Experimental Conditions That Enhance Potency of an Antibacterial Oligo-Acyl-Lysyl

Synthesis and purification.The OAK was synthesized by the solid-phase method using 9-fluorenylmethyloxycarbonyl active ester chemistry (17) using a 433A peptide synthesizer (Applied Biosystems), as reported previously (39). After cleavage from the resin, the crude OAKs were purified to >95% chromatographic homogeneity by reverse-phase high-pressure liquid chromatography (Alliance-Waters). Product identity was confirmed by subjecting the purified OAKs to electrospray mass spectrometry (ZQ-Waters). OAKs were stored as a lyophilized powder at −20°C. Prior to experimentation, fresh solutions were prepared in distilled water (1 mg/ml), briefly vortexed, and sonicated, and these were used as stock solutions in all experiments.

Bacteria. Escherichia coli O157:H7 (ATCC 43894) and Staphylococcus aureus (ATCC 25923, ATCC 29213, methicillin-resistant S. aureus [MRSA] ATCC 43300, and MRSA clinical isolate 15903) cells were grown overnight in LB medium (85 mM NaCl, 0.5% yeast extract, 1% tryptone, pH 7) and Listeria monocytogenes Li2 (ATCC 19115) cells were grown on Bacto brain heart infusion (BHI) medium (Difco Labs, France) (also containing 85 mM NaCl at pH 7), diluted to 2 × 10⁷ to 4 × 10⁷ CFU/ml, and incubated at room temperature for 60 min prior to being assayed. The stock cultures (of all strains) were maintained in 50:50 glycerol-LB broth at −80°C.

Antibacterial assays.Unless otherwise stated, antibacterial activities were evaluated by comparing the MIC, determined using the microdilution assay in sterilized 96-well plates in a final volume of 200 μl as follows. A stock solution of the OAK was diluted 10-fold in culture medium. One hundred microliters of medium containing bacteria (2 to 4 × 10⁵ cells/ml) was added to 100 μl of medium containing OAK (serial 2-fold dilutions). Growth under standard conditions (medium containing 85 mM NaCl, pH 7, at 37°C) was determined by optical density measurements at 620 nm. The MIC was considered the lowest peptide concentration that showed no increase in optical density after 24 h of incubation (72 h in the 4°C assay). Alternatively, to determine the MIC under nonstandard conditions, bacteria and OAK were preconditioned for 15 min prior to passing to the specified incubation conditions. Results were routinely obtained from at least two independent assays performed in duplicates, whereas data points displaying a 2-fold difference in Table 1 were submitted to a second round of additional experiments to verify statistical significance.

To determine the bactericidal kinetics under standard conditions, the assay was performed in test tubes in a final volume of 1 ml. One hundred microliters of bacterial suspension was added to 900 μl of medium containing no OAK or various OAK concentrations. After exposure to peptide at 37°C (up to 240 min), cultures were subjected to serial 10-fold dilutions (up to 1/10⁶) by adding 20 μl of sample to 180 μl saline (140 mM NaCl). Cell counts were determined using the drop plate method (three 20-μl drops onto LB or BHI agar plates). CFU were counted after 16 to 24 h of incubation at 37°C. To assess the effect of temperature variations, the assays were performed as described above but bacteria and test tubes containing the culture medium were first incubated for 15 min at the specified temperatures (i.e., 4, 25, and 48°C). 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. Results are expressed as mean CFU ± standard deviations (SD) obtained from two independent experiments performed in duplicates. A result of zero CFU indicates negative cultures.

Binding assays.OAK binding to phospholipidic membranes was determined using an optical biosensor system (BIAcore 2000, Uppsala, Sweden) based on the principles of surface plasmon resonance. The sensor chip L1 (a carboxymethyldextran hydrogel derivatized with lipophilic alkyl chain anchors) was used to prepare a lipid bilayer as described previously (19). To prepare model membranes for the binding studies, small unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (PC/PG) (3:1 molar ratio) were prepared by the sonication method (according to instructions from Avanti Polar) in the buffer corresponding to the incubation conditions used in the antimicrobial assays. The vesicles had a mean diameter of 20 nm, as determined by dynamic light scattering. The liposome solution was used as a stock solution for the surface plasmon resonance (SPR) experiments. The binding assay and analysis were performed as described previously (19), by injecting peptide solutions in phosphate-buffered saline (PBS) at five different concentrations (2-fold dilutions from 50 to 3 μM) in duplicates at a flow rate of 5 ml/min at 25°C. PBS variants in pH and salt concentration were also tested.

Organization in solution.The OAKs’ self-assembly in solution was assessed by light-scattering measurements, using a Jobin-Yvon Horiba Fluorolog-3 system with FluorEssence. Briefly, serial 2-fold dilutions of the OAKs were prepared in PBS starting at a 100 μM concentration and then incubated for 2 h at room temperature. 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 scattered light was plotted against the total OAK concentrations. 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, and a slope value above unity indicates the presence of an aggregative form.

Effects of pH, salt, and temperature on MIC value.Table 1 summarizes the MIC values obtained under the different incubation conditions tested, as assessed against three pathogenic bacterial species: the Gram-negative bacterium Escherichia coli O157:H7 and the Gram-positive bacteria Listeria monocytogenes Li2 and Staphylococcus aureus (MRSA 15903). Namely, the data obtained with E. coli showed that OAK's activity was significantly reduced (MIC of >50 μM) at extremely acidic conditions (pH ≤ 3.5); activity was recovered at higher acidic pH values and eventually even slightly improved at alkaline pH. Extremely high salt concentrations have somewhat reduced OAK's potency (e.g., by up to 4-fold at 1,030 mM NaCl), but a lower high-salt concentration had negligible effects. In contrast, MgCl₂ was more deleterious to OAK's inhibitory activity. Interestingly, the MIC values were hardly affected over a wide range of temperatures in which a 2-fold fluctuation (at most) was obtained between 4°C and 48°C.

Table 1 also shows an essentially similar outcome when activity was assessed against the Gram-positive bacterium L. monocytogenes, although a few differences were noted with S. aureus (MRSA 15903). Thus, basic pH and high temperature exerted a significantly higher potentiating effect, whereas conversely, high NaCl levels more readily reduced OAK's potency. Essentially similar results were observed with 3 additional S. aureus strains (MRSA 43300, 25923, and 29213, for which MIC values under standard conditions were 25, 50, and 50 μM, respectively), although MIC values were not determined for low temperatures as bacteria did not grow in the untreated control experiments (data not shown).

Effects of pH, salt, and temperature on bactericidal kinetics.The effects of the different incubation conditions on the rate of bacterial viability were assessed after exposure to a single concentration (12 μM) of C₁₂K-7α₈, a concentration representing the MIC value against S. aureus but 4 and 8 multiples of the respective MIC values against E. coli and Listeria. Figure 2 shows the time-kill curves obtained for each variation. The upper panels of Fig. 2 show the time-kill curves obtained upon pH variations. Although extremely acidic pH conditions (pH 3.5) have reduced bacterial growth even in the absence of OAK, the data showed that varying the pH from 3.5 to 8.5 has drastically affected OAK's mode of action, which reverted from a growth-inhibitory effect (at pH 3.5) to an increasingly potent bactericidal effect with increasing pH values. The middle panels of Fig. 2 show the time-kill curves obtained in the presence of various NaCl concentrations. Clearly, OAK's bactericidal mode of action was increasingly hampered with increasing NaCl levels. The lower panels of Fig. 2 show that low temperatures (4 and 25°C) were associated with an essentially growth-inhibitory effect, whereas bactericidal activity was favored as temperatures increased.

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FIG. 2.

Effects of pH, NaCl, and temperature on OAK's bactericidal kinetics. Bacteria were incubated at the specified conditions in the presence of 12 μM OAK. Plotted are mean CFU values ± standard deviations obtained from two independent experiments performed in duplicates. Standard incubation conditions are highlighted by solid symbols.

Effects of pH and salt on binding properties and organization in solution.The interactions of C₁₂K-7α₈ with a model cationic phospholipid membrane were compared using the surface plasmon resonance (SPR) technology. Note that only some of the conditions were assayed because of membrane instability at extreme pH and temperature values. Representative traces of association/dissociation curves are shown in Fig. 3A, demonstrating faster association kinetics upon increasing the pH value from 7 to 8.5. Figure 3B summarizes the binding parameters as analyzed using the two-step binding model (19, 39, 44), demonstrating the increase in the affinity constant (K_apparent) upon increasing pH values (from 5.5 to 8.5) at physiological NaCl concentration (140 mM) and the reduced affinity constant at high salt concentration (pH 7). This correlated well with the outcome from the activity assays, linking OAK's higher potency to its higher propensity to be incorporated within the bilayer at alkaline pH, while the opposite was observed for low pH values or extremely high salt concentrations.

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FIG. 3.

Effects of pH and ionic strength on membrane-binding properties and OAK's aggregation. (A) SPR trace for a representative run showing the dose-dependent binding curves to a PC/PG (3:1) membrane; (B) apparent affinity constants determined from the SPR traces using the two-step binding model. Changes in pH and salt conditions were assessed at 140 mM NaCl and pH 7, respectively. Error bars represent SD values from independent experiments. The lack of bars indicates reproducibility. (C). Dose dependence of the light-scattering intensities of PBS solutions of C₁₂K-7α₈ (square, pH 3.5; circle, pH 8.5; triangle, pH 7.0 at an NaCl concentration of 140 mM; and inverted triangle, pH 7.0 at an NaCl concentration of 1,030 mM) and its analog C₁₂K-7α₁₂ (diamond, pH 7.0 at an NaCl concentration of 140 mM).

To investigate how the extreme incubation conditions might affect OAK's availability in solution, its aggregative state was assessed at the relevant range of concentrations. A hydrophobic analog (C₁₂K-7α₁₂) known to form self-assemblies at low concentrations (38) was used as a positive control. Figure 3C demonstrates that unlike C₁₂K-7α₁₂, C₁₂K-7α₈ did not display a detectable aggregation at least up to 50 μM under all conditions used, suggesting that the observed properties were not biased by self-assembly phenomena. In this respect, previous studies have shown that C₁₂K-7α₈ lacks secondary structure at the active concentration range, including in its membrane-bound state (38, 39), whereas self-assemblies of C₁₂K-7α₈ (at >100 μM) demonstrated thermostability over a wide range of temperatures (at least between 10 and 60°C) (15).

Effects of combined optimal conditions.OAK was tested under the conditions considered as optimal, specifically, at pH 8.5, a temperature of 48°C, and a minimal salt concentration of 85 mM NaCl. As shown in the upper panels of Fig. 4, the new incubation conditions endowed a 16-fold enhanced potency against E. coli (MIC was reduced from 3.1 to 0.2 μM). Similarly, the MICs against L. monocytogenes and S. aureus were reduced by 32- and 16-fold (i.e., from 1.6 to 0.05 μM and from 12.5 to 0.8 μM, respectively).

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FIG. 4.

Effects of OAK treatment on MIC and bactericidal kinetics under standard and optimal conditions. The upper panels show the dose dependence of growth inhibition plots comparing standard (solid symbols) versus optimal (open symbols) conditions. The middle and lower panels compare time-kill curves determined under standard and optimal conditions, respectively, where the open and solid symbols represent the untreated control and OAK treatment, respectively. Plotted are mean values ± SD obtained from two independent experiments performed in duplicates.

This increase in OAK's potency was also reflected in terms of the time-kill curves (Fig. 4, compare the middle and lower panels for standard and optimal conditions, respectively); effective reductions of 99% of the bacterial population were observed within about 15 min instead of 2 h for E. coli and L. monocytogenes and >4 h for staphylococci.