INFECTION AND IMMUNITY, June 2010, p. 2793–2800 Vol. 78, No. 60019-9567/10/$12.00 doi:10.1128/IAI.00688-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Human �-Defensin 3 Inhibits Cell Wall Biosynthesis in Staphylococci�

Vera Sass,1† Tanja Schneider,1† Miriam Wilmes,1 Christian Korner,1 Alessandro Tossi,2Natalia Novikova,3 Olga Shamova,3 and Hans-Georg Sahl1*

Institute of Medical Microbiology, Immunology and Parasitology—Pharmaceutical Microbiology Section, University of Bonn,53105 Bonn, Germany1; Department of Life Sciences, University of Trieste, I-34127 Trieste, Italy2; and Department of

General Pathology and Pathophysiology, Institute for Experimental Medicine, 19736 St. Petersburg, Russia3

Received 17 June 2009/Returned for modification 5 August 2009/Accepted 28 March 2010

Human �-defensin 3 (hBD3) is a highly charged (�11) cationic host defense peptide, produced by epithelialcells and neutrophils. hBD3 retains antimicrobial activity against a broad range of pathogens, includingmultiresistant Staphylococcus aureus, even under high-salt conditions. Whereas antimicrobial host defensepeptides are assumed to act by permeabilizing cell membranes, the transcriptional response pattern ofhBD3-treated staphylococcal cells resembled that of vancomycin-treated cells (V. Sass, U. Pag, A. Tossi, G.Bierbaum, and H. G. Sahl, Int. J. Med. Microbiol. 298:619–633, 2008) and suggested that inhibition of cell wallbiosynthesis is a major component of the killing process. hBD3-treated cells, inspected by transmissionelectron microscopy, showed localized protrusions of cytoplasmic contents, and analysis of the intracellularpool of nucleotide-activated cell wall precursors demonstrated accumulation of the final soluble precursor,UDP-MurNAc-pentapeptide. Accumulation is typically induced by antibiotics that inhibit membrane-boundsteps of cell wall biosynthesis and also demonstrates that hBD3 does not impair the biosynthetic capacity ofcells and does not cause gross leakage of small cytoplasmic compounds. In in vitro assays of individualmembrane-associated cell wall biosynthesis reactions (MraY, MurG, FemX, and penicillin-binding protein 2[PBP2]), hBD3 inhibited those enzymes which use the bactoprenol-bound cell wall building block lipid II asa substrate; quantitative analysis suggested that hBD3 may stoichiometrically bind to lipid II. We report thatbinding of hBD3 to defined, lipid II-rich sites of cell wall biosynthesis may lead to perturbation of thebiosynthesis machinery, resulting in localized lesions in the cell wall as demonstrated by electron microscopy.The lesions may then allow for osmotic rupture of cells when defensins are tested under low-salt conditions.

Host defense peptides (HDPs) are important effector mol-ecules of the innate immune system, which provides effectivebarriers against a wide range of invading microorganisms.HDPs can be found in high concentrations mainly on epithelialsurfaces and in the granules of neutrophils and have two keyfunctions: (i) direct antimicrobial activities which lead to ef-fective killing of microorganisms and (ii) immunomodulatoryfunctions such as recruitment of immune cells, induction ofcytokine release, and alteration of gene transcription (5, 6, 13,14, 44).

Generally, HDPs are cationic peptides which can be foundin all groups of organisms, from bacteria to vertebrates andmammals. They are gene encoded, consist of 10 to 50 aminoacids (15, 50), and include structurally heterogenous groupssuch as the cecropins, magainins, bactenecins, protegrins, de-fensins, and cathelicidins (11, 15, 16, 21, 47), with the last twogroups constituting the most relevant HDPs in mammals.

Defensins sensu stricto were discovered in mammals and arecharacterized by six conserved cysteine residues which formthree disulfide bridge bonds. Two types are most prominent inhumans: the �-defensins, which are produced primarily in neu-trophils and in granules of Paneth cells of the small intestine,and the �-defensins, which are mainly expressed in epithelial

cells (10, 22). Four human �-defensins have been described sofar, with human �-defensin 3 (hBD3) possessing the highestpositive charge of �11 (31, 35, 40, 43). In contrast to manyother defensins, hBD3 is “salt insensitive” in that it kills mi-crobes even at physiological salt concentrations (4, 15).

An important feature of cationic HDPs is the amphiphiliccharacter, i.e., they are able to adopt three-dimensional struc-tures in which polar and apolar residues are clustered on op-posite sides of the molecule’s surface. This structural featureappears to be important for their relatively selective interac-tions with microbial cell envelopes (48). It is generally assumedthat, through patches of positive charges on the surface of themolecule, HDPs interact with negatively charged microbial cellenvelopes and subsequently disrupt membrane barrier func-tions via pore formation or generalized perturbation of thebilayer (23, 45).

HDPs have retained their antimicrobial activity throughoutevolution without selecting for high-level resistance, and yetpathogenic microbes have evolved mechanisms to reduce theirsusceptibility toward HDPs (33). A well-studied adaptationmechanism is based on reduction of the negative charge of thebacterial cell surface by introduction of D-alanine into teichoicacids or of L-lysine into phosphatidylglycerol of staphylococcalcell membranes (32, 34, 46). In Gram-negative organisms, e.g.,in Salmonella enterica serovar Typhimurium, a two-componentsystem, PhoPQ, senses the presence of cationic peptides and inresponse modulates lipopolysaccharide (LPS) structures andother membrane components. However, such adaptations didnot lead to high-level resistance, as occurs for many human-

* Corresponding author. Mailing address: Pharmaceutical Microbi-ology, University of Bonn, Meckenheimer Allee 168, 53115 Bonn,Germany. Phone: 49-228-73-5272. Fax: 49-228-73-5267. E-mail: sahl@microbiology-bonn.de.

† The two authors contributed equally to this work.� Published ahead of print on 12 April 2010.

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designed antibiotics. More research on the molecular mode ofaction of HDPs on bacterial cells could help us to understandwhat has allowed these peptides to retain their activity overmillions of years without eliciting high-level resistance. Thiscould yield valuable information for the future design of newanti-infective drugs.

For such applications it is essential to better understand onthe molecular level the mode of bactericidal activities of de-fensins. hBD3 probably interacts with membranes as a dimerforming a platform which remains floating on the surface withtwo long helices underneath sinking into the membrane inter-face (26). Consistent with such a model, we recently obtainedevidence that hBD3 may not cause membrane disruption inStaphylococcus aureus but rather may interfere with the cellwall biosynthesis machinery (30, 38). This interpretation wasalso supported by early observations by Harder et al. (17), whofirst described hBD3 and reported cell wall perforations inhBD3-treated S. aureus cells reminiscent of those in penicillin-treated cells. To get further insight into such a mechanism, wehere report on a series of cellular and biochemical in vitro cellwall synthesis experiments which suggest that inhibition of cellwall biosynthesis is a defined activity of hBD3. On the mole-cular level, it resembles cell wall biosynthesis inhibitors whichdirectly interfere with the membrane-bound cell wall precursorlipid II.

MATERIALS AND METHODS

Bacterial strains and peptides used in this study. Staphylococcus aureusSG511 was maintained on blood agar (Becton-Dickinson GmbH, Heidelberg,Germany). All whole-cell assays were performed in half-concentrated Mueller-Hinton (MH) broth (Oxoid, Basingstoke, United Kingdom), except in one ex-periment in which we used the assay medium as described by Dorschner et al. (9)to mimic in vivo-like growth conditions This medium contained 20% Trypticasesoy broth (TSB), 10% fetal bovine serum (FBS; Invitrogen GmbH, Karlsruhe,Germany), and 150 mM NaCl in 70% minimal essential medium (MEM; In-vitrogen GmbH, Karlsruhe, Germany) adjusted to pH 7.4. We used the mediumwith and without carbonate as described previously (9) and confirmed a 4-foldincrease of hBD3 potency in the presence of carbonate. hBD3 and LL37 weresynthesized and purified as described previously (4, 51).

MIC determination. Determination of MICs was performed in 96-wellpolypropylene microtiter plates (Nunc brand). Serial 2-fold peptide dilutions inhalf-concentrated MH broth were prepared from a stock solution of the respec-tive peptide. Bacterial strains were grown to an optical density at 600 nm (OD600)of 1.0 and diluted 1:104-fold. Subsequently, 100 �l of this suspension was mixedwith 100 �l of the peptide dilution in each well. After incubation for 18 h at 37°C,the MIC was read as the lowest concentration of antimicrobial agent resulting inthe complete inhibition of visible growth, and results given are the means of atleast two independent experiments performed in duplicate.

Antagonization assays. Antagonization of the antimicrobial activity of hBD3by potential target molecules was monitored using polypropylene microtiterplates and half-concentrated Mueller-Hinton broth (Oxoid, Basingstoke, UnitedKingdom). To a conventional MIC determination putative target, moleculeswere added in a fixed concentration, yielding an increasing molar ratio of targetto peptide. After 18 h of incubation at 37°C, the lowest ratio leading to completeantagonization of hBD3 activity was read.

Intracellular accumulation of the final soluble cell wall precursor UDP-N-acetylmuramyl pentapeptide (UDP-MurNAc-pp). For analysis of the cytoplasmicpeptidoglycan nucleotide precursor pool of S. aureus SG511, we used the methodof Kohlrausch and Holtje (18), elaborated for Bacillus subtilis, with slight mod-ifications. Cells were grown in 5 ml of half-concentrated Mueller-Hinton broth toan OD600 of 0.5 and then supplemented with 130 �g/ml of chloramphenicol andincubated for 15 min. Chloramphenicol is necessary to prevent, under the impactof the antibiotic under investigation, induction of autolytic process and de novosynthesis of enzymes hydrolyzing the nucleotide-activated sugars interfering withdetermination of the soluble precursor (e.g., the work of Dai and Ishiguro [8]).Then peptides were added at 5� MIC as determined under standard conditions

described above, and the cells were incubated for another 30 min. Subsequently,cells were rapidly cooled on ice and spun down (15,000 � g, 5 min, 4°C),resuspended in cold water, and, under stirring, treated with 2 volumes of boilingwater. Cell debris was removed (48,000 � g, 30 min), and the supernatant waslyophilized. For C18 reverse-phase high-pressure liquid chromatography(HPLC), lyophilizates were dissolved in water and acidified to pH 2 with 20%(vol/vol) phosphoric acid; insoluble material was removed (15,000 � g, 5 min);aliquots of the supernatants, adjusted to identical cell wet weights for the dif-ferently treated cultures, were applied to the column. Separation was achievedunder isocratic conditions with 50 mM sodium phosphate, pH 5.2, as solvent; theidentity of the UDP-linked wall precursor was confirmed by matrix-assisted laserdesorption ionization–time of flight (MALDI-TOF) mass spectrometry using thenegative mode and 6-aza-2-thiothymine dissolved in 50% (vol/vol) ethanol-20mM ammonium citrate as matrix.

In vitro peptidoglycan synthesis with isolated membranes. In vitro lipid IIsynthesis was performed using membranes of Micrococcus luteus as previouslydescribed (7, 42). In short, synthesis was performed in a total volume of 150 �lcontaining 300 to 400 �g of membrane protein, 10 nmol of undecaprenylphos-phate (C55P), 100 nmol of UDP-MurNAc-pp, 100 nmol of UDP-N-acetylglu-cosamine (UDP-GlcNAc) in 60 mM Tris-HCl, 5 mM MgCl2, pH 7.5, and 0.5%(wt/vol) Triton X-100. UDP-MurNAc-pp was purified as described elsewhere(18). Bactoprenol-containing products were extracted with butanol-pyridine ac-etate (2:1, vol/vol, pH 4.2), purified as described previously, and analyzed bythin-layer chromatography (TLC), using phosphomolybdic acid (PMA) staining(42). For testing the impact of hBD3, the defensin was added in molar ratios withrespect to C55P. For purification of milligram quantities of lipid II to be used assubstrate in the enzyme assay, the analytical procedure was scaled up by a factorof 500 and lipid II was purified as described previously (42). Radiolabeled lipidII was synthesized using [14C]UDP-GlcNAc as substrate. Purification of lipid Ifollowed the same protocol except that UDP-GlcNAc was omitted.

Cloning, expression, and purification of cell wall biosynthesis enzymes. Clon-ing, expression, and purification of the cell wall biosynthesis enzymes penicillin-binding protein 2 (PBP2), MraY, MurG, and FemX of S. aureus NCTC 8325were performed as described previously without any modifications (41).

In vitro peptidoglycan synthesis reaction using purified proteins and sub-strates. To determine the enzymatic activity of purified MraY-His6, assays werecarried out in a total volume of 50 �l containing 5 nmol C55P or [3H]C55P (14.8GBq/mmol; Biotrend, Cologne, Germany), 50 nmol of UDP-MurNAc-pp in 100mM Tris-HCl, 30 mM MgCl2, pH 7.5, and 10 mM N-lauroylsarcosine. Thereaction was initiated by the addition of 7.5 �g of the enzyme, and the reactionmixture was incubated for 1 h at 37°C.

The MurG activity assay was performed in a final volume of 30 �l containing2.5 nmol purifed lipid I, 25 nmol UDP-GlcNAc or [14C]UDP-GlcNAc in 200 mMTris-HCl, 5.7 mM MgCl2, pH 7.5, and 0.8% Triton X-100 in the presence of 0.45�g of purified MurG-His6 enzyme. The reaction mixture was incubated for 30min at 30°C.

The assay for the synthesis of lipid II-Gly1 catalyzed by FemX was performedas described previously without modifications, including the presence of 0.8%Triton X-100 (42). In a second series of experiments Triton X-100 was omittedin order to test the effect of detergent on the action of hBD3.

Enzymatic activity of PBP2 was determined by incubating 2.5 nmol lipid II or1 nmol [14C]lipid II in 20 mM morpholineethanesulfonic acid (MES), 5 mMMgCl2, pH 5.5, in a total volume of 50 �l. The reaction was initiated by theaddition of 7.5 �g PBP2-His6, and the reaction mixture was incubated for 1.5 hat 30°C. In all in vitro assays hBD3 was added in molar ratios ranging from 0.5 to2 with respect to the amounts of C55P, lipid I, and lipid II, respectively.

Synthesized bactoprenol-containing intermediates were extracted from thereaction mixtures with n-butanol-pyridine acetate, pH 4.2 (1:1, vol/vol), andanalyzed by TLC. Quantification of enzyme reactions was routinely performed byPMA staining and subsequent evaluation of spot density using ImageQuant TLsoftware (Amersham); all experiments were performed at least three times. Toincrease accuracy, the Fem and PBP2 assays were confirmed at least once using14C-labeled glycine (for the Fem reactions) and lipid II with a 14C-labeledGlcNAc moiety; radiolabeled bands were quantified using a storage phosphorscreen to visualize radioactivity in a Storm imaging system (GE Healthcare). Forquantification of the PBP2-catalyzed lipid II polymerization, reaction mixtureswere applied directly onto TLC plates developed in solvent B (butanol-aceticacid-water-pyridine [15:3:12:10, vol/vol/vol/vol]), and quantification of residualradiolabeled free lipid II was determined using phosphorimaging.

Transmission electron microscopy. Mid-logarithmic-phase S. aureus SG511(approximately 5 � 108 CFU/ml) was exposed to 15 �M hBD3 in 10 mM sodiumphosphate buffer, pH 7.4, with 20% Mueller-Hinton broth for 10, 30, or 60 minat 37°C. At intervals, bacterial cells were collected by centrifugation at 2,000 �

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g, resuspended in 1 ml of freshly prepared 2.5% glutaraldehyde in 0.2 M sodiumcacodylate buffer, pH 7.4, and fixed for 2.5 h at room temperature. Samples werethen washed with cacodylate buffer and postfixed in 1% osmium tetroxide in 0.1M cacodylate buffer, pH 7.4 (the osmolarity of the buffer was adjusted to 450osM by addition of sucrose), for 40 min. Samples were washed three times incacodylate buffer, and cell pellets were placed in 1% agarose and then dehy-drated in a graded (50 to 100%) ethanol series and embedded in Araldite-Eponresin mix using acetone as an intermediate solvent; polymerization was carriedout subsequently at 37, 45, and 60°C for 3 days. Ultramicrotome sections werecontrasted with uranyl acetate and lead citrate and examined with a JEM 1011electron microscope operated at 80 kV with digital image acquisition.

RESULTS

Effect of hBD3 on living cells. It is generally accepted that, atthe very high concentrations locally occurring in the host, cat-ionic antimicrobial peptides may destroy bacterial cells by firstbinding to the surface and subsequently disrupting the mem-brane bilayer structure through mechanisms such as that de-scribed by the carpet model (28, 36, 45). However, we recentlydemonstrated, using micromolar concentrations in the MICrange, that hBD3 hardly affected the membrane integrity butstrongly induced genes of the core cell wall stress stimulon ofstaphylococci (38) as defined by McAleese et al. (24). Suchgenes included those for the two-component system VraRS, acell wall stress sensor system (12, 20), and putative detoxifica-tion ABC transporters such as VraDE (38).

To obtain evidence that inhibition of cell wall biosynthesis isa relevant component of the antibiotic activity of hBD3, wefirst determined the cytoplasmic levels of UDP-N-acetylmu-ramyl pentapeptide (UDP-MurNAc-pp) in Staphylococcus au-reus SG511. Antibiotics such as vancomycin, bacitracin, orbeta-lactams, which interfere with the late, membrane-boundstages of peptidoglycan synthesis, trigger an accumulation ofthe final soluble peptidoglycan precursor (Fig. 1) in the cyto-

plasm (18). After hBD3 treatment (27.8 mg/liter, correspond-ing to 5� MIC as determined earlier [38]) for 30 min, signif-icant accumulation of the precursor was observed (Fig. 2D),similar to the accumulation seen with vancomycin-treated con-trols (Fig. 2B). Comparable results were obtained both in thestandard test medium (half-concentrated MH broth) and inthe carbonate-containing medium of Dorschner et al. (9)which mimics in vivo-like growth conditions (Fig. 2E). In con-trast, treatment of cells with LL37, a linear cationic HDP witha stronger impact on the bilayer structure (27), did not lead toan accumulation of UDP-MurNAc-pp (Fig. 2C). This result issignificant and indicative in two ways: (i) hBD3 obviously doesnot depolarize the cytoplasmic membrane to such an extentthat cells are unable to perform biosynthesis reactions and (ii)hBD3 should not induce gross membrane leakage since theprecursor was retained in the cytoplasm and did not leak fromtreated cells. This interpretation is further substantiated bypreviously published data that hBD3 (38) and a template �-de-fensin (37) only slightly affected the membrane potential ofgrowing staphylococcal cells (reduction by 15 to 20 mV at 5�MIC). In contrast, in control experiments with 5� MIC ofLL37 a substantial decrease of the membrane potential by 50to 60 mV was observed (data not shown), indicating that thispeptide intrinsically may have a higher capacity for bilayerperturbation than hBD3 or may directly interfere with energygeneration.

When growing cells of S. aureus strain SG511 were inspectedby transmission electron microscopy, small membrane protru-sions filled with cytoplasmic contents were observed after 30min of treatment with hBD3. Such evaginations were not de-tectable after 10 min and became more frequent after 60 minof treatment (Fig. 3). The protrusions were rather well definedin shape and obviously occurred through small lesions in the

FIG. 1. Cartoon of the membrane-associated cell wall biosynthesis reactions in S. aureus. The biosynthesis reactions at the inner face of thecytoplasmic membrane catalyzed by MraY, MurG, and FemXAB produce the membrane-bound precursor molecule lipid II (undecaprenylpyro-phosphate-MurNAc[pentapeptide]-GlcNAc) with the pentaglycine interpeptide bridge attached. The cell wall building block is then translocatedacross the membrane by an unknown mechanism and subsequently incorporated into the growing peptidoglycan network. When bactoprenolcycling is inhibited by antibiotics at any stage, the ultimate soluble precursor UDP-MurNAc-pentapeptide (boxed) accumulates in the cytoplasm.

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cell wall, in agreement with observations made by Harder et al.(17). Generalized and rapid cell wall lysis as mediated byautolytic enzymes, particularly in the septum area, was notdetectable; such lytic events were described for Staphylococcussimulans after treatment with cationic lantibiotics (3) and witha template defensin (37). Only after 60 min of treatment couldcells with progressive cell wall damage be identified (Fig. 3,panel 9).

Effects of hBD3 on membrane-bound cell wall biosynthesisreactions in vitro. To obtain information on the molecularactivities of hBD3, we performed a series of in vitro cell wallbiosynthesis assays using membrane preparations for the over-all reaction and purified enzymes for testing the individualsteps of the membrane-bound synthesis cycle (Fig. 1). Mem-brane preparations of Micrococcus luteus contain sufficientMraY and MurG activity for the in vitro formation of the cellwall precursors lipid I and lipid II when the soluble precursorUDP-MurNAc-pp is added (49). Addition of hBD3 to such atest system did not lead to inhibition of lipid I and II biosyn-thesis (data not shown). Similarly, testing the individual reac-tions of MraY (by adding UDP-MurNAc-pp to the bactopre-nol carrier to form lipid I), MurG (by adding GlcNAc to formlipid II), and FemX, which adds to lipid II the first glycineresidue of the interpeptide bridge, did not indicate any inhibi-tion by hBD3 (data not shown). All of these assays have incommon that 0.8% (by volume) Triton X-100 is routinely in-cluded in the assay mixture, which we suspected to interferewith hBD3 activity. Among those assays, we found that onlywith FemX can Triton be omitted without affecting enzymeactivity, allowing us to study a possible detergent effect onhBD3 inhibitory activity (Fig. 4). Indeed, in the absence ofTriton, hBD3 almost completely inhibited the FemX reactionwhen added in equimolar concentrations with regard to lipidII, indicating that the defensin may stoichiometrically bind tothe substrate of the enzyme (Fig. 4). In contrast, no inhibition

was observed in the presence of Triton X-100, even with a2-fold molar excess of hBD3, whereas vancomycin and nisinwere fully inhibitory under these conditions (Fig. 4). The rea-sons for the difference between hBD3 and the antibiotics areunclear; besides a lower affinity of hBD3 for lipid II, it may bepossible that Triton also affects dimerization of hBD3, whichappears necessary for antibiotic activity (40).

For further analysis of the inhibitory nature of hBD3, wechose another lipid II-consuming reaction, the transformationof monomeric lipid II into a polymeric peptidoglycan as cata-lyzed by the bifunctional penicillin-binding protein 2 (PBP2) ofS. aureus, which occurs on the outside of the membrane andtherefore is the likely target reaction of hBD3. Using this assay,both the disappearance of lipid II and the appearance of oneproduct, the liberated bactoprenol carrier C55P, can be de-tected simultaneously. Purified, recombinant PBP2-His6 wasincubated with lipid II and hBD3, and the lipids were subse-quently extracted and separated by TLC (Fig. 5A). For reliablequantification of residual lipid II, we also used radiolabeledlipid II as substrate (Fig. 5B). In the presence of hBD3, thePBP2-catalyzed reaction was clearly blocked. Again, at a molarratio of hBD3 to lipid II of 1:1, only 19% lipid II was transg-lycosylated; at a ratio of 2:1, inhibition of the reaction wascomplete. Thus, the inhibitory potency of hBD3 was compa-rable to that of nisin (96% inhibition at a nisin/lipid II molarratio of 2:1).

Since the Triton experiments indicated that hBD3 may bindlipid II less tightly than nisin or vancomycin, we tried to obtaininformation on the specificity of the inhibition and the involve-ment of ionic interactions between lipid II with two phosphategroups and hBD3 (net charge, �11). Interactions of hBD3 andlipid II solely based on electrostatics should be weakened byother cationic peptides which could also interact with the neg-ative charges in the lipid II molecule. We chose the lantibioticPep5 with overall similar physicochemical properties (molec-

FIG. 2. Intracellular accumulation of the ultimate soluble cell wall precursor UDP-N-acetylmuramyl pentapeptide (UDP-MurNAc-pentapep-tide) in S. aureus SG511. Cells were treated with 5� MIC of the respective antibiotic compound, incubated for 30 min, and subsequently extractedwith boiling water. The intracellular nucleotide pool was analyzed by applying standardized aliquots to reversed-phase HPLC. (A) Untreatedcontrol cells. (B) Vancomycin-treated (4 mg/liter) positive-control cells. (C) LL37-treated (73.625 mg/liter) cells. (D) hBD3-treated (27.8 mg/liter)cells. (E) hBD3-treated cells growing in cell culture broth (27.8 mg/liter). (Inset) Mass spectrum of the peak containing UDP-MurNAc-pentapeptide; the calculated mass is 1,149.43; in addition, the mono- and disodium salts are detectable.

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ular mass of 3,488, net charge of �8), which effectively killsstaphylococci through pore formation (19) and which does notinterfere with cell wall biosynthesis, as well as the synthetictripeptide Lys-Lys-Lys (net charge, �3), and conducted thesame assay. Neither Pep5 (molar ratio, 2:1) nor the tripeptide(molar ratios of 20:1 and 50:1) was able to inhibit the PBPreaction and therefore may not bind to lipid II with the sameaffinity as that with hBD3. On the other hand, both were ableto some extent to interfere with the inhibitory activity of hBD3when incubated with lipid II before hBD3 and the enzymewere added (Fig. 6). These data suggest that ionic forces areinvolved in hBD3 binding to lipid II and yet are not sufficientto fully describe the interaction between the defensin and thecell wall precursor.

Antagonization of hBD3 activity by putative target mole-cules. To obtain further information on the lipid II-hBD3interaction, we performed antagonization assays, based onconventional broth microdilution MIC determination in thepresence of lipid II and its structural components and relatedcompounds. For this, the potential antagonist was added indifferent molar ratios with respect to hBD3 to a cell suspensionof S. aureus SG511 and supplemented with a bactericidal con-

centration of hBD3 (1� to 8� MIC). Growth of bacteria after18 h of incubation indicated an antagonizing effect of theputative target structure by binding of hBD3, thus making itunavailable for interaction with staphylococcal cells. As poten-tial antagonists, we used lipid II, undecaprenylphosphate(C55P, the lipid carrier of the disaccharide-pentapeptide unitof lipid II), two derivatives of C55P with shorter lipid tails(C20P and C15P, consisting of 4 and 3 isoprenoid units, respec-tively), pure phospholipids (dioleoylphosphatidylglycerol[DOPG]), UDP-GlcNAc, UDP-MurNAc, and glucosamine-6-phosphate, respectively. Of these putative antagonists, onlythose with negative charges and the potential to form micellesin aqueous solutions were able to interfere with hBD3 activitybut, however, with different potencies as indicated by differentmolar ratios necessary for antagonization. Phospholipids(DOPG), C55P, and the shortened carrier C20P antagonized ata molar excess of 2 to 5, whereas only lipid II was able to bindhBD3 at a 1:1 molar ratio. Activated cell wall sugars such asUDP-GlcNAc, UDP-MurNAc, and glucosamine-6-phosphate,as well as C15P, which is water soluble and unable to formmicelles, did not have any antagonizing effect. Obviously, be-sides a negatively charged membrane-like environment which

FIG. 3. Transmission electron microscopy micrograph of S. aureus SG511 treated with 15 �M hBD3. (Panel 1) Cells treated for 10 min; cellsdid not show any sign of damage and did not differ from untreated controls (not shown). (Panels 2 to 5) Cells treated for 30 min; in approximately5 to 10% of cells, membrane protrusions with cytoplasmic contents were observed (panels 3 and 4; panel 4 shows a magnification of the areaindicated by an arrow in panel 2); additionally, 5 to 10% of the cells showed aberrant septum formation, as in panel 5. (Panels 6 to 9) Cells treatedfor 60 min; the number of cells with membrane protrusions (panels 6 and 7) and with additional septa (panel 8) increased, and more damaged cellswith signs of general lysis (panel 9) occurred. Scale bar, 200 nm.

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is known to attract amphiphilic cationic peptides, lipid II pro-vides additional structural elements, possibly in the disaccha-ride moiety, for intensified interactions with hBD3.

DISCUSSION

Amphiphilic cationic host defense peptides are generallyassumed to kill microbes by disruption of the membrane bi-

layer. Here we present evidence that for hBD3 in concentra-tions close to its MIC inhibition of cell wall biosynthesis is amajor component of its antibiotic activity against staphylococciand that interactions with the membrane-bound cell wall build-ing block lipid II appear to be involved in inhibition. Theseconclusions are based on transcriptional response analysis aspublished earlier (38) and on experiments with intact cells,including electron microscopy and a series of in vitro assays

FIG. 4. Impact of detergent on the lipid II-hBD3 interactions. Effect of hBD3 on the FemX-catalyzed addition of glycine to lipid II in vitro inthe presence (dark gray bars) or absence (light gray bars) of Triton X-100.

FIG. 5. Inhibition of the PBP2-catalyzed reaction by hBD3 in vitro. (A) The conversion of lipid II into polymeric peptidoglycan in the presenceof increasing concentrations of hBD3 was qualitatively analyzed using TLC and PMA staining. (B) For quantitative analysis of the reaction,[14C]lipid II was used as a substrate; reaction mixtures were applied directly onto TLC plates and developed in solvent B (butanol-aceticacid-water-pyridine [15:3:12:10, vol/vol/vol/vol]) with subsequent detection and quantification of residual [14C]lipid II using storage phosphortechnology.

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with lipid II-consuming enzyme reactions. Electron microscopystrongly suggests that through the activity of hBD3, local le-sions in the cell wall layer are created through which eventuallymembranes and cytoplasmic contents protrude as a result ofosmotic pressure. These local lesions could thus represent thetesserae proposed in the “hydro-osmotic transtesseral extru-sion and rupture (HOTTER)” mechanisms of AMP action(29).

Where in the cell could those local lesions occur? There isincreasing evidence that bacterial cell walls are synthesized byhighly organized multienzyme machineries which assemble atparticular sites, i.e., the septum and septum initiation sites incoccoid cells and in rod-shaped bacteria and additionally atsites which follow the helically arranged cytoskeleton, to en-able elongation growth (for a review, see, e.g., the work ofScheffers and Pinho [39]). These sites have been shown to berich in negatively charged phospholipids such as phosphatidyl-glycerol and cardiolipin, an essential feature for the correctassembly of the division machinery for which a cationic helicalstretch in the division protein MinD seems important (1).Necessarily, these sites are also rich in lipid II and thereforerepresent ideal binding sites for hBD3. It seems reasonable toargue that the presence of hBD3 at such an exposed site wouldinterfere, like “sand in a gearbox,” with the coordinated as-sembly and function of the machinery, resulting in localizedinhibition of cell wall biosynthesis and subsequent lesions asobserved by electron microscopy in this study and in others(e.g., the work of Harder et al. [17]).

Transcriptional response data with hDB3, but also withother antimicrobial peptides (30, 34), clearly indicate that in-duction of the cell wall stress stimulon is the most prominentresponse of staphylococcal cells to treatment with HDPs. Nev-ertheless, membrane depolarization to various degrees (mildwith hBD3 and rather strong with LL37) and the concomitantimpact on energy-consuming cellular processes certainly con-

tribute to killing as reported earlier (36). It remains to bestudied whether the observed membrane depolarization resultsfrom destabilizing the lipid bilayer or from a direct impact ofthe peptides on energy generation processes. Most strikingly, adirect interference with electron transport and components ofrespiratory chains has been reported for a linear cationic pep-tide derived from the human bactericidal/permeability-increas-ing protein (BPI [2]) and for gramicidin S (25). If such activ-ities can be identified more frequently, it appears that not onlycell wall biosynthesis but also other multiprotein machineriesorganized in the cytoplasmic membrane, e.g., the electrontransport chain, may be affected. Such pleiotropic effects ofcationic HDPs may explain why antibiotic peptides remainedactive throughout evolution and why target microbes may havebeen able to develop mechanisms for reducing susceptibility(for a review, see, e.g., the work of Peschel and Sahl [33]) butscarcely developed high-level resistance mechanisms as ob-served with clinically used antibiotics.

ACKNOWLEDGMENTS

The financial support by the German Federal Ministry of Educationand Research (BMBF, SkinStaph project), the German ResearchFoundation (DFG, Sa292/13-1), the BONFOR program of the Medi-cal Faculty, University of Bonn, and the European Commission (NAMproject, 218191) is gratefully acknowledged.

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FIG. 6. Impact of cationic peptides on the lipid II-hBD3 interactions. In vitro inhibition of the PBP2-catalyzed reaction by Pep5 (�8) orLys-Lys-Lys (�3) and by hBD3 (�11) in the presence of Pep5 or Lys-Lys-Lys.

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