looked for induction of gelE and sprE gene expression. despite carrying a functional vanA operon in the pAT80 plasmid. faecalis JH2-2∆croR. We thus asked if the cell-wall perturbations. when added with vancomycin. being dependent on simultaneous induction of other genes responding to cell-wall perturbations. TCS06 could be responding to vancomycin indirectly. resulting in down-regulation of the system and the genes it controls. tive bactericidal antibiotics induce intracellular oxidative stress . faecalis JH2-2. as croR promoter was previously shown to be induced by vancomycin in this strain (Figure 4A). Fsr Two-component system The Fsr system was repressed following the addition of vancomycin (Table S1). they had never been associated before with vancomycin or cell-wall stress response. which had a MIC value for vancomycin of 1 µg/ml. but is unable to produce GBAP . At the late exponential growth phase when the cells were collected. This sensory and regulatory system has not been previously associated with vancomycin exposure or other cellwall stressors. which carries PRvanRSPHHAXcat construction  into JH2-2. Both croR and croS were induced following exposure to vancomycin conﬁrming previous data from Comenge et al. measured by semi-quantitative RT-PCR. neither err06 nor ehk06 responded to vancomycin. neither gelE nor sprE were induced (Figure 5A). JH2-2DcroR strain has neither VanRS nor CroR. with and without vancomycin. and as expected. and message corresponding to genes known to be regulated by this system. E. One explanation would be that vancomycin affected the ability of FsrC to sense the external GBAP. and our results suggest that it also responds to cell-wall stress. C. The SAVE 11 strain. Effect of vancomycin on the expression of croRS and err06-ehk06 genes. Expression of croR and croS genes in JH2-2 served as a positive control. and interfering with Fsr autoinduction. sensed by CroRS.iMedPub Journals 2013 ARCHIVES OF CLINICAL MICROBIOLOGY Vol. TCS06 has been implied in response to oxidative stress in E. Concerning the other two TCS. only for the regulator gene. demonstrating that TCS06 responds to vancomycin in susceptible E. We therefore compared the expression of err06 and ehk06 (ef1261) genes in JH2-2. E. SAVE 9 and SAVE 11 strains.com/ . we used the V583DfsrB strain. Collectively. remained susceptible to vancomycin. expression of both genes was increased upon GBAP addition to V583DfsrB cells. faecalis JH2-2. we would have expected that GBAP was accumulating outside the cells and thus activating the Fsr system . E. faecalis. A. namely gelE and sprE. Similar changes were observed for TCS06 (Figure 4B). and into its isogenic croR deletion mutant. under exposure to vancomycin. could be linked to TCS06 induction. which can sense GBAP and activate fsr and gelE-sprE transcription. a vancomycin susceptible strain with an MIC value of 3 µg/ml. As shown in Figure 4C. 4 No. . B. these results suggest that the presence of an intact CroRS system is important for induction of the TCS06 system by vancomycin. We wondered if TCS06 response to vancomycin could also be generalized to susceptible strains.3823/274 Our Site: http://www. faecalis JH2-2. was less abundant. To test this hypothesis. The reduction in mRNA for the Fsr system was unexpected. respectively.
by both sensing cellwall perturbing agents and by regulating genes involved in cell-wall synthesis and in the metabolic events that support cell-wall synthesis. active against E.com/ Figure 5. (2008)  has shown that vancomycin is not the activating signal for VanS phosphorylation.imedpub. iii) were involved in metabolic pathways and cell-wall synthesis and modiﬁcation.iMedPub Journals 2013 ARCHIVES OF CLINICAL MICROBIOLOGY Vol. which likely includes some of the genes detected in this study. which are known to be key players in transducing environmental information into bacterial phenotypes. The aim of this study was to ﬁnd speciﬁc genes that contribute to the response of the bacterium to vancomycin. which is clearly needed for both VanA and VanB phenotypes. (2011) have shown that vancomycin binds ef0911. B. indicates that for reasons unknown.12]. as illustrated in a very simple way in Figure 6. vanB gene was. our results indicate that both CroRS and TCS06 control unknown steps that are crucial for E. This sensory and regulatory system has been associted with oxidative stress response in JH2-2 strain  and with resistance to heat and SDS in V583 . The immediate repression of the Fsr system upon exposure to vancomycin. The transversal effect of CroRS impairment in resistance to cell-wall antibiotics puts this TCS in the ﬁrst line of E. FsrC becomes blind to the quorum signal GBAP. corroborating the microarray data and also validating our results with GBAP and the V583DfsrB strain. induced by vancomycin. responding to its quorum signal. A. 9 . It is possible that other cell-wall active antibiotics may produce the same effect. faecalis cells to tolerate vancomycin and allow for the activities of vanA and vanB to protect the cell. sprE and vanB genes in strain V583. The quorum-sensing disrupting activity of vancomycin would be predicted to limit the contributions of GelE and SprE proteases to bacterial virulence. Recent work from Ma et al. Discussion As previously seen with other transcriptome studies of the effect of antibiotics on strain V583 [11. Although VanRS is responsible for induction of the vancomycin resistance operon. directly inhibits autophosphorylation of FsrC kinase . Our ﬁndings also suggest that cell-wall perturbations sensed by CroS may be providing the signal for TCS06 induction. ii) were part of TCSs. CroRS and TCS06 may represent important targets for future research on antibiotic therapy against both VRE and VSE. In this experiment. We found genes that i) had very high fold-changes. faecalis and previously found to inhibit both gelatinase and GBAP production . vancomycin prevents FsrC phosphorylation. can be involved in regulation of genes which expression is essential for phenotypic expression of the VanA and © Copyright iMedPub VanB types of resistance. Possibly.3823/274 Our Site: http://www. prior to addition of GBAP. Effect of GBAP and vancomycin on the expression of gelE. The molecule actually sensed by the VanS sensor that regulates the van operon is currently controversial. as expected. This binding likely induces a cascade of events which involve the genes identiﬁed in this work. as is the case for the antimicrobial peptide siamycin I. Siamycin I. 5:3 doi: 10. Recently. vancomycin was added at T0. faecalis defense. one component of an oligopeptide transport system in V583 . substantial global changes in mRNA abundance occur. CroRS. GBAP was added alone at T0. 4 No. Eirich et al.
aureus it has been reported that genes involved in sugar distribution for cell-wall synthesis and glycolysis. is involved in pH homeostasis and in regulation of reducing power from NAD(P)H [��]���������������������������������� ���������������������������������� . It is possible that these important metabolic traits are involved in activation/deactivation of metabolic pathways. of FtsW/RodA/ SpoE family proteins and the MreD and MreC proteins. Regulators EF1815. which hydrolyses 8-oxo-dGTP to 8-oxo-dGMP. further studies are needed to clarify this. is also crucial in the events produced by the therapeutic dose of vancomycin. information leading to increased expression of these regulators is transduced from the outside to the inside compartment. as demonstrated by the up-regulation of PBP coding genes. The citrate gene cluster. loci likely involved in either (or both) vancomycin mode of action and VRE response to a therapeutic dose of this antibiotic. In S. or here demonstrated. which are obviously affected by the presence of vancomycin and the need to produce a different cell-wall precursor. in VRE.imedpub. 5:3 doi: 10. 42]. Apparently.. the increased expression of their genes. namely the isoprenoid synthesis and utilization. The high repression of ef1503 suggests that FBP. EF1302 and EF1303 are represented “outside” the cell in order to simplify this representation. it is possible that. Although the concentration of vancomycin used in our experiments with V583 was bacteriostatic (Figure 1). two enzymes GlmS (ef2151) and Fbp (ef1503). it is possible that the induction of the resistance mechanism also produces a burst of ROS. 4 No. which probably leads to acetyl-CoA being directed to mevalonate pathway and cell-wall-synthesis. recently found to be regulated by the citO gene product. The down-regulation of the citrate transport and utilization enzymes may imply that the cell stops using this pathway to produce pyruvate. namely glmS. Representation of the main. vanY and VanX.iMedPub Journals 2013 ARCHIVES OF CLINICAL MICROBIOLOGY Vol. ﬁlled lines represent previously known. which in turn induces the production of hydroxyl radicals responsible for cell damage and death. namely VanB.e. and the deviated flux of fructose into glycolysis in order to provide pyruvate for lactate synthesis and incorporation into the nascent cell-wall precursors. and in particular. Supporting this prospect is the observation that ef1587 was also up-regulated. and thus glycolysis. VanH. Kohanski et al. Other pathways which are likely providing the substrates for cellwall synthesis were also induced. D-Ala-Dlac instead of D-Ala-DAla. kill bacteria by depletion of reducing power from NAD(P)H. As we detected increased expression of a gene coding for a DNA repair enzyme (ef0972). required for the induced production and activity of Van genes. (2007)  have proposed that bactericidal antibiotics. regardless of their cell target. Cell-wall synthesis-associated enzymes were induced by vancomycin. fructose metabolism. namely ef0746. have an impact in susceptibility to cell-wall active antibiotics . Down-regulation of the citrate and malate utilization enzymes. Dotted lines represent putative or unconﬁrmed direct or indirect interaction.3823/274 Our Site: http://www. or most affected. inside the cell. will result in the reduction of NAD(P)H availability [23. The key metabolite in the activation of glycolysis is fructose-1. potentially due to decreased NAD(P)H. by a yet unknown source or pathway which induces. of Mur proteins. However. Likely. are key players in linking cell-wall stress/ synthesis to metabolism. an event that can prevent the misincorporation © Copyright iMedPub 10 . induction of the vanB operon by vancomycin may actually increase cell damage by ROS. interactions. ef1587 encodes a MutT protein.com/ Figure 6. i.6-biphosphate (FBP).
co-ﬁnanced through FEDER. Funding This work was partially supported by Fundação para a Ciência e Tecnologia (POCTI/CVT/59636/2004 and PTDC/ CVT/67270/2006. hypothesise that vancomycin induced in E. but also in regulation of autolysis. This warrants additional study. PEst-OE/ EQB/LA0004/2011. 5:3 doi: 10. It is possible that E. faecalis. be able to stand therapeutic doses of vancomycin.imedpub.com/ of 8-oxoguanine opposite adenine in DNA. Another important trait highly affected by the presence of vancomycin was LTA synthesis. through LTA synthesis. faecalis strain to vancomycin.3823/274 Our Site: http://www. faecalis is well suited to accommodate and metabolically support the expression of the Van operon genes. Further studies will be required to clarify the relationship between vancomycin exposure and oxidative damage in enterococci. SFRH/BD/21535/2005 to TR and SFRH / BD / 65750 / 2009 to NT). faecalis cells genes involved in LTA synthesis and thus may have implications on the ability of VRE to increase pathogenicity and antibiotic resistance under the presence of the antibiotic. faecalis may. It will be of interest to determine whether these molecules also disrupt TCS systems. © Copyright iMedPub 11 . a damaged form of guanine (G) generated by reactive oxygen species. 8-oxoguanine (8oxoG). Two LtaS proteins are predicted in the V583 genome. We found that vancomycin possesses surprising ability as a disruptor of signaling systems in VRE. Modiﬁed vancomycin molecules are being examined for activity against both VSE and VRE . and contribute to an understanding of the success of this pathway in enterococci. and NIH research grant AI072360). is known to have highly mutagenic potency because of its mispairing with adenine. Portions of the work conducted in the US were supported by the Harvard-wide Program on Antibiotic Resistance (AI083214). based on the actual knowledge. which also involves the up-regulation of ef0559. (2007)  suggest that bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage.iMedPub Journals 2013 ARCHIVES OF CLINICAL MICROBIOLOGY Vol. Recently. and Isabel Marques for helping with the bioinformatic analysis of the hypothetical proteins. 4 No. perhaps making enterococci especially conducive for hosting and spreading this resistance. Acknowledgements The authors acknowledge Kelli Palmer for assisting in the microarrays analysis and RNA extraction protocol. as quorum-sensing is not only involved in bioﬁlm and virulence. Here we examined the cascade of events that follows exposure of a vancomycin resistant E. ef1813 and ef1264. but we can. ef1746 (GalU) is one of the enzymes in the cytoplasm responsible for precursor synthesis for the LTA biosynthesis. Competing interests There are no competing interests. which may have the corresponding activity to LtaA. If any of these effects is related with LTA biosynthesis and activity of ef1813 and ef0559 is yet to be determined. Our results suggest that E. therefore limiting the impact of the Van operon on the ﬁtness of E. Some of these genes may represent candidates for future studies aimed at sensitizing VRE to vancomycin. faecalis has multiple effects on antibiotic resistance and virulence. Kohanski et al. ef1815 may regulate positively the expression of LTA synthesis in response to vancomycin. (2011)  showed that impairing this gene in E. Rigottier-Gois et al. and the ﬁrst was highly induced by vancomycin. This allowed us to identify genes that may play important supportive roles for the expression of the cell wall modifying activity of the vancomycin resistance operon. including proteins involved in triggering the DNA damage response.
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