Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2011 Jan 20:11:16.
doi: 10.1186/1471-2180-11-16.

Induction kinetics of the Staphylococcus aureus cell wall stress stimulon in response to different cell wall active antibiotics

Vanina Dengler  1 , Patricia Stutzmann Meier, Ronald Heusser, Brigitte Berger-Bächi, Nadine McCallum
Affiliations
Comparative Study

Induction kinetics of the Staphylococcus aureus cell wall stress stimulon in response to different cell wall active antibiotics

Vanina Dengler et al. BMC Microbiol. .

Abstract

Background: Staphylococcus aureus activates a protective cell wall stress stimulon (CWSS) in response to the inhibition of cell wall synthesis or cell envelope damage caused by several structurally and functionally different antibiotics. CWSS induction is coordinated by the VraSR two-component system, which senses an unknown signal triggered by diverse cell wall active agents.

Results: We have constructed a highly sensitive luciferase reporter gene system, using the promoter of sas016 (S. aureus N315), which detects very subtle differences in expression as well as measuring > 4 log-fold changes in CWSS activity, to compare the concentration dependence of CWSS induction kinetics of antibiotics with different cell envelope targets. We compared the effects of subinhibitory up to suprainhibitory concentrations of fosfomycin, D-cycloserine, tunicamycin, bacitracin, flavomycin, vancomycin, teicoplanin, oxacillin, lysostaphin and daptomycin. Induction kinetics were both strongly antibiotic- and concentration-dependent. Most antibiotics triggered an immediate response with induction beginning within 10 min, except for tunicamycin, D-cycloserine and fosfomycin which showed lags of up to one generation before induction commenced. Induction characteristics, such as the rate of CWSS induction once initiated and maximal induction reached, were strongly antibiotic dependent. We observed a clear correlation between the inhibitory effects of specific antibiotic concentrations on growth and corresponding increases in CWSS induction kinetics. Inactivation of VraR increased susceptibility to the antibiotics tested from 2- to 16-fold, with the exceptions of oxacillin and D-cycloserine, where no differences were detected in the methicillin susceptible S. aureus strain background analysed. There was no apparent correlation between the induction capacity of the various antibiotics and the relative importance of the CWSS for the corresponding resistance phenotypes.

Conclusion: CWSS induction profiles were unique for each antibiotic. Differences observed in optimal induction conditions for specific antibiotics should be determined and taken into account when designing and interpreting CWSS induction studies.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Schematic representation of the enzymatic steps involved in S. aureus cell wall synthesis and the targets of cell wall active antibiotics. Fosfomycin inhibits the enzyme MurA (UDP-N-acetylglucosamine-3-enolpyruvyl transferase) that catalyses the addition of phosphoenolpyruvate (PEP) to UDP-N-acetyl-glucosamine (GlcNAc) to form UDP-N-acetyl-muramic acid (UDP-MurNAc) [34]. D-cycloserine prevents the addition of D-alanine to the peptidoglycan precursor by inhibiting D-alanine:D-alanine ligase A and alanine racemase [35]. Tunicamycin is a glycoprotein antibiotic that inhibits the transfer of peptidoglycan precursor (phospho-MurNAc-pentapeptide) to the lipid carrier undecaprenyl pyrophosphate (or C55-isoprenyl pyrophosphate), catalysed by the translocase MraY [36,37]. Sub-lethal concentrations of tunicamycin also inhibit TarO, the first enzyme in the wall teichoic acid pathway [38,39]. Bacitracin forms a metal-dependent complex with the lipid carrier undecaprenyl pyrophosphate, thereby preventing dephosphorylation and the recycling of the lipid carrier required for cell wall synthesis [40,41]. Flavomycin (a moenomycin complex) is a phosphoglycolipid antibiotic that inhibits transglycosylation through binding of the transglycosylase domain of penicillin-binding protein 2 (PBP2) [42]. Glycopeptide antibiotics, such as vancomycin and teicoplanin, inhibit cell wall synthesis by binding the D-ala-D-ala of the lipid II and sterically hindering transglycosylation and transpeptidation. Teicoplanin activity is enhanced through its interaction with the cytoplasmic membrane [43]. ß-lactam antibiotics, such as oxacillin, bind the transpeptidase active domain of penicillin-binding proteins (PBPs) by mimicking the D-ala-D-ala end of the pentapeptide [44]. The mode of action of daptomycin is not fully known, it causes calcium-dependent disruption of membrane function and potassium efflux [45], but was also predicted to directly or indirectly inhibit peptidoglycan systhesis [9]. Lysostaphin is a zinc metalloenzyme that cleaves the pentaglycine crosslinking bridge specific for the cell wall of S. aureus [46]. (Adapted from [47]).
Figure 2
Figure 2
Induction kinetics of three CWSS promoters in response to varying concentrations of oxacillin. Luciferase activities and growth curves of strain BB255 containing: A, psas016-luc+; B, psa0908-luc+; and C, ptcaA-luc+; after addition of 0, 0.5, 1, 2 or 5-fold the MIC of oxacillin at time point zero.
Figure 3
Figure 3
Northern blot analysis of sas016 and sas016p-luc+ transcript induction BB255 psas016p-luc+. RNA was harvested from cultures after 20 and 60 min of induction with 0, 0.2, 0.5, 1, 2 or 5-fold MIC concentrations of oxacillin. Transcripts hybridising to sas016 and luc+-specific DIG and their approximate sizes are indicated. Approximate transcript sizes are indicated on the left side of the blots. Ethidium bromide stained 16S rRNA bands are shown below Northern blots as an indication of RNA loading.
Figure 4
Figure 4
Antibiotic dependent induction of the cell wall stress stimulon. The upper graph shows relative light units (RLU) measured upon induction of BB255 psas016p-luc+ of cultures stressed with 1x MIC of different antibiotics. The corresponding OD values at each sampling point are presented below. The graphs shown are representative results of between two and four induction experiments performed for each antibiotic.
Figure 5
Figure 5
Concentration-dependent cell wall stress stimulon induction kinetics of different cell wall active antibiotics. Graphs show relative light units (RLU) measured upon induction of BB255 psas016p-luc+ with five different antibiotic concentrations and the corresponding OD values at each sampling point. The graphs shown are representative results of between two and four induction experiments performed for each antibiotic. A, concentration-dependent induction kinetics of antibiotics scored as high- or medium-level inducers. B, concentration-dependent induction kinetics of antibiotics scored as low-level inducers and the fluoroquinolone antibiotic ciprofloxacin.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jordan S, Hutchings MI, Mascher T. Cell envelope stress response in Gram-positive bacteria. FEMS Microbiol Rev. 2008;32(1):107–146. doi: 10.1111/j.1574-6976.2007.00091.x. - DOI - PubMed
    1. Kuroda M, Kuroda H, Oshima T, Takeuchi F, Mori H, Hiramatsu K. Two-component system VraSR positively modulates the regulation of cell-wall biosynthesis pathway in Staphylococcus aureus. Mol Microbiol. 2003;49(3):807–821. doi: 10.1046/j.1365-2958.2003.03599.x. - DOI - PubMed
    1. Utaida S, Dunman PM, Macapagal D, Murphy E, Projan SJ, Singh VK, Jayaswal RK, Wilkinson BJ. Genome-wide transcriptional profiling of the response of Staphylococcus aureus to cell-wall-active antibiotics reveals a cell-wall-stress stimulon. Microbiology. 2003;149(Pt 10):2719–2732. doi: 10.1099/mic.0.26426-0. - DOI - PubMed
    1. Belcheva A, Golemi-Kotra D. A close-up view of the VraSR two-component system. A mediator of Staphylococcus aureus response to cell wall damage. J Biol Chem. 2008;283(18):12354–12364. doi: 10.1074/jbc.M710010200. - DOI - PubMed
    1. Belcheva A, Verma V, Golemi-Kotra D. DNA-binding activity of the vancomycin resistance associated regulator protein VraR and the role of phosphorylation in transcriptional regulation of the vraSR operon. Biochemistry. 2009;48(24):5592–5601. doi: 10.1021/bi900478b. - DOI - PubMed

Publication types

MeSH terms

Substances