Project description:Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase with reduced susceptibility to beta-lactam antibiotics. Here, we show that preventing the expression of wall teichoic acids (WTAs) genetically or with a TarO inhibitor sensitizes MRSA strains to beta-lactams although PBP2A is still expressed. Using S. aureus microarrays and array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center) we have characterized the transcriptomes of S. aureus COL. in order to further understand the sensitization of strain COL to methicillin by tunicamycin we determined the tunicamycin and methicillin transcriptomes alone and in combination. Methicillin treatment of COL at 500 µg/mL had almost no effect on cell growth rate and, remarkably, the only gene in the transcriptome that showed a more than two-fold change in expression was lytM, which was downregulated. The tunicamycin transcriptome of COL, acquired at 0.4 µg/mL, shows modest changes compared to the untreated control both in terms of the total numbers of affected genes and in the degree of up- or downregulation. Several of the genes upregulated upon tunicamycin treatment are part of the cell wall stress stimulon.COL was grown with methicillin to an OD600 ~0.4, and challenged with tunicamycin for 2 hrs whereas the control culture contained methicillin alone. transcriptome for COL growing in the presence of both agents showed extensive changes in gene expression. , the cell wall stress stimulon, which was not induced by methicillin when tunicamycin was absent, was clearly induced in its presence and the changes were far more dramatic than observed with tunicamycin alone. ). vraS and vraR, which encode a two component signaling system dedicated to the cell wall regulon, were upregulated 3.8 and 3.7 fold, respectively. Other upregulated cell wall stress stimulon genes include pbp2, fmtA, mvaD (mevalonate diphosphate decarboxylase), crtN (dehydrosqualene desaturase) mvak1 (mevalonate kinase), recU, SAV1424 (methionine sulfoxide reductase A), prsA (peptidyl-prolyl cis/trans isomerase), tcaA (Tca protein) and cwrA. A considerable number of genes were also downregulated upon challenge of COL with the combination of tunicamycin and methicillin. These included sspB, lrgA, dltA, capL, SAS0988, sspA, pflB, and spa. Several of these genes have been found to be downregulated in previous studies of cell wall-active antibiotic challenge of S. aureus.

Project description:Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase with reduced susceptibility to beta-lactam antibiotics. Here, we show that preventing the expression of wall teichoic acids (WTAs) genetically or with a TarO inhibitor sensitizes MRSA strains to beta-lactams although PBP2A is still expressed. Using S. aureus microarrays and array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center) we have characterized the transcriptomes of S. aureus COL. in order to further understand the sensitization of strain COL to methicillin by tunicamycin we determined the tunicamycin and methicillin transcriptomes alone and in combination. Methicillin treatment of COL at 500 Âµg/mL had almost no effect on cell growth rate and, remarkably, the only gene in the transcriptome that showed a more than two-fold change in expression was lytM, which was downregulated. The tunicamycin transcriptome of COL, acquired at 0.4 Âµg/mL, shows modest changes compared to the untreated control both in terms of the total numbers of affected genes and in the degree of up- or downregulation. Several of the genes upregulated upon tunicamycin treatment are part of the cell wall stress stimulon.COL was grown with methicillin to an OD600 ~0.4, and challenged with tunicamycin for 2 hrs whereas the control culture contained methicillin alone. transcriptome for COL growing in the presence of both agents showed extensive changes in gene expression. , the cell wall stress stimulon, which was not induced by methicillin when tunicamycin was absent, was clearly induced in its presence and the changes were far more dramatic than observed with tunicamycin alone. ). vraS and vraR, which encode a two component signaling system dedicated to the cell wall regulon, were upregulated 3.8 and 3.7 fold, respectively. Other upregulated cell wall stress stimulon genes include pbp2, fmtA, mvaD (mevalonate diphosphate decarboxylase), crtN (dehydrosqualene desaturase) mvak1 (mevalonate kinase), recU, SAV1424 (methionine sulfoxide reductase A), prsA (peptidyl-prolyl cis/trans isomerase), tcaA (Tca protein) and cwrA. A considerable number of genes were also downregulated upon challenge of COL with the combination of tunicamycin and methicillin. These included sspB, lrgA, dltA, capL, SAS0988, sspA, pflB, and spa. Several of these genes have been found to be downregulated in previous studies of cell wall-active antibiotic challenge of S. aureus. Growth conditions for microarray analysis- For transcriptional profiling, an overnight-grown COL culture was diluted (2% vol/vol) to 20 ml TSB medium in a 50-ml Erlenmeyer flask and grown at 30Â°C with shaking at 200 rpm. For the sensitization experiment, COL was grown with methicillin to an OD600 ~0.4, and challenged with tunicamycin for 2 hrs whereas the control culture contained methicillin alone. Total bacterial RNA was isolated as previously described from S. aureus COL (30Â°C, 200 rpm). The RNA samples were then converted to fluorescently-labeled cDNA and hybridized to S. aureus microarrays version 7 (NIAID's Pathogen Functional Genomics Resource Center). Hybridization signals were scanned using an Axon4000B scanner (Molecular Devices, Sunnyvale, CA ) with Acuity 4.0 software and scans were saved as TIFF image. Scans were analyzed using TIGR-Spotfinder (www.tigr.org/software/) software and the local background was subsequently subtracted. The data set was normalized by applying the LOWESS algorithm using TIGR-MIDAS (www.tigr.org/software/) software. The normalized log2 ratio of test/reference signal for each spot was recorded. Genes with less than three data points were considered unreliable, and their data points were discarded. The averaged log2 ratio for each remaining gene on the four replicate slides was ultimately calculated. Significant changes of gene expression were identified with SAM (significance analysis of microarrays; www.tat.stanford.edu/~tibs/SAM/index.html. Genes analyzed using these programs were further sorted and grouped based on their function using our in-house software Staphylococcus aureus Gene Sorter (SAGS). Several controls were employed to minimize the technical and biological variations and to ensure that the data obtained were of good quality. First, each ORF was present in triplicate on the array. Second, each RNA preparation was used to make probes for at least two separate arrays for which the incorporated dye was reversed. Contents of raw data files: Channel A = Cy3 dye Channel B = Cy5 dye File names ending with S1,S3 = Cy3 untreated, Cy5 treated File names ending with S2,S4 = Cy5 untreated, Cy3 treated

Project description:Staphylococcus aureus (S. aureus) constantly evolves under host and environment pressures. The monitoring network is essential in assessing the epidemiology of S. aureus infections. A total of 555 S. aureus isolates were collected from five hospitals in three different geographical regions of China for the investigation of molecular characteristics, antibiotic resistance, virulence gene, and wall teichoic acid (WTA) glycosyltransferase gene profiles. 233 (42.0%) isolates were identified as MRSA, and 323 (58.2%) were defined as multidrug-resistant (MDR) isolates. MRSA prevalence showed no significant difference among the three regions. In contrast, the MDR prevalence was significantly higher in central China than that in northern China (63.5% vs. 50.8%, P < 0.05). Thirty-eight sequence types (STs) belonging to 17 clone complexes (CCs) and 126 distinct spa-types were identified. The most prevalent clone was ST59-t437 (9.7%, 54/555), followed by ST22-t309 (7.6%, 42/555) and ST5-t2460 (7.2%, 40/555). Most ST59-t437 and ST5-t2460 were MRSA isolates, whereas most ST22-t309 was MSSA isolates. The predominant clones varied in different geographical areas. The distribution of the pvl, etb, tsst, clfb, sdrC, sdrD, hlg, fnbA, and hla genes showed significant differences among different regions. We found five WTA glycosyltransferase gene profiles, with tarP-/tarS+/tarM-/tagN- being the most common combination. Remarkably, the tarP gene was identified in more CCs than just CC5 and CC398. All of 16 tarP-positive isolates also contained the tarS. Moreover, tarS was present in almost all S. aureus isolates except 10 ST630 isolates. The tagN gene was only detected in 10 of 12 ST630 S. aureus isolates without tarS. The tarM gene was absent in CC5 and CC398. In brief, there were regional differences among molecular characteristics, antibiotic resistance, and virulence gene profiles. The tarS-negative ST630 lineage carried the tagN, which was never found before, indicating that it may be capable of expressing GroP-α-GalNAc WTA and exchanging mobile genetic elements with coagulase-negative staphylococci (CoNS).

Project description:UnlabelledThe methicillin resistance factor encoded by fmtA is a core member of the Staphylococcus aureus cell wall stimulon, but its function has remained elusive for the past two decades. First identified as a factor that affects methicillin resistance in S. aureus strains, FmtA was later shown to interact with teichoic acids and to localize to the cell division septum. We have made a breakthrough in understanding FmtA function. We show that FmtA hydrolyzes the ester bond between d-Ala and the backbone of teichoic acids, which are polyglycerol-phosphate or polyribitol-phosphate polymers found in the S. aureus cell envelope. FmtA contains two conserved motifs found in serine active-site penicillin-binding proteins (PBPs) and ?-lactamases. The conserved SXXK motif was found to be important for the d-amino esterase activity of FmtA. Moreover, we show that deletion of fmtA (?fmtA) led to higher levels of d-Ala in teichoic acids, and this effect was reversed by complementation of ?fmtA with fmtA. The positive charge on d-Ala partially masks the negative charge of the polyol-phosphate backbone of teichoic acids; hence, a change in the d-Ala content will result in modulation of their charge. Cell division, biofilm formation, autolysis, and colonization are among the many processes in S. aureus affected by the d-Ala content and overall charge of the cell surface teichoic acids. The esterase activity of FmtA and the regulation of fmtA suggest that FmtA functions as a modulator of teichoic acid charge, thus FmtA may be involved in S. aureus cell division, biofilm formation, autolysis, and colonization.ImportanceTeichoic acids are involved in cell division, cell wall synthesis, biofilm formation, attachment of bacteria to artificial surfaces, and colonization. However, the function of teichoic acids is not fully understood. Modification by glycosylation and/or d-alanylation of the polyol-phosphate backbone of teichoic acids is important in the above cell processes. The intrinsic negative charge of teichoic acid backbone plays a role in the charge and/or pH of the bacterial surface, and d-alanylation represents a means through which bacteria modulate the charge or the pH of their surfaces. We discovered that FmtA removes d-Ala from teichoic acids. We propose FmtA may provide a temporal and spatial regulation of the bacterial cell surface charge in two ways, by removing the d-Ala from LTA to make it available to wall teichoic acid (WTA) in response to certain conditions and by removing it from WTA to allow the cell to reset its surface charge to a previous condition.

Dataset Information

Structure and Mechanism of Staphylococcus aureus TarS, the Wall Teichoic Acid ?-glycosyltransferase Involved in Methicillin Resistance.

Publications

Structure and Mechanism of Staphylococcus aureus TarS, the Wall Teichoic Acid β-glycosyltransferase Involved in Methicillin Resistance.

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