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| Status |
Public on Nov 30, 2006 |
| Title |
Microarray analysis of the effect of Cipro on wt and lexA mutant S. aureus cells |
| Organism |
Staphylococcus aureus |
| Experiment type |
Expression profiling by array
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| Summary |
Staphylococcus aureus is a leading cause of human disease and can be difficult to treat due to both multi-drug resistance and the organism’s remarkable ability to persist in the host, which is thought to result from several complex regulatory networks that modify transcription in response to environmental stress. In this study, we characterize the global transcriptional response of S. aureus strain 8325 to the antibiotic ciprofloxacin. We find that ciprofloxacin induces prophage mobilization and also significant alterations in metabolism, most notably an upregulation of the tricarboxylic acid cycle. In addition, ciprofloxacin induces the SOS response, which based on a comparison of the wild-type and lexA mutant strains, we show includes the de-repression of sixteen genes. In addition to RecA, LexA, and the hypothetical proteins encoded by SACOL0436, SACOL1375, SACOL1986 and SACOL1999, the S. aureus SOS genes encode proteins involved in DNA metabolism and induced mutation. Finally, we show that rendering LexA uncleavable significantly sensitizes the pathogen to ciprofloxacin therapy in vivo. These observations suggest that the SOS response may play a critical role in the pathogenicity of S. aureus.
Keywords: time course
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| Overall design |
Sample preparation for transcriptional analysis. For each strain, 3 clones were inoculated in TSB and incubated for 18 hours. Cultures were diluted 1:100 and incubated until they reached early log phase (OD600 ~0.5-0.6) at which point ciprofloxacin was added to a final concentration of 0.8 •g/ml. Immediately prior to ciprofloxacin addition, and again 30 and 120 minutes following addition, appropriate volumes from each of the 3 cultures per strain were pooled and added to 2 volumes of RNAprotect reagent (Qiagen). Cultures were centrifuged and cell pellets were stored at 4 ºC until RNA extraction. During the experiment, OD600 and viable cfu per ml were monitored for each of the cultures (Figure 2A and 2B). Total RNA was extracted using the RNeasy Mini kit (Qiagen) at the end of the sample collection period. This procedure was repeated 3 independent times to generate 3 samples at each time point for each strain.
Generation of probes for microarray experiments. cDNA probes for microarray experiments were generated as follows. Briefly, 2 μg of total RNA in a mixture containing 6 μg of random hexamers (Invitrogen); 0.01 M dithiothreitol; an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amino-allyl-dUTP (aa-dUTP) (Sigma); reaction buffer (Clontech); and 400 units of Powerscript reverse transcriptase (Clontech) were incubated at 42 °C overnight. The resulting RNA template was hydrolyzed by adding NaOH and EDTA to a final concentration of 0.2 and 0.1 M, respectively, and incubating at 65 °C for 15 min. Unincorporated aa-dUTP was removed with a Minelute column (Qiagen). The cDNA probes were eluted with phosphate buffer (4 mM KPO4, pH 8.5, in ultrapure water), dried, and resuspended in 0.1 M sodium carbonate buffer (pH 9.0). To couple the amino-allyl cDNA with fluorescent labels, normal human serum-Cy3 or normal human serum-Cy5 (Amersham) was added at room temperature and incubated for 2 h. Uncoupled label was removed using a Minelute column (Qiagen).
Microarray hybridization, scanning, image analysis, normalization, and
analysis. Epoxy-coated slides were prehybridized in 5´ SSC (1´ SSC, 0.15 M NaCl plus 0.015 M sodium citrate) (Invitrogen), 0.1% sodium dodecyl sulfate, and 1% bovine serum albumin at 42 °C for 60 min. The slides were then washed at room temperature with distilled water, dipped in isopropanol, and spun dry. Equal volumes of the appropriate Cy3- and Cy5-labeled probes were combined, dried and then resuspended in a solution of 40% formamide, 5´ SSC, and 0.1% sodium dodecyl sulfate. Resuspended probes were denatured by heating to 95ºC prior to hybridization. The probe mixture then was added to the microarray slide and allowed to hybridize overnight at 42 °C. Hybridized slides were washed sequentially in solutions of 1´ SSC-0.2% SDS, 0.1´ SSC-0.2% SDS, and 0.1´ SSC at room temperature, dried, and then scanned with an Axon GenePix 4000 scanner. Individual TIFF images from each channel were analyzed with TIGR Spotfinder (available at http://www.tigr.org/software/tm4). Microarray data were normalized by LocFit (LOWESS) Normalization in block mode using TIGR MIDAS software (available at http://www.tigr.org/software/tm4). Median spot intensity values were used to calculate the log2 ratio and fold change. Oligos with intensities less than 10,000 units were subtracted from the analysis to rule out background. All hybridizations were performed with a minimum of 1 flip dye experiment. Each hyb has 3 inslide replicates per oligo to rule out bias data.
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| Citation(s) |
17085555 |
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| Submission date |
Nov 09, 2006 |
| Last update date |
Aug 17, 2012 |
| Contact name |
Marcus Jones |
| E-mail(s) |
mjones@jcvi.org
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| Organization name |
JCVI
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| Street address |
4120 Torrey Pines Road
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| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platforms (1) |
| GPL4324 |
JCVI PFGRC Staphylococcus aureus 22K v4.1 array designed primarily based on strain N315 [FULL_FEATURE_LAYOUT] |
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| Samples (13)
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| GSM143514 |
Cipro treated lexA S. aureus cells 30 min replicate 3 |
| GSM143527 |
Cipro treated lexA S. aureus cells 30 min replicate 2 |
| GSM143528 |
Cipro treated lexA S. aureus cells 30 min replicate 1 |
| GSM143529 |
Cipro treated S. aureus cells 30 min replicate 1 |
| GSM143530 |
Cipro treated S. aureus cells 120 min replicate 4 |
| GSM143531 |
Cipro treated S. aureus cells 30 min replicate 2 |
| GSM143532 |
Cipro treated S. aureus cells 120 min replicate 2 |
| GSM143533 |
Cipro treated S. aureus cells 120 min replicate 3 |
| GSM143534 |
Cipro treated S. aureus cells 30 min replicate 6 |
| GSM143535 |
Cipro treated S. aureus cells 120 min replicate 1 |
| GSM143536 |
Cipro treated S. aureus cells 30 min replicate 3 |
| GSM143537 |
Cipro treated S. aureus cells 30 min replicate 4 |
| GSM143538 |
Cipro treated S. aureus cells 30 min replicate 5 |
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| Relations |
| BioProject |
PRJNA100541 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE6258_RAW.tar |
13.3 Mb |
(http)(custom) |
TAR (of MEV) |
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