Transcriptional Profiling of Daptomycin Induced Staphylococcus aureus. Staphylococcus aureus

Daptomycin is a lipopeptide antibiotic that has recently been approved for treatment of Gram-positive bacterial infections. The mode of action of daptomycin is not yet entirely clear. To further understand the mechanism transcriptomic analysis of changes in gene expression in daptomycin-treated Staphylococcus aureus was carried out. The expression profile indicated that cell wall stress stimulon member genes (B. J. Wilkinson, A. Muthaiyan, and R. K. Jayaswal. 2005. Curr. Med. Chem. Anti-Infective Agents 4: 259-276) were significantly induced by daptomycin, and by the cell wall-active antibiotics vancomycin and oxacillin. Comparison of the daptomycin response of a two-component cell wall stress stimulon regulator VraSR mutant, S. aureus KVR, to its parent N315 showed diminished expression of the cell wall stress stimulon in the mutant. Daptomycin has been proposed to cause membrane depolarization, and the transcriptional responses to carbonyl cyanide m-chlorophenylhydrazone (CCCP) and nisin were determined. Transcriptional profiles of the responses to these antimicrobial agents showed significantly different patterns compared to those of the cell wall-active antibiotics, including little or no induction of the cell wall stress stimulon. However, there were a significant number of genes induced by both CCCP and daptomycin that were not induced by oxacillin or vancomycin, such that the daptomycin transcriptome was probably reflecting a membrane depolarizing activity of this antimicrobial also. The results indicate that inhibition of peptidoglycan biosynthesis, either directly or indirectly, and membrane depolarization are parts of the mode of action of daptomycin. Keywords: mode of action, transcriptional profiling Overall design: S. aureus strain ATCC 29213 was provided by Cubist Pharmaceuticals and S. aureus vraSR- mutant KVR derived from S. aureus N315 was provided by K. Hiramatsu. Strain SH1000 was also used in some experiments. Daptomycin (Cubist Pharmaceuticals, Lexington, MA), CCCP and oxacillin (Sigma Chemicals, St. Louis, MO), vancomycin (Lilly Laboratories), nisin (Danisco Beaminster Ltd, UK.) were used for transcriptional profiling. Overnight grown S. aureus ATCC 29213 was inoculated in MHBc medium (20 ml) in a 50 ml Erlenmeyer flask and incubated at 37°C, with shaking at 200 rpm. Growth was measured at regular intervals at 600 nm until OD= ~0.4. Based on the GIC study the following concentrations of antimicrobial agents were added separately for 15 min of challenge: daptomycin, 4 µg ml-1; vancomycin , 10 µg ml-1 ; CCCP, 2 µg ml-1 ; nisin, 20 µg ml-1 . These concentrations were chosen because they gave a similar degree of inhibition of growth. For comparison purposes strain SH1000 was challenged with 1.2 µg oxacillin ml-1 for 15 min. 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