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Published as doi: 10.1096/fj.07-105015.
 (The FASEB Journal. 2008;22:2652-2661.)
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Analogous oligo-acyl-lysines with distinct antibacterial mechanisms

Shahar Rotem*,1, Inna S. Radzishevsky*,1, Dmitry Bourdetsky*, Shiri Navon-Venezia{dagger}, Yehuda Carmeli{dagger} and Amram Mor*,2

* Department of Biotechnology and Food Engineering. Technion-Israel Institute of Technology; and

{dagger} Division of Epidemiology, Tel Aviv Sourasky Medical Center, Israel

2Correspondence: Laboratory of Antimicrobial Peptides Investigation (LAPI), Department of Biotechnology and Food Engineering, Technion—Israel Institute of Technology, Haifa, Israel. E-mail: amor{at}tx.technion.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bactericidal properties were recently shown to emerge from hydrophobicity and charge buildup in oligo-acyl-lysine (OAK) peptide mimetics. Toward understanding the attributes that govern the activity of this novel antimicrobial system, we compared the functional and mechanistic properties of a known octamer and a newly generated hexamer analog. The data provide strong evidence for multiple similarities that included high tissue stability, low hemolysis, large-spectrum antibacterial activity in vitro, and the ability to prevent Escherichia coli-induced mortality in vivo. Despite these similarities, however, the octamer mode of action involved membrane disruption, unlike the hexamer, which acted predominantly through inhibition of DNA functions with characteristically slower bactericidal kinetics. Collectively, the data support the view that the analogous OAKs induced bacterial death by distinct mechanisms and further suggest that relatively minor differences in the sequence of host defense peptides are responsible for selecting one mechanism over another, possibly in conjunction with differential binding affinities to the external and/or cytoplasmic membrane.—Rotem, S., Radzishevsky, I. S., Bourdetsky, D., Navon-Venezia, S., Carmeli, Y., Mor, A. Analogous oligo-acyl-lysines with distinct antibacterial mechanisms.


Key Words: host defense peptides • peptidomimetics • mechanism of action • intracellular targets


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HOST DEFENSE PEPTIDES (HDPs; ref. 1 ) have been widely investigated in the past two decades namely for their potential as new antimicrobial agents (2 , 3) despite multiple drawbacks such as reduced activity in presence of salt (4 5 6) , plasma components and proteases (7 , 8) , poor pharmacokinetic issues, high systemic toxicity, and high production costs (9 , 10) . To address these issues, various synthetic peptide mimetics, such as β-peptides (polyamides with conformational freedom greater than {alpha}-peptides; ref. 11 ), peptoids (oligomers of N-substituted glycines; ref. 12 ), and facially amphiphilic arylamide polymers (13) , have been proposed to improve activity and/or to endow metabolic resilience. Although some of these HDP-mimetic compounds were reported to exhibit nonhemolytic potent antibacterial activity against gram-positive and gram-negative bacteria (14 , 15) , no natural or modified peptide has obtained Food and Drug Administration (FDA) approval yet for any medical indications (2) .

A major pitfall to improving the antimicrobial properties of these highly promising compounds is the yet ill understood mode of action, a task rendered overwhelming namely due to the presence of too many variables. Thus, while various HDPs are believed to act in concert with the host innate and/or specific immune system (16 , 17) , many HDPs are believed to directly affect viability of microorganisms by nonspecific mechanisms that culminate in irreversible damage to the cytoplasmic membrane functions (18 , 19) and/or by inhibiting intracellular processes (20 21 22) , some of which, via specific interaction with microbial proteins. Although some aspects of the HDP mode of action are becoming comprehensible, various specific details pertaining to the molecular basis for potency and selectivity are yet to be fully established. Moreover, the reasons for a particular peptide to "select" one particular mechanism of action over another are not understood. There is thus a clear need for a simple antimicrobial system to provide some clearer answers on particular aspects of the HDP mechanism of action. The main aim of this study is to address the molecular requirements for mechanism selection. This question is of significance namely due to the intriguing fact that host defense peptides are often produced as a series of closely related analogs that can differ with but one or two residues (23 , 24) . A few studies (23 24 25) have suggested that this is possibly linked to increasing potency of the host antimicrobial response through synergistic action between the isoforms and/or through enlarging the spectrum of sensitive organisms. To the best of our knowledge, there are no studies that have addressed the question of how analogous peptide sequences might be related to target selection (e.g., membrane disruption vs. another defined target such as proteins or nucleic acids), although earlier studies (26) reported a correlation between antibacterial activity (e.g., of buforin II) and cell-penetrating ability, suggesting nucleic acids as potential targets.

Among the various classes of antimicrobial peptide (AMP) mimics proposed, the OAK class is quite interesting mainly due to its simplicity (27 , 28) . OAKs are composed of a small number of building blocks, acyl-lysine subunits (referred to as {alpha}n subunits, where n specifies the acyl length). Their flexible chain arrangement represents a novel intercalation of hydrophobic and hydrophilic residues, the sequence of which can be brought to vary in a myriad of alternatives. For instance, while increasing the number of acyl-lysine subunits revealed antibacterial activity, increasing hydrophobicity through a single acyl substitution (from C8K-7{alpha}8 to C12K-7{alpha}8) was sufficient to broaden the activity spectrum of OAK (28) much as observed with conventional AMPs (29 30 31) .

In the present study, we further define the OAK properties, namely by generating new closely related analogs and by demonstrating the consequences on the mechanism of action by comparing interactions of two analogous OAKs at three levels of suspected bacterial targets: the external membrane, the cytoplasmic membrane, and the intracellular components.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptides
OAKs and control peptides were produced by the solid phase method (32) as described previously (28) .

Antibacterial activity
Minimal inhibitory concentrations (MICs) were determined as described previously (28) . Bactericidal kinetics were assessed using the drop plate method (33) modified as described previously (34) . Statistical data for each experiment were obtained from at least two independent assays performed in duplicates.

Hemolytic activity
Human blood was obtained from a healthy donor in accordance to a protocol approved by the Technion Institutional Review Board. The ability of OAKs to induce hemolysis of human red blood cells was assessed according to Antibacterial Peptides Protocols (35) , where hemoglobin leakage was determined after 1 h of incubation in PBS at 37°C using a 10% hematocrit. Bivalirudin was used as a negative control. In addition, LC50 values listed in Table 1 were determined after 3 h of incubation with 1% hematocrit (7) . Reported results are from two independent experiments performed in duplicates.


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  		Table 1. Biophysical properties of {alpha}8 OAK derivatives

Dansyl-polymixin assay
The dansyl-polymixin assay was performed basically as described previously (36) . Reported results are from two independent experiments performed in duplicates.

Surface plasmon resonance
OAK binding properties to phospholipid membranes were investigated using the optical biosensor system (BIAcore 2000, Uppsala, Sweden). Experimental procedure and analysis of the binding kinetics and the resulting affinity constants were performed as described previously (37) . Briefly, the sensor chip L1 was used to prepare a lipid bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPC:POPG, 3:1 M ratio) as described below. The binding assay was performed by injecting peptide solutions in PBS at five different concentrations (3.12, 6.25, 12.5, 25, and 50 µM) in duplicates at a flow rate of 5 µl/min at 25°C. Surface regeneration between consecutive binding cycles included a sequential injection of NaOH (50 mM) and HCl (50 mM). To determine binding properties to lipopolysaccharide (LPS), various concentrations of OAKs were preincubated with 100 µg/ml of LPS (Escherichia coli O127B8), and then the assay proceeded as above. Reported results are from two independent experiments performed in duplicate. For liposome preparation, small unilamellar vesicles composed of POPC:POPG (3:1 M ratio) were sonicated in PBS as per the manufacturer (Avanti Polar, Alabaster, AL, USA) instructions using a G112SP1 bath sonicator (LASBCO; Laboratory Supplies Co., Louisville, KY, USA) to give a translucent solution of vesicles with a mean diameter of 20 nm as measured by dynamic light scattering using a BI-200SM Research Goniometer System (Brookhaven Instruments Corp., Holtsville, NY, USA). Liposomes were suspended in PBS buffer (pH 7.4), and measurements were performed at 25°C.

Cytoplasmic membrane permeation assays
The effect of OAKs and S4(1–16) on bacterial envelope integrity was determined by measurement of β-galactosidase activity in E. coli ML35 using O-nitrophenyl-β-D-galactopyranoside (ONPG), a nonmembrane-permeative chromogenic substrate essentially as described previously (38) . To assess leakage of β-galactosidase from the cytoplasm to the medium, midlogarithmic phase E. coli cells (optical density at 600 nm=0.05) were washed in PBS (pH 7.4) and incubated with OAK samples at 6x MIC value for different time intervals. At the designated time points, bacteria were centrifuged and discarded. The supernatant was incubated with ONPG (1.5 mM) for 10 min after the addition of sodium carbonate (0.2 M) to stop the reaction and enhance light absorbance. The hydrolysis of ONPG to O-nitrophenol was monitored spectrophotometrically at 420 nm. Reported results are from three independent experiments.

Cytoplasmic membrane depolarization
The assay was performed using E. coli ATCC 35218 as described previously (39) .

Intracellular localization using DNA binding assay
The assay was performed using E. coli K-12 containing pUC19 plasmid and S. typhimurium ATCC 14028 containing pGFP plasmid. Midlogarithmic phase bacterial cultures [grown on Luria-Bertani (LB) containing ampicillin] were washed in PBS, pH 7.4, and resuspended in the same buffer. C12K-4{alpha}8, C12K-5{alpha}8, C12K-6{alpha}8, and C12K-7{alpha}8 were incubated (at various concentrations) with the bacterial cells [106 colony-forming units (CFU)/ml] for 30 min and 1 h. After this preincubation step, bacteria were thoroughly washed (4 wash-spin cycles to prevent carry over of unbound OAK). Pellets from the last wash were submitted to the plasmid purification procedure using a miniprep plasmid DNA purification kit (Qiagen, Valencia, CA, USA). The plasmid was then incubated for 1 h at 37°C with DNase that opens the plasmid in one point (XbaI) according the manufacturer’s procedure (New England Biolabs, Beverly, MA, USA). The plasmids and marker ({lambda}-HindIII) were run in 1% agarose gel for 40 min. Control experiments performed to assess the OAKs binding to DNA were carried out as described above but instead of the OAKs being incubated with bacteria they were incubated with 150 ng of clean pUC19 (extracted from E. coli K-12 and purified with plasmid DNA purification kit after 1 h incubation with the OAKs). Reported results are from three independent experiments.

Inhibition of bacterial biosynthesis
Macromolecular synthesis and bacterial killing assays were performed as described (22) . Briefly, overnight cultures of E. coli CGSC 5895 were diluted in synthetic media and allowed to grow to the exponential phase (optical density at 600 nm=0.3). The cultures were spun down and resuspended in warm synthetic M9 medium, and 500 µl aliquots were incubated with 15 µl of [3H]thymidine and an excess of the remaining supplements. After 5 min of incubation at 37°C, the peptides were added at the specified concentrations. Samples of 50 µl were removed immediately before peptide addition (0 min) and at 5, 30, 60, 120, 180, and 360 min after peptide addition and added to 5 ml of ice-cold 10% trichloroacetic acid with excess unlabeled precursors to precipitate the macromolecules. After 40 min on ice and 15 min at 37°C, the samples were collected over vacuum on Whatman 47 mm GF/C glass microfiber filters (Whatman, Maidstone, UK) and washed twice with 10 ml of ice-cold 10% trichloroacetic acid. The filters were dried and placed in 7 ml scintillation vials with ReadySafe liquid scintillation cocktail (Beckman, Fullerton, CA, USA), and counts were obtained in a Packard Tri-Carb 2100TR (Packard, Meriden, CT, USA) scintillation counter for 5 min for each filter. At the same time points, 5 µl aliquots were removed from nonradioactive parallel cultures otherwise identical to those containing a radioactive precursor, diluted in 1 ml of buffer, and plated onto plates with Luria-Bertani medium and supplements to obtain a viable count. The aliquots were diluted immediately on withdrawal from the experimental samples, thus preventing further peptide action by radically reducing its concentration. This ensured that the viable counts corresponded to the respective time points. Reported results are from three independent experiments.

Whole blood assay
Thirty microliters of Klebsiella pneumoniae CI 1276 in saline (0.85% NaCl; 106 CFU/ml) and 4 µl of OAK solution in double-distilled water (final concentration of 16 and 4 multiples of the MIC value) were added to 270 µl whole blood and incubated at 37°C for 2 h. After the incubation period, the samples were plated on LB agar. Reported results are from two independent experiments.

In vivo studies
All procedures and care and handling of the animals were reviewed and approved by the Technion and Tel-Aviv Medical Center Institutional Animal Care and Use Committees. Prevention of E. coli-induced mortality in an acute peritonitis/sepsis model (40) was assessed using neutropenic mice as described previously (28) . Briefly, adult female imprinting control region (ICR) mice (Harlan, Rehovot, Israel) were rendered neutropenic. Infection was induced by intraperitoneal administration of a logarithmic-phase culture of extended spectrum β-lactamase-producing strain of E. coli (CI 3504, isolated from a patient with peritonitis and bacteremia) with a mean bacterial inoculum 5 x 106 CFU in 0.5 ml brain heart infusion broth. The intraperitoneal treatments included four injections (administered 1, 6, 20, and 28 h after infection) in 0.5 ml of vehicle (PBS) with imipinem as control antimicrobial compound C12K-7{alpha}8 or C12K-5{alpha}8. Mice (n=10 per group) were monitored for survival over a 6 day period after infection. Survival data were obtained from two independent experiments.

For quantitative analysis in blood, each test compound was administered in a single intraperitoneal dose (100 µg/mouse) to pathogen-free, male BALB/c mice (18–20 g). At various time points (0, 10, 20, 30, 40, 50, 60, and 120 min), 2 mice were euthanized by CO2 asphyxiation, blood samples were collected from the portal vein and centrifuged for 2 min at 6000 g, and the plasma was lyophilized. Samples were rehydrated with 0.2 ml water, vigorously vortexed, sonicated, centrifuged (10 min at 14,000 g), and analyzed by liquid chromatography-mass spectrometry (LC-MS). For quantitative calibration, standard curves were established (0.4–50 µg OAK) in 0.2 ml water (limit of detection 20 ng) as well as in plasma samples as described above. The LC-MS apparatus was as described previously (28) . Data acquisition was in the single ion recording (SIR) mode using transitions corresponding to z = +3.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The basic structure of a typical OAK is shown in Fig. 1 . Listed in Table 1 , for comparison purposes, are the sequences of a series of five {alpha}8 OAKs and their lauryl derivatives. To assess the effect of octyl to lauryl substitution, the MIC of each OAK was determined against four test microorganisms representing gram-positive (Staphylococcus aureus and Bacillus cereus) and gram-negative (E. coli and Pseudomanas aeruginosa) bacteria. Also shown in Table 1 are the hemolytic concentrations along with the HQ properties of the compounds: H is a hydrophobicity measure and Q is the calculated charge, as determined by reversed-phase HPLC.


Figure 1
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  		Figure 1. Molecular structure of a typical OAK. Parentheses define {alpha}8 (aminooctanoyl-lysyl) subunits composing the OAK (n≥1).

As previously observed with C8K-7{alpha}8 (28) , laurylation of shorter OAKs, C8K-6{alpha}8 and C8K-5{alpha}8, resulted in improved antibacterial activity. Thus, despite the charge reduction, increasing hydrophobicity through elongation of the amino-terminal acyl significantly increased antibacterial potency on all four test bacteria but not hemolytic activity. Laurylation of the pentamer C8K-4{alpha}8 resulted in a quasi-similar outcome in that potency was enhanced again but to a lesser extent even though hydrophobicity increased proportionally. Finally, laurylation of the tetramer C8K-3{alpha}8 had no apparent effects. None of these modifications seemed to affect the hemolytic properties as verified below (Fig. 2 F).


Figure 2
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  		Figure 2. Comparing hexamer and octamer properties in vitro and in vivo. A) Time-kill curves of E. coli cultured in LB medium in the presence of 3 (white) and 6 (black) multiples of the MIC value. Circles = C12K-5{alpha}8; squares = C12K-7{alpha}8; triangles = normal bacterial growth. B) Survival of infected mice. Neutropenic mice (n=10 per group) were inoculated intraperitoneally with 5 x 106 CFU of extended spectrum β-lactamase producing E. coli and treated intraperitoneally with 4 doses (1, 6, 20, and 28 h after infection) of imipenem at 2 mg/kg (triangles), PBS (inverted triangles), or C12K-5{alpha}8 (circles) and C12K-7{alpha}8 (squares) at 4 mg/kg. C) Mean plasma concentrations determined after intraperitoneal administration (5 mg/kg body weight). Each time point represents values obtained from 2 mice. Data sets were obtained from LC-MS analysis of known concentrations of OAK in plasma. Plot shows integrated peak area for each known quantity of OAK, as monitored with SIR mode for M/z corresponding to z = +3. Dotted line = limit of detection. D) MIC experiments on E. coli before and after incubation in 50% murine plasma of C12K-5{alpha}8 (black), C12K-7{alpha}8 (gray), and dermaseptin S4(1–16) (white). Stars = MIC >50 µM. E) Viability reduction of blood-resistant bacteria (K. pneumonia) after 2 h incubation with C12K-5{alpha}8 (black) and C12K-7{alpha}8 (gray) in 90% human whole blood (initial inoculum 106 CFU/ml). Dashed line = detection limit. F) Hemolytic activity. Hemoglobin release by C12K-5{alpha}8 (black), C12K-7{alpha}8 (gray), and bivalirudin (white) determined after 1 h incubation in PBS at 37°C with human red blood cells (10% hematocrit). In all panels, plotted values represent means ± SD obtained from at least 2 independent experiments performed in duplicate (lack of SD indicates consistency).

These results therefore revealed two new analogs with an apparent potency comparable with that of C12K-7{alpha}8 but with reduced activity against P. aeruginosa. Indeed, further assessment of the shortest most active derivative against a larger panel of bacteria (Table 2 ) established a rather poor activity of C12K-5{alpha}8 on certain bacteria such as P. aeruginosa as verified on 10 different strains despite potent activity (MIC=3.1–6.2 µM) over many of the 50 bacteria tested.


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  		Table 2. Growth inhibitory activity against a panel of bacteria

To find out what other discrepancies might be ascribed to the sequence difference between the OAKs (2{alpha}8 subunits), C12K-5{alpha}8 was submitted to a series of functional and mechanistic studies and compared with C12K-7{alpha}8 that was previously partly characterized (28) . Figure 2A shows time-kill curves of the analogous OAKs determined at a concentration corresponding to three and six multiples of the MIC value. Although both OAKs exerted a bactericidal effect, the shorter derivative consistently reduced the viable bacterial population at slower rates.

Next, we compared therapeutic efficacy in vivo as assessed using a murine peritonitis-sepsis model. In this model, drugs were administered intraperitoneal starting 1 h after intraperitoneal infection. As evident from Fig. 2B , both OAKs have significantly prevented mortality of mice infected with a lethal inoculum. In these representative experiments, treatment with a conventional antibiotic, imipenem (4x2 mg/kg), resulted in survival of 100% of the infected mice, while treatment with the OAKs was responsible for 100 and 90 for C12K-7{alpha}8 and C12K-5{alpha}8, respectively (4x4 mg/kg each), compared with 30% in the vehicle-treated control group (P<0.005). Note that neither the dermaseptin or magainin derivatives were efficient in this model (27) .

To compare the stability of OAKs, we assessed their ability to reach circulation after intraperitoneal administration to mice (5 mg/kg body weight). As shown in Fig. 2C , LC-MS analysis of blood provided evidence that the OAKs reached the bloodstream within minutes while their respective concentration dropped over time rather slowly. Nevertheless, the data suggested that C12K-5{alpha}8 was subject to somewhat faster elimination. Note that under these experimental conditions a control peptide [dermaseptin S4(1–16)] did not reach a detectable level.

To compare the ability of OAK to withstand activity in complex media, we assessed the MIC evolution against E. coli after incubation in presence of 50% human plasma (Fig. 2D ) and the bactericidal properties on incubation in 90% whole blood (Fig. 2E ). Figure 2D shows that 3 h of incubation of a conventional AMP, the dermaseptin derivative S4(1–16), led to activity loss, as reflected by MIC increase from 3 to >50 µM. Under these inactivating conditions, the OAKs remained practically as active after 18 h. Figure 2E shows that they also exerted a bactericidal effect in a dose-dependent manner when added to infected human whole blood. These results predict that the hexamer, like the octamer (28) , could maintain direct antibacterial activity in vivo with minimal loss to nonspecific interactions with tissue components.

As shown in Fig. 2F , C12K-5{alpha}8 was devoid of hemolytic activity. Namely, incubation with human red blood cells did not yield the characteristic dose-response profile observed with conventional AMPs (7) . In fact, both OAKs behaved similarly to the non-AMP bivalirudin, a thrombin inhibitor approved by FDA for intravenous administration (41) . Bivalirudin and the OAKs displayed a noise-like background level at least up to 156 µM, a concentration that corresponds to 50-to 100-fold the MIC value observed on various bacteria (Table 2) .

Figure 3 summarizes mechanistic experiments in which the analogous OAKs displayed distinct behaviors. The OAKs were first assessed for their ability to affect the cytoplasmic membrane integrity by comparing their ability to induce cleavage of the chromogenic substrate ONPG by the cytoplasmic enzyme β-galactosidase using the E. coli mutant ML35, as compared with the dermaseptin S4 derivative, known for its membrane-active properties (28) . Normally, because of the distinct localizations of the enzyme and substrate, any significant ONPG hydrolysis would be indicative of damage to the cytoplasmic membrane; presumably, the membrane discontinuities generated by the test compound are large enough to allow enzyme leakage. E. coli cultures were first exposed to the test compounds, and the cleavage reaction was allowed to occur after the addition of ONPG to the supernatant. The results shown in Fig. 3A indicated that only dermaseptin and C12K-7{alpha}8 but not C12K-5{alpha}8 afflicted damage to the bacterial cytoplasmic membrane.


Figure 3
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  		Figure 3. Interference with cytoplasmic membrane integrity and with DNA functions. A) Leakage kinetics of intracellular β-galactosidase to medium measured as a function of ONPG hydrolysis. E. coli ML35 (2.1x106 CFU/ml) were incubated with test compounds at 6 multiples of the MIC value. Changes in absorbance were recorded after ONPG was added to suspension supernatants. Circles = C12K-5{alpha}8; squares = C12K-7{alpha}8; triangles = dermaseptin S4(1–16); inverted triangles = bacterial suspension without treatment. Data in A-C are means ± SD obtained from 3 independent experiments performed in duplicate (lack of SD indicates consistency). B) Cytoplasmic membrane depolarization assay using the fluorescent dye diSC3-5 in E. coli permeabilized with EDTA. Changes in fluorescence were recorded after test compounds were added to cell suspensions at 6 x MIC. Note that B depicts continuous recordings where symbols were added for clarity. C) Macromolecule synthesis in E. coli CGSC 5895 using [3H]thymidine incorporation assay in the presence of 6 x MIC of test compounds. D) Evidence for translocation through cytoplasmic membrane and interaction with DNA. Shown (on left) are representative agarose gel runs of the E. coli plasmid pUC19 (150 ng) in its native state and after exposure to XbaI. Middle depicts pUC19 after incubation with 6 x MIC of C12K-7{alpha}8 and C12K-5{alpha}8 followed by plasmid extraction procedure and exposure to XbaI. Right shows similar gel runs resulting from OAKs pretreatment of E. coli (at 6xMIC, 1 h incubation in PBS at 37°C) followed by pUC19 extraction and exposure to XbaI. E) Dose-dependence demonstration of translocation, essentially as described in D, using either E. coli (top gels) or S. typhimurium (bottom gels). Shown (on left) are representative gel runs of pUC19 and pGFP in native state and after exposure to XbaI. Middle and right gels show similar runs but starting with OAK pretreatments of bacteria (1 h incubation in PBS at 37°C) and then proceeding to plasmid extraction and exposure to XbaI as per left lanes. F) Macromolecule synthesis in E. coli CGSC 5895 using [3H]thymidine incorporation assay in the presence of 1 (black), 3 (black and white), and 6 (white) multiples of the MIC value of C12K-5{alpha}8 (left panel) and C12K-7{alpha}8 (right panel).

In addition, we assessed the ability of the compounds to induce membrane depolarization in E. coli cells. The rapid increase in fluorescence shown in Fig. 3B indicated that the octamer was nearly as potent as the conventional AMP in inducing membrane depolarization but clearly more potent than the hexamer. Time-kill curves obtained under the specific experimental conditions (data not shown) showed a strong correlation in that both dermaseptin and C12K-7{alpha}8 but not C12K-5{alpha}8 induced a rapid and concomitant reduction in bacterial viability (essentially as observed in Fig. 2A ), thus also suggesting that the mechanism of action of C12K-7{alpha}8 but not C12K-5{alpha}8 involved destabilization of the cytoplasmic membrane.

In accordance with the outcome from Fig. 3A, B , Fig 3C shows that bacteria exposed to dermaseptin or to the octamer OAK were subject to rapid inhibition in carrying out bacterial biosynthesis as assessed by their ability to incorporate radiolabeled thymidine. Furthermore, as shown in Fig. 3F , both OAKs behaved similarly, inhibiting bacterial biosynthesis in a dose-dependent manner. Interestingly, however, the hexamer was practically as potent an inhibitor despite its apparent inability to afflict damage to the cytoplasmic membrane (Fig. 3A, B ).

To compare the ability of OAK to affect bacterial viability after translocation across the cytoplasmic membrane, we developed a simple assay exploiting the fact that bacteria often contain easily extractable plasmids, such as pUC19 in E. coli K-12, which can be used as a natural reporter for OAK interactions with bacterial DNA. When run in a 1% agarose gel, extracted pUC19 normally displays three bands (topoisomers), which, as shown in Fig. 3D , left, can easily be converted to a single band on treatment with the specific DNase, XbaI (selected due to the presence of a single restriction site on the plasmid). Figure 3D , middle, shows that pUC19 pretreatment with either the hexamer or octamer (at 6x the MIC value) has protected the plasmid from XbaI digestion, indicating that, in principle, both OAKs have the ability to interact with pUC19. It is assumed that nonspecific interactions with bacterial DNA will alter the enzyme binding affinity to its site of action either by the physical occupation of OAK of the binding site on the plasmid or by altering its chemical environment in a manner that will prohibit proper Xbal reaction.

The question now is what actually happens in live bacteria? To answer this, E. coli cultures were pretreated with one of four OAK analogs, including the octamer and hexamer but also the heptamer that displayed a similar MIC value and the pentamer that was significantly less active (Table 1) . OAK pretreatment was followed by a thorough wash to minimize OAK carryover, and then pUC19 was extracted and exposed to the restriction enzyme. As shown in Fig. 3D (right), when DNA extraction was preceded by OAK treatment, only the hexamer was able to affect the enzymatic activity of Xbal. The fact that the octamer did demonstrate the direct ability to bind pUC19 and affect activity of Xbal similarly to the hexamer (Fig. 3D , middle) supports the view of the reduced ability of the octamer to translocate into the cytoplasm. In contrast, the hexamer was found to inhibit Xbal activity as soon as after 30 min incubation (and even sooner; data not shown). This finding supports the view of cytoplasmic localization of the hexamer despite its inefficacy in disrupting the plasma membrane structure. To verify whether the OAKs would display indeed the same behavior under different sets of conditions, the analogs were assayed under different concentrations and against either E. coli or S. typhimurium. As shown in Fig. 3E (middle), the hexamer protected both plamids (i.e., translocated into the cytoplasm of both bacteria) at concentrations of 6x and 3x the MIC value. Such protection was not evidenced at 1 x MIC and was indeed least efficient in inhibiting bacterial proliferation (Fig. 3F ). Under these conditions, the octamer did not protect to any observable extent (i.e., displayed reduced cell-penetrating ability of either bacteria; Fig. 3E , right). Note that one cannot exclude the possibility that the octamer also translocated and affected DNA (even to a small extent). The apparent lack of effect on DNase activity might be linked to the membrane disruption property of the octamer, which hypothetically could have allowed OAK-bound plasmids to be removed during the wash steps. Nevertheless, as this argument does not hold for the hexamer, the combined data therefore support the notion that C12K-5{alpha}8 could affect bacterial viability though interference with DNA maintenance and expression. Interestingly, out of the series of four OAK analogs tested, only the hexamer exhibited this capacity.

To obtain insight into possible reasons for the postulated differential mechanisms of action, the analogous OAKs were compared for their binding affinity for the outermost potential targets of E. coli, anionic phospholipid membranes and LPS, the major components of the outer membrane. Data shown in Fig. 4 A, B using the dansyl-polymixin assay indicated that both OAKs can displace dansyl-polymixin from their binding sites on LPS, suggesting that LPS-binding might represent a step in their mechanism of antibacterial action. The OAK performance was rather comparable with that of unmodified polymixin.


Figure 4
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  		Figure 4. Binding properties of the analogous OAKs. A, B) Comparison of the ability of OAKs and polymixin to displace LPS-bound dansyl-polymixin. Squares = C12K-7{alpha}8; circles = C12K-5{alpha}8; triangles = polymixin B. Plotted values for both panels are means ± SD obtained from 2 independent experiments. C, D) Comparison of the binding properties to POPC:POPG bilayers (3:1) as determined by SPR. Shown are typical association/dissociation curves used to calculate affinity constants listed in G. Each panel displays binding curves obtained with 5 concentrations (3, 6, 12, 25, and 50 µM) of C12K-5{alpha}8 (C) and C12K-7{alpha}8 (D). Curves with greater intensity correspond to higher concentrations. E, F) Maximum resonance for OAKs association in presence (dashed line) and absence (solid line) of LPS. G) Summary of affinity constants of the interactions analyzed by local fitting using 2-stage binding model. Parentheses represent affinity constants in presence of LPS. BST = below sensitivity threshold. Values are means ± SD obtained from 2 independent experiments performed in duplicate.

Finally, we compared the binding properties with a phospholipid membrane model that mimics bacterial cytoplasmic membrane using the surface plasmon resonance (SPR) technology. Figure 4C, D depicts typical dose-dependent accumulation on the PC:PG bilayer (association) resulting in a return to baseline (reflecting dissociation from the membrane into the medium). These kinetic curves were used to derive the binding constants shown in Fig. 4G , using the two-step binding model (37) . The OAKs displayed binding parameters rather comparable with those observed with potent AMPs such as magainin and dermaseptin derivatives (28 , 37) . The octamer displayed a higher overall binding constant (Kapparent) with a 5-fold higher affinity for the first step (Kadhesion), whereas the values obtained for Kinsertion indicated that both analogs displayed a tendency for insertion approximately 2-fold (1.7 and 2.3) higher than for remaining superficially adhering to the membrane surface. Also, as shown in Fig. 4E, F , the binding affinities of the OAKs were significantly affected in presence of LPS, supporting the outcome from the dansyl-polymixin experiments (Fig. 4A, B ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Based on previous findings on the structure-activity relationships of OAKs (27 , 28) , this study aimed to further assess the implication of HQ properties in antibacterial activity of this novel peptide mimetic antimicrobial system. As previously observed with C12K-7{alpha}8, we show here that the mere substitution of the N-terminal acyl, from octyl to lauryl, was sufficient to endow potency and broader activity spectrum on shorter analogs, including the heptamer C12K-6{alpha}8 and the hexamer C12K-5{alpha}8, both bearing similar H but reduced Q values compared with C12K-7{alpha}8 (Table 1) . The data suggest that nonhemolytic antibacterial activity was maintained as long as the analogs remained within an optimal HQ window, since beyond the hexamer activity gradually dropped then vanished. Of note is the fact that C12K-5{alpha}8 inhibited growth of most of the strains tested but displayed weaker activity against certain bacteria such as Vibrio cholera and P. aeruginosa, highlighting the significance of higher Q values in potency against these clinically important bacteria.

In addition, by comparing functional and mechanistic properties of two analogous OAKs (C12K-7{alpha}8 and C12K-5{alpha}8), this study has provided evidence for the implication of an HQ window also in the mechanism of action. Thus, the OAKs displayed similar properties with many respects, suggesting a priori a similar mode of action: both OAKs exerted potent antibacterial activity in vitro and demonstrated in vivo efficacy in a representative model of lethal infection. Moreover, both OAKs were devoid of hemolytic activity, while displaying rather similar tissue stability. These results, which undoubtedly advocate optimistic prospects as to the potential use of the new (shorter) analog C12K-5{alpha}8 in fighting bacterial infections, suggested, furthermore, a similar mechanism of action with C12K-7{alpha}8.

However, a variety of experimental evidence argued for rather distinct mechanisms of action: both OAKs exerted a bactericidal effect but at different rates. This might not necessarily reflect different mechanisms of action but could rather reflect different efficiencies namely due to differential affinities to target sites of action. We therefore investigated the binding properties to model systems that mimic external and cytoplasmic membranes as well as with plasmidial DNA, representing an intracellular target. We interpret the data that emerged from these experiments as follows: both OAKs are initially attracted to the bacteria, driven by electrostatic forces, and interact sequentially with the external as well as the cytoplasmic membranes as proposed by the self-promoted uptake hypothesis (42) . However, due to their higher affinities, relatively more octamer molecules adhere to these external components reducing de facto the number of octamer molecules that can translocate across the cytoplasmic membrane. Conversely, relatively more hexameric molecules would be available for crossing the cytoplasmic membrane and to interact with intracellular targets. Moreover, this study showed that the faster bactericidal kinetics of the octamer correlated with faster depolarization of bacterial membrane potential and faster leakage of an intracellular enzyme. These results support the view that the octamer induced bacterial death through disruption of the cytoplasmic membrane, whereas the slower rates observed with the hexamer seem inconsistent with death caused by a membrane disruptive event. In contrast, only the hexamer displayed the unambiguous ability to translocate across the cytoplasmic membrane and interact with nucleic acids. This behavior is likely to be linked to its reduced interactions with cell wall components, as shown in Fig. 4 . Furthermore, once the octamer has reached the cytoplasmic membrane and induced its disruption, protons and ions equilibrate and the difference in trans-membrane potential is canceled out and so is the driving force for translocation (43 , 44) . Conversely, the reduced Q value of the hexamer is likely to reduce all OAK electrostatic interactions on the way to the cytoplasmic membrane where the undamaged trans-membrane potential difference can be used as a driving force for internalization and interaction with intracellular targets such as DNA.

Taken together, the data support the view that both the hexamer and octamer OAKs undergo multiple interactions with cell wall components but only the octamer is endowed with the capacity of inducing bacterial death through disruption of the cytoplasmic membrane, whereas the hexamer kills bacteria predominantly as a result of its unique capacity to translocate across an apparently intact cytoplasmic membrane, which enables its interaction with DNA and mediate inhibition of vital functions. The finding that analogous OAKs can use distinct mechanisms of action was therefore surprising although not unprecedented in conventional HDPs. While many HDPs are believed to act by destabilizing the plasma membrane, recent studies (21 , 26 , 45 , 46) report that a growing list of peptides, such as PR-39, buforin II, pyrrhocoricin, tPMP-1, and HNP-1, all proposed to affect bacterial viability through interaction with intracellular targets. Therefore, the novelty here includes the finding that minor differences (such as a single charge, slight hydrophobicity, and/or backbone length) are sufficient to make an OAK select one mechanism over another. These findings therefore suggest a parallel with HDPs and thereby raise the possibility that the reason for the simultaneous production of multiple closely related host defense peptides might be linked to an evolutionary advantage that provides the producing cell/tissue with a highly efficient attack system composed of a battery of peptides acting on multiple targets and using multiple mechanisms (as per the present study) and acting in synergy and/or against a larger spectrum of bacteria as previously proposed (23 24 25) . The fact that dermaseptins S3, which belongs to a family of well-known membrane disrupting peptides (47 48 49) , was recently shown to induce apoptosis (50) supports this view. Also, a recent study (20) reached a similar conclusion concerning human β-defensins HBD2 and HBD3.


   ACKNOWLEDGMENTS
 
This research was supported by the Israel Science Foundation (grant 387/03) and BiolineRx (grant 2006992).


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication December 19, 2007. Accepted for publication March 13, 2008.


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 DISCUSSION
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