Daptomycin versus Friulimicin B: In-Depth Profiling of Bacillus subtilis Cell Envelope Stress Responses

The related lipo(depsi)peptide antibiotics daptomycin and friulimicinB show great potential in the treatment of multiply resistantgram-positive pathogens. Applying genome-wide in-depth expressionprofiling, we compared the respective stress responses of Bacillussubtilis. Both antibiotics target envelope integrity, basedon the strong induction of extracytoplasmic function ${sigma}$ factor-dependentgene expression. The cell envelope stress-sensing two-componentsystem LiaRS is exclusively and strongly induced by daptomycin,indicative of different mechanisms of action in the two compounds.

INTRODUCTION

Top

INTRODUCTION

Staphylococcus aureus is a leading cause of nosocomial infections,especially in mechanically ventilated patients. Its remarkablepotential to acquire and accumulate high-level resistance againstmost of the classical antibiotics (including vancomycin) usedfor the treatment of gram-positive infections is one of thereasons for the ongoing mortality caused by hospital-acquiredS. aureus infections (7, 17).

Daptomycin is the first of a new class of cyclic lipodepsipeptideantibiotics (Fig. 1A) with strong bactericidal activities againstgram-positive pathogens (2). It interferes with cell envelopeintegrity, and cell death occurs presumably by either membranedepolarization or membrane perforation (19, 20). FriulimicinB, an acidic, cyclic lipopeptide produced by Actinoplanes friuliensis,shows structural similarities to daptomycin (Fig. 1B) and isalso active against multidrug-resistant gram-positive bacteria(1, 22).

View larger version (26K):
[in this window]
[in a new window]

FIG. 1. Chemical structures of the lipodepsipeptide antibiotic daptomycin (A) and the lipopeptide antibiotic friulimicin B (B).

As part of a coordinated effort to study and characterize itsmode of action, we have performed comparative in-depth expressionprofiling for both antibiotics. This technique is a powerfulapproach to elucidate the inhibitory mechanisms of novel antimicrobialcompounds (4, 9) and has been successfully applied to characterizeand differentiate antimicrobial actions, often using Bacillussubtilis as a model organism (3, 10). B. subtilis is particularlywell suited for studying cell wall antibiotics, since the regulatorynetwork orchestrating its cell envelope stress response (CESR)is well characterized. It consists of four two-component systemsand at least four extracytoplasmic function (ECF) ${sigma}$ factors (11).

Here, we present results from an in-depth analysis of the expressionsignature provoked by the treatment of B. subtilis with sublethalamounts of daptomycin and friulimicin B. Our data show thatboth antibiotics specifically target cell envelope integrity.But significant differences in the corresponding CESRs, as clearlydocumented by transcriptomics, proteomics, and detailed geneexpression profiling, strongly suggest different modes of actionof the two structurally related antibiotics.

(This study was presented in part at the 47th InternationalConference on Antimicrobial Agents and Chemotherapy, Chicago,IL, 17 to 20 September 2007 [25]).

Transcriptomics and proteomics.

Top

Transcriptomics and proteomics.

For microarray experiments, midlogarithmic cultures of B. subtiliswere challenged with 1 µg/ml (sublethal amounts) of eitherdaptomycin or friulimicin B. The cells were harvested 10 minpostinduction, and cell pellets were directly snap-frozen inliquid nitrogen. RNA preparation and microarray experimentswere performed essentially as described previously (13, 23).To validate the gene expression profiles, we also performedtwo-dimensional gel electrophoresis of the cytoplasmic proteomeof B. subtilis cells, quantifying de novo protein synthesisafter the addition of daptomycin or friulimicin B by incubatingthe cultures in the presence of L-[³⁵S]methionine, as describedpreviously (3). The results are summarized in Table 1 and Fig.2. The complete microarray data sets can be found in the supplementalmaterial and, together with additional supporting information,at http://microbial-stress.iab.kit.edu/87.php.

View this table:
[in this window]
[in a new window]

TABLE 1. Marker genes induced by daptomycin and/or friulimicin B

View larger version (53K):
[in this window]
[in a new window]

FIG. 2. Synthesis patterns of marker proteins after induction with daptomycin (DAP) or friulimicin B (FRI) compared to patterns of untreated control cells. Details from the two-dimensional gels of the cytoplasmic proteome ("spot albums" of marker proteins) are shown for two time points postinduction with each of two compounds. (A) Proteins induced by both antibiotics. (B) Daptomycin-specific spots. (C) Friulimicin B-specific spots. See text for details.

Both antibiotics induced a limited number of genes, most ofwhich could be assigned to known CESR regulons. Daptomycin specificallyand strongly activated the LiaRS two-component system, withmore than 200-fold induction of its primary target genes, liaIH.This induction has also been observed recently in an independentstudy (9a) and is in good agreement with data from the orthologousVraSR system of S. aureus which was also induced by daptomycin(16). Moreover, a strong LiaH induction was also observed withproteomics analysis, where it was identified in three strongneighboring spots (differing in their isoelectric points), indicativeof posttranslational modifications (Fig. 2).

Both compounds induced numerous genes known to be regulatedby ECF ${sigma}$ factors. This ECF-dependent response was much strongerfor friulimicin B (Table 1). In addition, only seven genes/proteinsof unknown regulation were differentially expressed (Table 1and Fig. 2), including the actin homolog mreBH, which was inducedabout three- to fourfold by both compounds. Five more geneswithout known regulator, some of which are potentially involvedin cell envelope biogenesis, specifically responded to friulimicinB (Table 1). All genes identified in our analysis have beenlinked to CESR of B. subtilis previously (data not shown). Whileno expression signature available so far resembles that of friulimicinB, both the transcriptome and the proteome profile for daptomycinclosely resemble those of bacitracin (3, 14).

In-depth gene expression profiling.

Top

In-depth gene expression...

The results of our microarray study led to three follow-up analyseson the specificity of the corresponding CESR. (i) We analyzedthe induction of all seven ECF ${sigma}$ factors by quantitative real-timereverse transcriptase PCR (RT-PCR), based on the known and highlyECF-specific autoregulation of their own genes, to determinethe respective inducer spectrum and strength. The primers usedfor amplification are listed in Table 2. Both antibiotics activate ${sigma}$ ^M and ${sigma}$ ^V, with friulimicin B provoking a significantly strongerresponse. In addition, friulimicin B also induced the uncharacterizedECF ${sigma}$ factor ${sigma}$ ^YlaC (Table 3).

View this table:
[in this window]
[in a new window]

TABLE 2. Strains and oligonucleotides used in this study

View this table:
[in this window]
[in a new window]

TABLE 3. Induction of ECF ${sigma}$ factors and liaH by daptomycin and friulimicin B

(ii) The much stronger activation of ECF target genes by friulimicinB was not due to the corresponding lack of liaIH induction,as demonstrated by the induction values of ECF genes in theliaIH mutant strain TMB0389, which were identical to those inthe wild type (data not shown). The stronger ECF response tofriulimicin B is therefore LiaIH independent and a true antibiotic-specificdifference in the corresponding gene induction profiles.

(iii) We also quantified the activity of the LiaR target promoterP_liaI as a function of the daptomycin/friulimicin B concentrationsover a range of 4 orders of magnitude by performing a β-galactosidaseassay (using strain BFS2470 as described previously) (15). P_liaIinduction was indeed only observed in the presence of daptomycinand in a very narrow window of antibiotic concentrations (between0.5 and 2 µg/ml) (data not shown). These results stronglysuggest different modes of action for daptomycin and friulimicinB.

Conclusions.

Top

Conclusions.

Our data clearly allowed the identification of cell envelopeintegrity as the site of daptomycin and friulimicin B action,but the results strongly suggest mechanistic differences betweenthe two compounds. This assumption is primarily based on thedramatic differences in the LiaRS response. Moreover, friulimicinB activates both ${sigma}$ ^M and ${sigma}$ ^V more strongly than daptomycin and,additionally, induces ${sigma}$ ^YlaC expression (summarized in Fig. 3).The strong similarities of CESR between daptomycin and bacitracinwere initially viewed as an indication that daptomycin mightinterfere with the lipid II cycle of cell wall biosynthesis.But a detailed biochemical mechanism of action study revealedthat friulimicin B, like amphomycin but in contrast to the membrane-interferingdaptomycin, inhibits cell wall biosynthesis by binding bactoprenolphosphate (18).

View larger version (11K):
[in this window]
[in a new window]

FIG. 3. Schematic representation of the regulatory networks orchestrating the daptomycin (DAP) and friulimicin B (FRI) stress responses. The thickness of the arrows corresponds to the strength of induction of the given regulators (see text and Table 1 for details). TCS, two-component system.

ACKNOWLEDGMENTS

This work was supported by grants from the Deutsche Forschungsgemeinschaft(to T.M.), the Fonds der Chemischen Industrie (to T.M. and M.H.),the Bundesministerium für Bildung und Forschung (to G.H.,U.M., and M.H.; project name, Unternehmen Region-Zentren fürInnovationskompetenz; project number from PtJ, 03ZIK012; projectrunning time, June 2005 to May 2010), and the Bildungsministeriumof the country Mecklenburg-Vorpommern (to M.H.). Funding forCombinature Biopharm AG (now Merlion Pharmaceuticals GmbH) forfriulimicin-related work was granted by the BMBF (project name,BioChancePLUS; project number from PtJ, 0313173; and projectrunning time, April 2004 to March 2007). T.W. was supportedby a Chemiefonds Ph.D. scholarship from the Fonds der ChemischenIndustrie.

We thank Anja Hoffmann and Susanne Paprotny for excellent technicalassistance, Anna-Barbara Hachmann, John D. Helmann, Tanja Schneider,and Hans-Georg Sahl for sharing data prior to publication, andthe Decodon GmbH (Greifswald, Germany) for their cooperation.

FOOTNOTES

* Corresponding author. Mailing address: Karlsruhe Institute of Technology (KIT), Institute of Applied Life Sciences, KIT Research Group 11-1 Microbial Stress Responses, Fritz-Haber-Weg 4, Building 30.43, Room 710, D-76131 Karlsruhe, Germany. Phone: 49 (0)721 608-3473. Fax: 49 (0)721 608-8932. E-mail: thorsten.mascher{at}kit.edu

Published ahead of print on 21 January 2009.

Supplemental material for this article may be found at http://aac.asm.org/.

^# These two authors contributed equally to this work.

Present address: BRAIN (Biotechnology Research And InformationNetwork) AG, Darmstädter Str. 34, D-64673 Zwingenberg,Germany.

REFERENCES

Top