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Antimicrob Agents Chemother. Aug 2009; 53(8): 3422–3429.
Published online Jun 1, 2009. doi:  10.1128/AAC.00010-09
PMCID: PMC2715605

Antibacterial Properties and Mode of Action of a Short Acyl-Lysyl Oligomer[down-pointing small open triangle]

Abstract

We investigated the potency, selectivity, and mode of action of the oligo-acyl-lysine (OAK) NC12-2β12, which was recently suggested to represent the shortest OAK sequence that retains nonhemolytic antibacterial properties. A growth inhibition assay against a panel of 48 bacterial strains confirmed that NC12-2β12 exerted potent activity against gram-positive bacteria while exhibiting negligible hemolysis up to at least 100 times the MIC. Interestingly, NC12-2β12 demonstrated a bacteriostatic mode of action, unlike previously described OAKs that were bactericidal and essentially active against gram-negative bacteria only. The results of various experiments with binding to model phospholipid membranes correlated well with those of the cytotoxicity experiments and provided a plausible explanation for the observed activity profile. Thus, surface plasmon resonance experiments performed with model bilayers revealed high binding affinity to a membrane composition that mimicked the plasma membrane of staphylococci (global affinity constant [Kapp], 3.7 × 106 M−1) and significantly lower affinities to mimics of Escherichia coli or red blood cell cytoplasmic membranes. Additional insertion isotherms and epifluorescence microscopy experiments performed with model Langmuir monolayers mimicking the outer leaflet of plasma membranes demonstrated the preferential insertion of NC12-2β12 into highly anionic membranes. Finally, we provide mechanistic studies in support of the view that the bacteriostatic effect resulted from a relatively slow process of plasma membrane permeabilization involving discrete leakage of small solutes, such as intracellular ATP. Collectively, the data point to short OAKs as a potential source for new antibacterial compounds that can selectively affect the growth of gram-positive bacteria while circumventing potential adverse effects linked to lytic compounds.

The widespread resistance of bacteria to conventional antibiotics continues to stimulate the search for alternative antimicrobials, such as the host defense antimicrobial peptides (AMPs). AMPs can demonstrate a broad spectrum of activities, including antibiotic activity against both gram-positive and gram-negative bacteria and antiviral and antifungal activities, as well as chemotactic activity and the ability to stimulate chemokine production (23). While the precise mechanism of action needs to be better understood, a multitude of observations suggest more than one mode of action (8, 60). These include abrupt disruption of the cell membrane, which is often concomitant with rapid depolarization of the transmembrane potential (30, 58), as well as intracellular targets, including inhibition of enzymatic activities (2, 10, 37) and biosynthesis (6, 27, 41, 53). Such a multitarget mode of action might significantly overcome various drug resistance mechanisms, which renders AMPs attractive in a variety of antimicrobial applications (21). Nevertheless, various drawbacks, including high susceptibility to proteolysis, toxicity, and high manufacturing costs, complicate the application of AMPs in systemic routes of administration and encourage de novo and rational design of novel compounds with improved properties.

The peptidomimetic approach has emerged in recent years as a powerful means for overcoming the inherent limitations of peptide physical characteristics, where the therapeutic potential can be improved by increasing selectivity and bioavailability (9, 40, 45, 49, 52). Peptidomimetic strategies consisting of peptide backbone modifications and incorporation of unnatural amino acids, aromatic rings (25), and amino fatty acids (43, 44) have been proposed as novel building blocks to improve potency by enhancing binding affinity to membranes and to reduce susceptibility to inactivation by serum proteases (39). Oligo-acyl-lysines (OAKs) are among the simpler AMP-mimetic designs, in which the two most important characteristics for AMP activity, hydrophobicity and charge, are represented as tandem repeats of amide-linked fatty acids and lysines (15, 42-44, 46, 48). This design was shown to overcome the limitations of conventional AMPs with respect to in vivo efficacy and toxicity (43-45). Two types of OAKs have been presented so far, each composed of acyl-lysyl (αn) or lysyl-acyl-lysyl (βn) subunits, where n defines the acyl length (43). In previous studies, the α-OAKs C12K-7α8 (44) and C12K-5α8 (46) showed potent antibacterial activity against most gram-negative strains tested, both in vitro and in vivo. Furthermore, the OAKs were shown to induce bacterial death by damaging the cytoplasmic membrane or by inhibiting DNA expression, respectively. Shorter OAK sequences also displayed potent antiplasmodial activity (42) but were too hemolytic to be considered for systemic applications.

To study the structure-activity relationships in OAKs, we recently used a rapid screening assay to test >100 OAK sequences for their hemolytic activities against human red blood cells (RBCs) and antibacterial activities against Escherichia coli and Staphylococcus aureus as representatives of gram-negative and gram-positive bacteria, respectively. The structure-activity relationships that emerged indicated that the self-assembly properties of excessively hydrophobic OAKs are responsible for poor antibacterial properties and for high hemolytic activity (43). Moreover, the study suggested that the minimal requirements for potent and selective antibacterial activity are embedded in the sequence aminolauryl-[lysyl-aminolauryl-lysyl]2 (designated NC12-2β12), the structure of which is depicted in Fig. Fig.1a.1a. Here, we tested this premise and further characterized the antimicrobial properties of NC12-2β12 to shed new light on the mechanism of action.

FIG. 1.
Biophysical characteristics of NC12-2β12. (a) OAK molecular structure (mass, 1,121.7 g/mol). (b and c) Effects of ionic strength and pH on antimicrobial activity, as assessed against two strains of S. aureus: ATCC 29213 (squares) and ATCC 25923 ...

MATERIALS AND METHODS

Synthesis.

OAKs and peptides were synthesized by the solid-phase method (16), applying the N-(9-fluorenyl) methoxycarbonyl active ester chemistry on a fully automated, programmable model 433A peptide synthesizer (Applied Biosystems) as described previously (44). 4-Methylbenzhydrylamine resin was used to obtain amidated compounds. The synthetic products were purified to chromatographic homogeneity in the range of 98 to >99% by reverse-phase high-pressure liquid chromatography (Alliance-Waters). The purified peptides were subjected to electrospray mass spectrometry (Micromass ZQ Waters) analysis to confirm their compositions and to amino acid analysis to determine the concentrations. The peptides and OAKs were then stored as lyophilized powders at −20°C. Prior to use, fresh solutions were prepared in distilled water (1 mg/ml), briefly vortexed, sonicated, and centrifuged, and these were used as stock solutions in all experiments.

Hemolytic activity.

The hemolytic potential was assessed as described previously (58). Briefly, the test compounds were incubated with human RBCs in phosphate-buffered saline (PBS) (10% hematocrit) at 37°C under agitation. After 1 h of incubation, hemoglobin leakage was determined by measuring the absorbance of the supernatants compared with that of RBCs exposed to PBS alone (baseline) or to 0.2% Triton X-100 (for 100% hemolysis).

Organization in solution.

The self-assembly of OAKs in solution was assessed by light-scattering measurements as described previously (54). Briefly, serial twofold dilutions of the OAKs were prepared in PBS (50 mM sodium phosphate, 150 mM NaCl, pH 7.4) and incubated for 2 h at room temperature, and the light scattering of each dilution was measured by holding both the excitation and the emission at 400 nm (slit width, 1 nm). To describe the dependence of the scattered signal on the OAK concentration, the intensity of the scattered light was plotted against the total OAK concentration. Since the light-scattering signal is proportional to the number of aggregated molecules and the size of the aggregate, the slope is indicative of the aggregation tendency and reveals the aggregation properties, where a slope value above unity indicates the presence of aggregative form.

Antibacterial assays.

Antibacterial activity was assessed against a panel of gram-positive bacteria—S. aureus (MRSA 15903, ATCC 25923, ATCC 29213, ATCC 43300, MSSA 15668, MSSA 15873, MSSA U-17309, MSSA B-20647, MSSA 16001, MSSA 15885, S.a 15886, MRSA 15819, MRSA 15852, MRSA B-20745, MRSA 15918, and MRSA U-17314), Staphylococcus epidermidis ATCC 12228, Staphylococcus xylosus ATCC 29971, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 35667, Listeria grayi ATTC 19120, Listeria ivanovi ATCC 19119, Listeria welshimeri ATCC 35897, Listeria seeligeri ATCC 35967, Listeria innocua ATCC 33090, Listeria monocytogenes ATCC 1915, Streptococcus agalactiae (ATCC 13813 and 27956), Streptococcus pneumoniae (ATCC 6303 and 49619), Streptococcus bovis ATCC 9809, Streptococcus pyogenes ATCC 19615, Bacillus subtilis ATCC 6633, Bacillus polymyxa ATCC 842, and Bacillus cereus ATCC 11778—and gram-negative bacteria—E. coli (ATCC 35218, ATCC 43894, ATCC 25922, U-16287, U-16223, U-16350, U-16377, U-16328, U-16147, and U-16327), Salmonella enterica serovar Typhimurium ATCC 14028, S. enterica serovar Choleraesuis ATCC 7308, and Pseudomonas aeruginosa (ATCC 9027 and CI 11662).

The bacteria were cultured in suitable broth medium (brain heart infusion or LB). The MIC was determined using a slightly modified NCLSI microdilution assay (44) to enable comparison with previously characterized OAKs (42-46). For pH and salt variations, the culture medium was brought to the desired pH by adding NaOH or HCl (1 N) or to the desired saline concentration by adding NaCl to the culture medium.

The bactericidal kinetic assays were performed in test tubes in a final volume of 1 ml as follows. One hundred microliters of suspension containing bacteria at 106 CFU/ml in culture medium was added to 1 ml of culture medium containing no peptide or various peptide concentrations (serial twofold dilutions). After 0, 30, 60, 120, 180, and 360 min of exposure to the peptide at 37°C with shaking, the cultures were subjected to serial 10-fold dilutions (up to 106) by adding 50 μl of sample to 450 μl saline (0.85% NaCl). Cell counts were determined using the drop plate method (three 20-μl drops on LB agar plates). The plates were incubated at 37°C for 16 to 24 h, and the colonies were counted. Data were obtained from at least two independent experiments performed in triplicate.

The cytoplasmic membrane permeability assay is based on ATP reaction with the enzymatic system luciferin-luciferase, which generates light detectable by a luminometer. The light produced (expressed in relative light units) is proportional to the ATP concentration and to the number of viable bacteria originally present in the sample (22). The assay was performed in two steps. In the first step, ATP was directly measured in the bacterial medium supernatant (a suspension of 5 × 106 bacteria with or without OAK or control peptide at four times the MIC), while in the next step, total ATP was extracted with dimethyl sulfoxide (DMSO) (sample-DMSO ratio, 1/9 [vol/vol]). The assay was performed using the Cell Titer-Glow luminescent microbial cell viability assay kit (Promega; G7570) according to the manufacturer's recommendations.

For the ethidium bromide (EtBr) uptake assay, cells were grown overnight in LB broth at 37°C to an optical density at 620 nm of 1, washed twice in 200 μl of 1× PBS, and resuspended in the same buffer containing 0.5% glucose. After an incubation of 10 min at 37°C, samples were placed into a 96-well plate containing EtBr (final concentration, 1.0 μg/ml), and then either NC12-2β12 (serial twofold concentrations), Triton (final concentration, 0.01%), or one of the antibiotics gentamicin, ciprofloxacin, or tetracycline (each at four times the MIC) was added. Fluorescence was recorded by a BioTeK synergy HT Microplate Reader (excitation, 530 nm; emission, 645 nm) (5).

Resistance studies were performed as described previously (13, 18, 33, 44) using S. aureus strain ATCC 29213 as representative of gram-positive bacteria. Bacteria in the exponential phase of growth were exposed to an antimicrobial agent for MIC determination basically as described above. Following incubation overnight, bacteria were harvested from wells that displayed near 50% growth inhibition, washed and diluted in fresh medium, grown overnight, and subjected again to MIC determination for up to 15 similar serial passages. In parallel, MIC evolution during these subcultures was compared concomitantly with each new generation, using bacteria harvested from control wells (wells cultured without an antimicrobial agent) from the previous generation. The relative MIC was calculated for each experiment from the ratio of the MIC obtained for a given subculture to that obtained for first-time exposure. Statistical data for each of these experiments were obtained from at least two repeats performed in duplicate.

The DNA binding assay was performed using pUC19 plasmids that were extracted from E. coli K-12, as previously described (46). Briefly, the plasmids were preincubated with the OAKs at the specified concentrations (1 h; 37°C), followed by washes and incubation with the DNase BamHI (1 h; 37°C). The plasmids and marker (λ-HindIII) were then run in a 1% agarose gel for 40 min. The reported results are from two independent experiments.

Surface plasmon resonance (SPR).

Peptide binding to phospholipid membranes was determined using an optical biosensor system (Biacore Life Science, Uppsala, Sweden). Experiments and analysis of the binding properties were performed as described previously (3, 17, 56), except that, because the levels of immobilized membrane might vary between successive injection cycles, the maximal response was set to a local parameter, i.e., was unique for each sensorgram. Small unilamellar vesicles composed of cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPG) (ratio, 1/9), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG)-1-palmitoyl-2 oleoylphosphatidyl ethanol-amine (PE) (ratio, 1/4), and POPG-cardiolipin (CL) (ratio, 1.4/1) were prepared in PBS by the sonication method (3, 56) according to the manufacturer's instructions (Avanti Polar Lipids, Inc.) using a G112SP1 bath sonicator (Laboratory Supplies Company Inc.). Briefly, the dry components were dissolved in CHCl3 in a glass vial, and the solvent was evaporated under a stream of nitrogen for at least 30 min. The dried lipids were resuspended in PBS to give a total lipid concentration of 0.5 mM. The suspension obtained was vigorously vortexed, briefly sonicated, and passed (21 times) through 100-nm polycarbonate membranes in a LiposoFast-Basic extrusion apparatus (Avestin, Inc.) to give a translucent solution of vesicles with a mean diameter of 100 nm.

Peptide insertion assays.

Insertion experiments were carried out to quantify the interactions of NC12-2β12 with membrane mimics as previously described (9, 20). Initially, Langmuir monolayers of dipalmitoyl-sn-glycero-3 phosphocholine (DPPC)-cholesterol (9:1) were deposited at the air-liquid interface from chloroform (high-performance liquid chromatography grade; Fisher Scientific), whereas 1,2-dipalmitoyl-sn-glycero-3 phosphoglycerol (DPPG) and DPPG-1,2-dipalmitoyl-sn-glycero-3 phosphoethanolamine (DPPE) (ratio, 1/4) (Avanti Polar Lipids, Inc.) were from 9%/1% (vol/vol) chloroform-methanol solution (high-performance liquid chromatography grade; Fisher Scientific). After equilibration for 15 min, the monolayers were compressed to a surface pressure of 30 mN/m, which is equivalent to the packing density of the cell membrane (9, 20, 34, 35). The surface pressure was kept constant via proportional-integral-derivative feedback control. The NC12-2β12 solution was then evenly injected underneath the monolayers using a microsyringe with an L-shaped needle (Hamilton, Reno, NV) to make up the final concentration of 5 μM/liter. The injected peptides interacted with the lipid monolayers and resulted in an increase in the surface pressure when incorporated into the membrane. To keep the surface pressure constant, the surface area would have to increase. The resulting relative change in area per molecule, ΔA/A, was monitored throughout the experiments to compare the degrees of NC12-2β12 insertion into DPPC/cholesterol (9/1), DPPG, and DPPG/DPPE (1/4) monolayers. Unless otherwise stated, the experiments were carried out on Dulbecco's PBS without calcium and magnesium (Invitrogen) at 30 ± 0.2°C.

Epifluorescence microscopy.

Epifluorescence microscopy was used concurrently with insertion experiments to monitor the morphology of the monolayers on insertion of NC12-2β12. The Langmuir trough used for insertion experiments was equipped with an epifluorescence microscope mounted to observe the phase morphology of the lipid monolayers. The epifluorescence microscopy techniques were carried out as previously described (34-36). In order to reduce evaporation, contamination, and convection effects, a resistively heated indium tin oxide-coated glass plate (Delta Technologies, Ltd.) was placed over the trough. Data for excitation between 530 and 590 nm and emission between 610 and 690 nm were gathered through the use of a HYQ Texas red filter cube. Lipid-linked Texas red dye (DHPE Texas Red; Molecular Probes, Eugene, OR; 0.5 mol%) was incorporated into the spreading phospholipid solutions. Due to steric hindrance, the dye partitions into the disordered phase, rendering it bright and the ordered phase dark.

RESULTS

As shown in Table Table1,1, NC12-2β12 displayed the highest growth-inhibitory activity against gram-positive bacteria, such as staphylococci, streptococci, enterococci, and Listeria spp., while Bacillus spp. and gram-negative bacteria in general were less susceptible. Since the mode of action of previously investigated OAKs was clearly linked to membrane-active properties, it is interesting that, unlike staphylococci, for example, whose membranes are composed mostly of the anionic lipids phosphatidylglycerol and CL (15, 28), the membranes of various Bacillus spp. are atypically composed of variable levels of zwitterionic lipids, such as phosphatidylethanolamine and phosphatidylcholine, a property generally found in gram-negative bacteria (8, 15, 58, 59). This might explain the activity profile observed in the present study, suggesting that the OAK targets bacteria having highly anionic membranes, as discussed below.

TABLE 1.
Activities against a panel of bacteria

The influence of ionic strength on antimicrobial activity was investigated by performing MIC experiments under varied incubation conditions. As shown in Fig. Fig.1,1, the potency of NC12-2β12 was maintained in the presence of rather high salt concentrations (Fig. (Fig.1b)1b) and remained practically unchanged from pH 5.5 to 8.5 (Fig. (Fig.1c).1c). The fact that activity was stable over a wide range of ionic strengths is also reminiscent of membrane-active peptides (47). When incubated with RBCs, NC12-2β12 exhibited very low hemolytic activity, even at >300 μg/ml, a concentration corresponding to 100 times the MIC (Fig. (Fig.1d),1d), unlike the previously reported short analog C16-K-β12 (48). Figure Figure1e1e shows the dose-dependent light scattering of PBS solutions of these OAKs, where the critical aggregation concentration was estimated upon divergence from linearity. The results suggested that NC12-2β12 was devoid of self-assembly properties at the biologically relevant concentrations, which agrees with the previous finding that hydrophobicity-based self-assembly and hemolytic properties are linked (43, 45). In addition, preliminary assessment of the OAK's acute toxicity revealed no signs of observable adverse effects (100% survival) upon single-dose intraperitoneal administration of up to 10 mg/kg of body weight to a group of seven BALB/c mice. Together, these results established NC12-2β12 as the smallest OAK sequence displaying potent and selective antibacterial activity.

To investigate the mechanism of action, the OAK's time-kill properties against staphylococci were determined at two concentrations representing two and four multiples of the MIC. Figure Figure2a2a shows the outcome using a representative strain of S. aureus (ATCC 29213), where NC12-2β12 was found to exert a bacteriostatic effect in that the OAK managed to reduce the CFU count by 1 log unit at most within 6 h of exposure. Similar results were obtained with two additional strains of S. aureus, MRSA 15903 and ATCC 25923, and with E. faecalis, as well (data not shown).

FIG. 2.
Mechanistic studies. (a) Ability of a representative S. aureus strain to form colonies upon exposure to the OAK. Open circles, control; open stars, NC12-2β12 at 14 μg/ml; closed stars, NC12-2β12 at 28 μg/ml. Bacteria were ...

Next, bacterial viability in the treated cultures was determined based on the ATP concentration, an indicator of metabolically active cells (22). Figure Figure2b2b shows the levels of extracellular ATP as measured directly in bacterial suspensions treated with 28 μg/ml (four times the MIC), whereas Fig. Fig.2c2c shows the total ATP in an equivalent sample. The results demonstrate that the positive control [the dermaseptin derivative S4(1-16), known for its membrane-disruptive properties (46)] induced an intensive and nearly immediate leakage of ATP, as indicated by the abrupt increase in luminescence. Unlike the control AMP, the OAK showed virtually no ATP leakage when it was monitored for 6 h. Nevertheless, zooming on the y axis (Fig. (Fig.2b,2b, inset) revealed a slight luminescence signal that began rising after ~1 h of treatment. Furthermore, as indicated in the complementing experiment (Fig. (Fig.2c),2c), total ATP increased at a normal rate within the first hour of treatment, and then the ATP levels stabilized and remained practically constant for hours afterward, consistent with a bacteriostatic effect. These findings therefore suggested that while the OAK was essentially unable to disrupt the plasma membrane (certainly not in an abrupt manner, the way dermaseptin did), it nevertheless managed to induce discrete leakage of small solutes, such as ATP.

To corroborate the potential membrane damage inflicted on OAK-treated staphylococci, we measured the intracellular accumulation of EtBr, known for its ability to spontaneously translocate across the plasma membrane and interact with nucleic acids, as evidenced by a time-dependent increasing fluorescence signal (31). As shown in Fig. Fig.2d,2d, NC12-2β12 enhanced in a dose-dependent manner the otherwise slow and limited EtBr uptake. Note that no such increase was observed upon exposure to non-membrane-active antibiotics (gentamicin, ciprofloxacin, and tetracycline). This provided further evidence in support of the OAK's capacity to slowly breach the plasma membrane permeability barrier in staphylococci.

Next, we investigated the OAK's properties of binding to two potential targets, phospholipid membranes and bacterial DNA, believed to represent major targets of OAKs (44, 46) and AMPs in general (12, 24).

The properties of binding to membrane models were assessed using a variety of technologies. Figure Figure33 portrays typical binding (association/dissociation) curves obtained with the SPR technology, using five concentrations of NC12-2β12 in PBS, for each of the three model membranes; the resulting binding parameters, analyzed by the two-step binding model (17), are summarized in Table Table2.2. Thus, no significant binding of NC12-2β12 to the cholesterol/PC membrane could be detected at the concentration range used (up to 56 μg/ml), reflecting the low affinity between the reactants. In sharp contrast, NC12-2β12 displayed markedly higher-affinity constants to the bilayer that mimicked the membrane of S. aureus. We interpret the data as follows. At first, the OAK adhered to the membrane with high affinity (Kadhesion, 2.5 × 106 M−1). After the adhesion step, the OAK displayed a tendency to insert within the bilayer that was higher rather than to remain superficially bound (Kinsertion, 1.32); hence, the global affinity constant, Kapp, was 3.67 × 106 M−1. On the other hand, the OAK displayed lower adhesion and insertion affinities to the bilayer that mimicked E. coli cytoplasmic membrane composition, leading to the nearly 10-fold-lower value obtained for the overall binding affinity (Kapp, 0.4 × 106 M−1). These findings therefore correlate well with the cytotoxicity results (Fig. (Fig.11 and Table Table1)1) and suggest that the observed potency and selectivity of NC12-2β12 are (at least partially) linked to its preferential interaction with the staphylococcal membrane.

FIG. 3.
Properties of binding to model membranes. Shown are overlays of typical sensorgrams for various concentrations of NC12-2β12 in PBS, using three different model membranes: POPG-CL (1.4/1) (a); POPG-PE (1/4) (b); cholesterol-PC (1/9) (c). In each ...
TABLE 2.
Binding parameters analyzed by the two-step binding model

Additional studies of the OAK interaction with lipid monolayers provided further support for this hypothesis (Fig. (Fig.4).4). Constant-pressure insertion experiments demonstrated little or no insertion of NC12-2β12 into zwitterionic cholesterol-DPPC (1/9) or DPPG-DPPE (1/4) monolayers, although the latter contained 20 mol% of anionic lipid species, whereas a substantial increase in anionic DPPG area per molecule (~63%) indicated incorporation of the OAK molecules into the monolayer structure (Fig. (Fig.4A).4A). Epifluorescence microscopy measurements were performed simultaneously with the insertion isotherms to monitor the effect of NC12-2β12 binding on the morphology of the monolayer. Fluorescence image contrast arises due to different phase densities and partitioning characteristics of the dye molecules in coexisting phases. Therefore, it is possible to gain insight into the structure of the lipid layer by imaging its lateral fluorescence distribution. Epifluorescence microscopy images of the DPPG and DPPG-DPPE (1/4) monolayers at 30 mN/m displayed an array of densely packed “dark-gray” domains of condensed phase with narrow, bright liquid-disordered portions (Fig. (Fig.4B).4B). In contrast, a cholesterol-DPPC (1/9) monolayer was uniformly bright (data not shown), which suggests that condensed domains are either absent or smaller than the resolution of an epifluorescence microscope (1 μm). With injection of the OAK, a significant increase in the area of the liquid-disordered phase was observed in 1 to 2 min, followed by complete elimination of condensed domains after 4 to 5 min. In the case of the DPPG-DPPE (1/4) monolayer, the disordering effect of NC12-2β12 was moderate, which implies that the OAK affected the ordering of only DPPG molecules, but not DPPE. These findings therefore complemented the SPR data in explaining the observed selective antibacterial activity by implicating both the binding affinity and the ability to insert within highly charged plasma membranes as parts of a plausible mode of action.

FIG. 4.
Properties of binding to Langmuir monolayers. (a) Insertion isotherms of DPPG, DPPG-DPPE (1/4), and cholesterol-DPPC (1/9) monolayers showing the percentage of change in area per molecule after injection of NC12-2β12 (5.6 μg/ml). (b) Epifluorescence ...

To investigate the option of the OAK targeting bacterial DNA, we next assessed the OAK's ability to protect plasmid (pUC19) DNA from digestion by the DNase BamHI. When run in a 1% agarose gel, pUC19 displayed one band that, upon treatment with BamHI, ran as a higher-molecular-weight band (Fig. (Fig.5,5, top). As shown in lanes 4 and 5, preincubation of pUC19 with NC12-2β12 at the relevant concentrations (14 and 28 μg/ml, respectively) did not inhibit the DNase action, unlike the positive control experiment using the OAK C12-K-7α8 (lane 6), known for its ability to bind DNA and thereby prevent DNase action (46). The inability of NC12-2β12 to similarly prevent BamHI action on the plasmid argues against the possibility of a mode of action involving DNA interactions. Moreover, the high binding affinity exhibited by the OAK toward the membrane model (demonstrated with both mono- and bilayers) also argues against the possibility of an effective OAK translocation across the plasma membrane for interaction with additional cytoplasmic targets, thereby suggesting that the inward progression of NC12-2β12 is likely to be stopped at the plasma membrane level, the “preferred” target of many AMPs.

FIG. 5.
DNA binding and resistance studies. (Top) Representative agarose gel runs of the bacterial plasmid pUC19 (150 ng) in its native state and after exposure to BamHII (lanes 2 and 3, respectively). Lanes 4 and 5, pUC19 after preincubation with NC12-2β ...

To compare bacterial abilities to develop resistance, we assessed the emergence of resistance following multiple exposures of staphylococci to subinhibitory concentrations of the OAK or the antibiotics oxacillin and ciprofloxacin. Figure Figure55 (bottom) shows that bacteria developed resistance to both antibiotics (as reflected by 8- and 16-fold increases in the relative MIC, respectively) but not to the OAK, even after 15 consecutive subcultures, indicating that despite its short sequence and its bacteriostatic mode of action, NC12-2β12 retained the property of long OAKs of circumventing many drug resistance mechanisms (44).

DISCUSSION

This study provides several lines of evidence establishing NC12-2β12 as a potentially interesting compound for limiting the growth of gram-positive bacteria, which are leading aggressive human pathogens in the community and health care settings (55) and are known for their aptitude to become resistant to multiple drugs, including methicillin and other β-lactam antibiotics (11, 32, 38). Therefore, although new antibiotics have recently been introduced (1, 7, 57), alternative chemotherapeutics are urgently needed. As suggested in previous preliminary studies (43), the present investigation established NC12-2β12 as the smallest OAK sequence that combines low hemolytic activity with potent antibacterial activity. These results therefore suggest NC12-2β12 as a potential candidate for drug development whose attributes could include high safety and stability, as well as low production costs, in addition to the known advantages of classical host defense peptides: molecular simplicity and low potential for emergence of resistance.

Another novelty provided by the present study pertains to the mode of action. The mechanism of action of AMPs has been intensely investigated for over 2 decades (6, 50). However, while these studies have established potential targets, as well as the general traits of potential mechanisms, various details are still not fully understood. Many AMPs are notorious for their swift killing/lysis of bacteria, while a bacteriostatic mechanism has been proposed for relatively few AMPs (4, 14, 19, 51). Still, the molecular basis for selecting one mechanism over the other is not understood. A complicating factor in this endeavor is the extremely high number of variables in the peptide characteristics. In that sense, simple yet robust AMP mimics might be useful in shedding new light on the mechanism(s) of AMP action, as was recently illustrated (43, 44). Furthermore, previously characterized OAKs were typically bactericidal, whereas NC12-2β12 exerted a bacteriostatic effect, which, moreover, did not involve either abrupt disruption of the plasma membrane or direct inhibition of DNA functions, the two major modes of action of AMPs. Therefore, in this sense, too, the present findings strengthen the notion that OAKs reflect both complexity and variability in the prevailing mechanisms of action, as expected from a legitimate mimic of natural host defense peptides. Thus, although the present study does not fully elucidate the mechanism of action of NC12-2β12, it provides strong evidence for a mode of action implicating high-affinity OAK interactions with the plasma membrane that modify the membrane properties, thereby altering processes linked to membrane functions that depend on fluidity and charge distribution, as proposed previously (15). Thus, by acting as a membrane “modifier,” NC12-2β12 may substantially impact the membrane barrier function, as reflected by the augmented entry of an exogenous agent, EtBr. This effect is likely to be more pronounced for gram-positive bacteria due to the lack of additional permeability barriers, particularly the outer membrane of gram-negative bacteria. Alternatively, NC12-2β12 might interact with the peptidoglycan layer or block the passage of solutes by forming a large supramolecular array, or it may also interfere with peptidoglycan biosynthesis. Future studies might clarify this issue.

Several related lipopeptides were recently introduced (26, 29). In comparison however, NC12-2β12 displayed distinct properties in terms of selectivity and/or mode of action.

In conclusion, this study established NC12-2β12 as the smallest OAK that combines potent antibacterial activity with low hemolytic activity. In addition, the data indicate short OAKs as a potential source for small antibacterial molecules that can selectively inhibit bacterial growth while circumventing potential adverse effects associated with cytolytic compounds.

Acknowledgments

This research was supported by the Israel Science Foundation (A.M.; grant 283/08) and the National Institutes of Health (D.G.; R01 AI073892).

Footnotes

[down-pointing small open triangle]Published ahead of print on 1 June 2009.

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