Unknown,Transcriptomics,Genomics,Proteomics

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NsaRS is a Cell-Envelope-Stress Sensing Two-Component System of Staphylococcus aureus

ABSTRACT: S. aureus possesses 16 two-component systems (TCS), two of which (GraRS & NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, conserved within the firmicutes. NsaRS has recently been documented as being important for nisin-resistance in S. aureus. In this study we present a detailed characterization of NsaRS and reveal that, as with other IM-HK TCS, it responds to disruptions in cell-wall stability. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-wall damaging antibiotics, including Phosphomycin, Ampicillin, Nisin and Penicillin G. Additionally; we reveal that NsaRS regulates a downstream transporter, NsaAB, during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall targetting antibiotic bacitracin. Microarray analysis reveals the transcription of 245 genes is altered in a nsaS mutant, with the vast majority down-regulated. Included within this list are genes involved in transport, drug-resistance, cell-envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using ICP-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low affinity conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in both cell-wall and cell-envelope structure. Finally, a variety of virulence related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus NsaRS is important in sensing cell-wall instability in S. aureus, and functions to reprogram gene expression to modify cell-envelope architecture, facilitating adaptation and survival. We did four hybridizations for this experiment, including a biological replicate and a dye-swap experiment for each replicate to account for dye-bias. Spots flagged as empty or bad were excluded and the raw data from each slide was normalized using LOWESS method, with background correction. The data from the replicates were combined (using the median value) and a one-sample t-test was performed. The volcano plot was used with a fold change cut-off of >= 2 and a p-value of <0.05, to filter the genes that were differentially expressed.

ORGANISM(S): Staphylococcus aureus

SUBMITTER: Vijayaraj Nagarajan

PROVIDER: E-GEOD-27061 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus.

Kolar Stacey L SL Nagarajan Vijayaraj V Oszmiana Anna A Rivera Frances E FE Miller Halie K HK Davenport Jessica E JE Riordan James T JT Potempa Jan J Barber David S DS Koziel Joanna J Elasri Mohamed O MO Shaw Lindsey N LN Shaw Lindsey N LN

Microbiology (Reading, England) 20110512 Pt 8

Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated th ...[more]

PMID: 21565927

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Project description:The two-component system (TCS) is a specific regulatory system in bacteria and plays an important role in sensing and adapting to the environment. In this study, we evaluated the roles of TCSs in the major cariogenic pathogen Streptococcus mutans in resistance to several types of bacteriocin. In a comprehensive analysis using individual TCS mutants, we found that two novel TCSs were associated with resistance against distinct lantibiotics nisin A (class I type A[I]) and nukacin ISK-1(class I type A[II]). One TCS, SMU.659-660 (designated as NsrRS), was related to resistance against nisin A produced by Lactococcus lactis ATCC 11454. The other TCS, SMU.1146-1145 (designated as LcrRS), was related to resistance against nukacin ISK-1 produced by Staphylococcus warneri ISK-1. NsrRS induced the expression of SMU.658 (designated as NsrX), which constitutes an operon with nsrRS, in response to nisin A. Inactivation of nsrX increased susceptibility to nisin A. Additionally, NsrX expression in S. mutans increased the binding affinity to nisin A compared to a no-expression strain. LcrRS induced the expression of SMU.1148-50 (lctFEG), which encodes an ABC transporter and is located upstream of lcrRS, in response to nukacin ISK-1. Inactivation of lctFEG significantly increased susceptibility to nukacin ISK-1. Electrophoretic mobility shift assay analysis revealed that NsrR and LcrR bound directly to regions upstream of nsrX and lctFEG, respectively. This is the first report that two distinct TCSs, NsrRS and LcrRS, are independently involved in resistance to nisin A and nukacin ISK-1 in S. mutans. Total of 14 samples were analyzed. Total RNA from each test strain and control described below were labeled with Alexa FluorÂ® 555 and Alexa FluorÂ® 647, respectively, and were cohybridized on a single array. Labeling and hybridization were performed once or twice independently. UA159 wild type as control vs. UA159 with nisinA or nukacin ISK-1, UA159 wild type with nisinA or nukacin ISK-1 vs. nsrRS or lcrRS deletion mutant in UA159 with nisinA or nukacin ISK-1.

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

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NsaRS is a Cell-Envelope-Stress Sensing Two-Component System of Staphylococcus aureus

Publications

NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus.

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