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. 2013 Sep;79(17):5179–5185. doi: 10.1128/AEM.01394-13

Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide αs2-Casein f(183–207)

Avelino Alvarez-Ordóñez a,b,✉, Máire Begley a,b, Tanya Clifford a,b, Thérèse Deasy a,b, Kiera Considine a,b, Colin Hill a,b,c
PMCID: PMC3753947  PMID: 23793637

Abstract

Template-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived αs2-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogens Listeria monocytogenes and Cronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the αs2-casein f(183–207) peptide also caused a significant reduction of the effectiveness against C. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of αs2-casein f(193–203) and αs2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency against L. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid αs2-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.

INTRODUCTION

Antimicrobial peptides (AMPs) are low-molecular-weight proteins that exhibit antimicrobial activity against bacteria, fungi, and viruses (1, 2). Proteins of food origin and milk proteins in particular have been identified as a rich source of antimicrobial peptides, many of which have been extensively characterized (3). Milk-derived AMPs include caseicins (4), kappacin (5), isracidin (6), lactoferricin (7), casocidin-I (8), and the fragment comprising residues 183 to 207 [f(183–207)] of bovine αs2-casein (9). The latter was identified in a peptic hydrolysate of bovine αs2-casein and shows high antibacterial activity against both Gram-positive and Gram-negative bacteria, with MICs in the μM range (9, 10). The precise mechanism by which the αs2-casein f(183–207) peptide acts remains still unclear, although López-Expósito and coauthors have postulated that initial binding sites of the αs2-casein f(183–207) peptide are lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative bacteria and that the peptide is able to permeabilize and generate pores in the outer membrane of Gram-negative bacteria and the cell wall of Gram-positive bacteria, causing the leakage of intracellular content (10).

Most AMPs are amphipathic and hydrophobic α-helical peptides, with a positively charged domain. These properties are thought to be essential for their biological activity, with the amphipathic character causing disruption of the negatively charged bacterial membrane (11). Template-based studies of peptide derivatives obtained through manipulation of the amino acid sequence have often been performed in order to identify properties that are important for AMP activity (12). Such studies have shed light on the importance of specific amino acids and residue positions in the activities of different peptides (13–15). However, it appears that the effect of particular manipulations is context dependent and varies according to the template sequence, in that analogous substitutions may have substantially different effects on different peptides or at different positions in the primary sequence (16).

Cronobacter sakazakii is a Gram-negative microorganism that has been identified as the causative agent of serious disease episodes in humans, especially infants, including meningitis, hydrocephalus, necrotizing enterocolitis, septicemia, and brain abscesses (17). C. sakazakii has been isolated from a wide variety of food sources, with powdered infant formula (PIF) being the most common vehicle involved in newborn infections (18–21). The food-borne pathogen Listeria monocytogenes also represents a concern for the dairy industry. L. monocytogenes is a Gram-positive bacterium that can cause serious invasive illness, mainly in certain high-risk groups, including elderly and immunocompromised patients, pregnant women, newborns, and infants (22).

The use of milk-derived antimicrobial peptides as food-grade additives, either alone or in combination with other antimicrobials, may be an attractive strategy to inhibit pathogenic or spoilage microorganisms in foods, and in particular, the αs2-casein f(183–207) peptide can be a good choice due to its potency; i.e., besides lactoferricin B, it is the most potent milk-derived antimicrobial peptide identified to date.

The aim of this study was to assess the structure-activity relationship of αs2-casein f(183–207) peptide variants using L. monocytogenes and C. sakazakii as test organisms. The experimental strategy included downsizing the peptide, alanine scanning, performance of amino acid substitutions, and hydrophobic end tagging. Finally, further investigations were carried out in order to assess the influence of different environmental factors (pH, temperature, and salinity) on the observed antibacterial activities.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

Listeria monocytogenes LO28 and Cronobacter sakazakii DPC6440, obtained from the University College Cork culture collection, were cultured aerobically for 24 h at 37°C in brain heart infusion (BHI) broth (Oxoid) and Luria-Bertani broth (LB; Oxoid), respectively. Bacteria were then subcultured into fresh media (2% inoculum) and incubated at 37°C for the time needed to reach the mid-log phase of growth (optical density at 600 nm [OD600] of 0.3 to 0.5). Aliquots of 1 ml of log-phase cells were then harvested by centrifugation at 12,000 rpm for 7 min, washed with 10 mM sodium phosphate buffer (SPB) (pH 7.4) (Fisher Scientific), and resuspended in 1 ml of 10 mM SPB. These bacterial suspensions were subsequently used for agarose well diffusion and broth inhibition assays.

Peptide synthesis and peptide properties.

The αs2-casein f(183–207) peptide (VYQHQKAMKPWIQPKTKVIPYVRYL) and the array of 30 variants obtained through peptide downsizing, alanine scanning, amino acid substitution, and hydrophobic end tagging (Table 1) were chemically synthesized by Metabion (Germany), resuspended in high-performance liquid chromatography (HPLC)-grade water at a 5 mM concentration, and stored at −80°C. Very hydrophobic peptides were initially dissolved in small volumes of dimethyl sulfoxide (DMSO) (Sigma), followed by the addition of the volume of HPLC-grade water required to reach the desired concentration, as recommended by the supplier. Peptides were thawed on ice prior to use. Properties of each peptide/variant were calculated by using the following websites: http://www.innovagen.se/custom-peptide-synthesis/peptide-property-calculator/peptide-property-calculator.asp and http://aps.unmc.edu/AP/main.php. HPLC and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analyses of the αs2-casein f(183–207) peptide and of six peptides of the array of variants tested [αs2-casein f(183–207)V1, αs2-casein f(183–207)V2, αs2-casein f(183–207)V3, αs2-casein f(183–207)V6, αs2-casein f(183–207)V22, and αs2-casein f(183–207)V27], performed by Metabion, showed high purity in all cases and allowed for the identification of the correct mass in abundance by mass spectrometry.

Table 1.

Properties and MICs of the αs2-casein f(183–207) peptide variants

Peptide Sequence Isoelectric point Net charge at pH 7.0 % hydrophobic residues MIC (μM)
C. sakazakii L. monocytogenes
αS2 f(183–207) VYQHQKAMKPWIQPKTKVIPYVRYL 10.46 +5.1 36 312.5 78.125
αS2 f(183–207)V1 KAMKPWIQPKTKVIPYVRYL 10.68 +5.0 40 39.063 39.063
αS2 f(183–207)V2 WIQPKTKVIPYVRYL 10.33 +3.0 40 78.125 39.063
αS2 f(183–207)V3 KAMKPWIQPKTKVIP 11.01 +4.0 40 >1,250 156.25
αS2 f(183–207)V4 AIQPKTKVIPYVRYL 10.33 +3.0 40 78.125 78.125
αS2 f(183–207)V5 WAQPKTKVIPYVRYL 10.33 +3.0 40 39.063 39.063
αS2 f(183–207)V6 WIAPKTKVIPYVRYL 10.33 +3.0 47 78.125 19.531
αS2 f(183–207)V7 WIQAKTKVIPYVRYL 10.33 +3.0 47 39.063 9.766
αS2 f(183–207)V8 WIQPATKVIPYVRYL 10.04 +2.0 47 156.25 39.063
αS2 f(183–207)V9 WIQPKAKVIPYVRYL 10.33 +3.0 47 78.125 19.531
αS2 f(183–207)V10 WIQPKTAVIPYVRYL 10.04 +2.0 47 312.5 39.063
αS2 f(183–207)V11 WIQPKTKAIPYVRYL 10.33 +3.0 40 78.125 19.531
αS2 f(183–207)V12 WIQPKTKVAPYVRYL 10.33 +3.0 40 78.125 19.531
αS2 f(183–207)V13 WIQPKTKVIAYVRYL 10.33 +3.0 47 39.063 2.441
αS2 f(183–207)V14 WIQPKTKVIPAVRYL 10.71 +3.0 47 78.125 39.063
αS2 f(183–207)V15 WIQPKTKVIPYARYL 10.33 +3.0 40 39.063 19.531
αS2 f(183–207)V16 WIQPKTKVIPYVAYL 9.94 +2.0 47 >625 156.25
αS2 f(183–207)V17 WIQPKTKVIPYVRAL 10.71 +3.0 47 78.125 19.531
αS2 f(183–207)V18 WIQPKTKVIPYVRYA 10.33 +3.0 40 78.125 39.063
αS2 f(183–207)V19 WIQPKTKVIPYV 10.18 +2.0 42 625 156.25
αS2 f(183–207)V20 WIQPKTKVIP 10.60 +2.0 40 >625 >156.25
αS2 f(183–207)V21 WIQPATAVIPYVAYL 5.87 0.0 60 >625 >156.25
αS2 f(183–207)V22 WIQPATAVIPYVRYL 9.59 +1.0 53 >625 156.25
αS2 f(183–207)V23 WIQPATKVIPYVAYL 9.52 +1.0 53 >625 >156.25
αS2 f(183–207)V24 WIQAKTKVIAYVRYL 10.33 +3.0 53 19.531 2.441
αS2 f(183–207)V25 TKVIPYVRYL 10.04 +2.0 40 156.25 78.125
αS2 f(183–207)V26 WIQPKTKVIPAVRAL 11.64 +3.0 53 312.5 156.25
αS2 f(183–207)V27 WIQPKTKVIPWWWWW 10.60 +2.0 60 78.125 4.883
αS2 f(183–207)V28 TKVIPYVRYLWWWWW 10.04 +2.0 60 >625 9.766
αS2 f(183–207)V29 WIQPKTKVIPFFFFF 10.60 +2.0 60 >625 4.883
αS2 f(183–207)V30 TKVIPYVRYLFFFFF 10.04 +2.0 60 >625 78.125
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Agarose well diffusion assays.

For well diffusion assays, 15 ml of underlay medium (0.03% LB or BHI, 1% ultrapure agarose [Invitrogen], 0.02% Tween 20 [Sigma], 10 mM SPB [pH 7.4]) was seeded with 75 μl of a bacterial suspension (prepared as described above), and when set, 6-mm holes were bored with the end of a sterile glass pipette. Fifteen microliters of each peptide/variant (5 mM stock) was added to the wells, and plates were incubated for 2 h at 37°C to allow diffusion to occur. Twenty milliliters of overlay medium (5% LB or 7.4% BHI, 1% ultrapure agarose in distilled water) was then added, and plates were incubated for 24 h at 37°C. After overnight incubation, zones of inhibition were measured by using a digital Vernier caliper (Fisher Scientific).

Broth inhibition assays.

Individual wells of a 96-well plate were inoculated with 50 μl of peptide solutions (2-fold serial dilutions of 5 mM stocks) and 50 μl of bacterial suspensions (prepared as described above). Plates were incubated for 2 h with shaking at 37°C. Subsequently, 100 μl of double-strength (2×) broth (LB for C. sakazakii and BHI broth for L. monocytogenes) was added to the wells, and growth was measured spectrophotometrically for 24 h at 37°C by determining the OD600 using a temperature-controlled automatic plate reader (Multiscan FC; Thermo Scientific). Positive-control wells (containing 50 μl of the bacterial suspension and 50 μl of SPB without peptide supplementation) and negative-control wells (containing 100 μl of SPB alone) were also included. The MIC was determined for each strain/peptide variant combination as the lowest peptide concentration that prevented bacterial growth.

Assignment of AAU.

In order to assess the overall impact of variant changes on the antimicrobial activity of the αs2-casein f(193–207) peptide, each peptide variant was assigned an arbitrary activity unit (AAU) value reflecting its MIC relative to that of the corresponding wild-type peptide. These values were as follows: 0, at least 8-fold-reduced activity; 1, 4-fold-reduced activity; 2, 2-fold-reduced activity; 3, wild-type activity; 4, 2-fold-increased activity; 5, 4-fold-increased activity; 6, ≥8-fold-increased activity (15).

Kinetics of inactivation experiments.

In order to assess the kinetics of bacterial killing by selected peptide variants, peptides were added at predetermined concentrations (up to 0.5 mM) to 500 μl of SPB. Subsequently, bacterial suspensions were inoculated to a final bacterial concentration of ∼106 CFU/ml. After incubation at room temperature, survival was monitored periodically. Tenfold serial dilutions were prepared in sterile SPB, and dilutions were plated in duplicate onto LB (C. sakazakii) or BHI (L. monocytogenes) agar plates. Viable cells were enumerated following incubation of the plates at 37°C for 48 h (longer incubation times did not have any influence on counts).

Effect of environmental factors on antimicrobial activity of the αs2-casein f(193–207) peptide.

The influence of pH (4.5, 5.5, 6.5, and 7.4), temperature (4°C, 25°C, and 37°C), and supplementation with NaCl (1%, 2%, and 3%) or CaCl2 (1%, 2%, and 3%) on the activity of the αs2-casein f(193–207) peptide was evaluated by using the well diffusion assay. Underlay medium was supplemented with HCl, NaCl, or CaCl2 at the desired levels and was then seeded with 75 μl of the bacterial suspension. After the addition of 15 μl of the peptide to the wells, agar plates were incubated for 2 h at different temperatures (4°C, 25°C, and 37°C) to allow diffusion to occur. Twenty milliliters of overlay medium was then added, and plates were incubated for 24 h at 37°C, after which zones of inhibition were measured.

Reproducibility.

All experiments were performed in triplicate using independent biological replicates. Results are presented as averages of the three independent experiments ± standard deviations.

RESULTS

The αs2-casein f(183–207) peptide is cationic, with an overall charge at pH 7.0 of +5.1, which is a consequence of the presence of six positively charged amino acids: H4, K6, K9, K15, K17, and R23. In order to understand the importance of these and other amino acids, a number of variants of the αs2-casein f(183–207) peptide were designed. First, the effects of downsizing of the peptide were investigated by the removal of groups of amino acids, i.e., amino acids 1 to 5 in αs2-casein f(183–207)V1, amino acids 1 to 10 in αs2-casein f(183–207)V2, and amino acids 1 to 5 and 20 to 25 in αs2-casein f(183–207)V3. Whereas removal of the first 10 amino acids at the N-terminal end of the peptide resulted in a reduction of the MIC against both L. monocytogenes and C. sakazakii, removal of the last 5 amino acids at the C-terminal end in αs2-casein f(183–207)V3 caused a loss of activity (Table 1). When testing the bactericidal potentials of the three variants and the wild-type peptide at a 0.5 mM concentration in SPB, αs2-casein f(183–207)V1 and αs2-casein f(183–207)V2 were the most inhibitory peptides, with at least 5 log cycles of bacterial inactivation after incubation at room temperature for 24 h. On the other hand, αs2-casein f(183–207)V3 was confirmed as the variant with the lowest level of activity against L. monocytogenes and C. sakazakii (Fig. 1).

Fig 1.

Fig 1

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Kinetics of killing activity of the αs2-casein f(183–207) peptide (wild type [WT]) and its variants αs2-casein f(183–207)V1, αs2-casein f(183–207)V2, and αs2-casein f(183–207)V3. C. sakazakii DPC6440 and L. monocytogenes LO28 were incubated with the peptides (0.5 mM). Samples for viable count determinations were collected directly after mixing at 1, 4, and 24 h of incubation.

The shortest peptide sequence which showed great bacterial killing, αs2-casein f(193–207), was used as a template for further structure-activity studies. To this end, a collection of derivatives was designed, synthesized, and tested (Table 1 and Fig. 2). First, alanine scanning was performed on the 15-amino-acid peptide. This involved the replacement of each amino acid by Ala to determine the contribution of each individual amino acid to antimicrobial activity [αs2-casein f(183–207)V4 to αs2-casein f(183–207)V18] (Table 1). Reduced activity against both L. monocytogenes and C. sakazakii was observed with the Ala substitution at position 13, where an Arg residue was replaced [αs2-casein f(183–207)V16]. Similar, but lesser, effects were observed for C. sakazakii when the other two positively charged residues (Lys at position 5 or 7) were replaced by Ala [αs2-casein f(183–207)V8 and αs2-casein f(183–207)V10]. On the other hand, replacement of Pro residues at position 4 or 10 of the amino acid sequence resulted in an enhanced activity of the respective variants [αs2-casein f(183–207)V7 and αs2-casein f(183–207)V13], especially against L. monocytogenes.

Fig 2.

Fig 2

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Arbitrary activity units (AAU) of αs2-casein f(193–207) variants against C. sakazakii DPC6440 and L. monocytogenes LO28. Colored residues represent those altered relative to the corresponding wild-type peptide, with the color in each case corresponding to the AAU of the peptide in question.

Additional manipulations of the αs2-casein f(193–207) peptide revealed that removal of 3 to 5 amino acids at the C-terminal end caused a reduction or loss of activity [αs2-casein f(183–207)V19 and αs2-casein f(183–207)V20]. It was also observed that simultaneous replacement of at least two of the positively charged amino acids by Ala gave rise to a reduction in activity [αs2-casein f(183–207)V21, αs2-casein f(183–207)V22, and αs2-casein f(183–207)V23], while simultaneous replacement of both Pro residues at positions 4 and 10 resulted in increased antibacterial potency [αs2-casein f(183–207)V24], with MICs as low as 2 μM and 20 μM for L. monocytogenes and C. sakazakii, respectively. Finally, hydrophobic end tagging of αs2-casein f(193–203) and αs2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their activity against L. monocytogenes but not against C. sakazakii.

Kinetics of inactivation experiments performed with selected variants at a 0.125 mM concentration confirmed that replacement of the two Pro residues, individually [αs2-casein f(183–207)V7 and αs2-casein f(183–207)V13] or simultaneously [αs2-casein f(183–207)V24], increased the bactericidal activity of the peptide, with more than 5 log cycles of bacterial inactivation after 1 h of incubation (Fig. 3). It was also observed that removal of the 3 to 5 amino acids at the C-terminal end [αs2-casein f(183–207)V19 and αs2-casein f(183–207)V20] and substitution of the positively charged amino acids reduced or eliminated bactericidal activity [e.g., the αs2-casein f(183–207)V21 variant, where all the positively charged residues had been replaced by Ala, did not reduce numbers of L. monocytogenes or C. sakazakii bacteria after 24 h of incubation] (Fig. 3).

Fig 3.

Fig 3

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Kinetics of killing activity of the αs2-casein f(183–207)V2 peptide and its variants αs2-casein f(183–207)V7, αs2-casein f(183–207)V13, αs2-casein f(183–207)V19, αs2-casein f(183–207)V20, αs2-casein f(183–207)V21, αs2-casein f(183–207)V22, αs2-casein f(183–207)V23, αs2-casein f(183–207)V24, and αs2-casein f(183–207)V26. C. sakazakii DPC6440 and L. monocytogenes LO28 were incubated with the peptides (0.125 mM). Samples were collected directly after mixing at 1, 4, and 24 h of incubation, and viable plate counts were performed.

The effects of pH, temperature, and salinity on the antibacterial activity of the αs2-casein f(193–207) peptide was evaluated by using agarose well diffusion assays (Fig. 4). The peptide was significantly more effective at pH values of 7.4 and 6.5 than at lower pH values (5.5 and 4.5). Nonetheless, at such low pH values, antibacterial activity was still evident. Refrigeration temperatures (4°C) also caused a similar reduction in antibacterial activity for both L. monocytogenes and C. sakazakii. Regarding salinity, the addition of sodium or calcium chloride to the medium at concentrations of 1%, 2%, and 3% completely abolished the antibacterial activity of αs2-casein f(193–207). Only a small level of bacterial inhibition was still observed for L. monocytogenes at 1% NaCl.

Fig 4.

Fig 4

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Effects of pH (4.5, 5.5, 6.5, and 7.4), temperature (4°C, 25°C, and 37°C), and supplementation with NaCl (1%, 2%, and 3%) or CaCl2 (1%, 2%, and 3%) on the antibacterial activity of the αs2-casein f(193–207) peptide, as determined by well diffusion assays. The zones of inhibition were measured in mm. The values presented include the diameter of the well (6 mm).

DISCUSSION

The milk-derived αs2-casein f(183–207) peptide was first described as an antimicrobial peptide (AMP) by Recio and Visser (9). Our results confirm that it is a peptide with significant antibacterial activity against the Gram-positive pathogen L. monocytogenes and the Gram-negative pathogen C. sakazakii. The mechanism of action of the αs2-casein f(183–207) peptide was previously studied by López-Expósito and coworkers (10), who reported that it binds to lipoteichoic acid in Gram-positive bacteria and the lipopolysaccharide in Gram-negative bacteria and that it permeabilizes and forms pores in the cellular envelope. However, to date, the importance of specific amino acids and peptide properties (charge and hydrophobicity) for the biological activity of the peptide has not yet been investigated. In the present study, this lack of knowledge was addressed by the design and generation of an array of synthetic variants in which specific changes in the amino acid sequence have been performed.

The hydrophobicity and isoelectric point of the peptide variants were analyzed (Table 1), as a number of studies have reported a correlation between both parameters and antimicrobial activity (23–26). However, no significant link between either parameter and activity could be observed for our collection of variants. The impact of the amino acid changes on the MICs varied between L. monocytogenes and C. sakazakii (Table 1). Nevertheless, when MIC results were analyzed through the assignment of arbitrary activity units (AAU), obvious patterns emerged (Fig. 2). Downsizing of the peptide demonstrated that some residues at the C-terminal end of the peptide are important for activity, since removal of the last 5 amino acids [in αs2-casein f(183–207)V3] caused a reduction in activity. Removal of the last 3 amino acids at the C-terminal end [in αs2-casein f(183–207)V19] and replacement of the Arg at position 23 of the peptide sequence with an Ala residue [in αs2-casein f(183–207)V16] also gave rise to a large decrease in activity. These findings suggest that this Arg23 residue is very important for optimal bacterial killing activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the αs2-casein f(183–207) peptide in the alanine scanning process also caused a significant reduction in effectiveness against C. sakazakii, which points toward the importance of positively charged residues for biological activity. Indeed, simultaneous replacement of various positively charged amino acids [in αs2-casein f(183–207)V21, αs2-casein f(183–207)V22, and αs2-casein f(183–207)V23] was linked to a notable loss of bactericidal activity. AMPs must interact with the membrane as part of their direct antimicrobial mechanism of action, leading to membrane perturbation, disruption of membrane-associated physiological events, and/or translocation across the membrane to interact with intracellular targets (11). It is generally assumed that the positive charge of cationic AMPs is essential for their interaction with the negatively charged lipid head groups of the outer surface of the cytoplasmic membrane lipid bilayer (12).

Replacement of Pro residues at positions 14 and 20 of the αs2-casein f(183–207) peptide resulted in a significantly increased antibacterial potency. Proline-rich AMPs, with a high content of Pro and Arg residues, are an important group of AMPs predominantly active against Gram-negative bacteria (27). However, previous studies have shown that when Pro residues are inserted into the sequences of α-helical AMPs, the ability of these peptides to permeabilize the bacterial cytoplasmic membrane decreases substantially as a function of the number of Pro residues incorporated (28), and this could explain our results.

Hydrophobic end tagging of AMPs with hydrophobic amino acid stretches was previously shown to be a way to achieve high AMP adsorption and to improve bactericidal potency (29). End tagging by hydrophobic amino acid stretches allows the primary AMP sequence to be retained at the same time as efficient but selective membrane anchoring is achieved. Although a number of hydrophobic amino acids may be used as end tags, Trp and Phe end tags have emerged as particularly potent choices (30). Through interaction with the phospholipid membrane, Trp/Phe residues are able to insert into the membrane, acting as an anchor for the peptide and resulting in increased bactericidal effects (31, 32). Our findings show that hydrophobic end tagging of the αs2-casein f(193–203) and αs2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their activity against L. monocytogenes, but this effect was not observed for C. sakazakii.

The effects of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the bactericidal activity of the 15 amino acids αs2-casein f(193–207) peptide were also determined. The lethal activity of the αs2-casein f(193–207) peptide was unaffected at pH 6.5, but it was significantly reduced at pH values below 5.5. Changes of pH can affect the net charge, structure, binding properties, and antimicrobial activity of AMPs (33). Thus, the decrease in the antimicrobial activities at low pH may be due to the instability of the AMP structures in these acidic environments (34). Similarly, refrigeration temperatures also gave rise to a reduction in antibacterial activity. The reduced activity at low temperatures could be attributed to changes in fatty acid composition and fluidity of bacterial cell membranes (35) or to changes in AMP solubility (36). Regarding the effects of sodium and calcium salts, their addition to the medium at concentrations of up to 3% completely abolished the antibacterial activity of the αs2-casein f(193–207) peptide. Only some degree of bacterial inhibition was observed in the presence of 1% NaCl for L. monocytogenes. These findings are in agreement with previous reports showing that AMPs are sensitive to high-salt environments (13, 37). This is probably linked to the fact that most of the natural cationic peptides are strongly antagonized by physiological concentrations of mono- and divalent cations (38). It has been suggested that high salt concentrations may mask electrostatic interactions between AMPs and the negatively charged surface of the cellular envelope (33). This may compromise the in vivo efficacy of the peptide in food systems. Indeed, previous reports have found either reduced AMP activity or the requirement for higher concentrations of AMPs in model food systems such as apple juice, milk, and meat products (33, 39, 40).

In conclusion, template-based studies on peptide derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties that are important for activity. This study sheds light on the importance of specific amino acids and residue positions to the activity of the milk-derived antimicrobial peptide αs2-casein f(183–207), which in turn may lead to the development of approaches to optimize its application as a food-grade antimicrobial for the control of food-borne pathogens such as L. monocytogenes and C. sakazakii.

ACKNOWLEDGMENT

We acknowledge the funding received by Food for Health Ireland under grant number CC20080001 from Enterprise Ireland.

Footnotes

Published ahead of print 21 June 2013

REFERENCES

  • 1.Hartmann R, Meisel H. 2007. Food-derived peptides with biological activity: from research to food applications. Curr. Opin. Biotechnol. 18:163–169 [DOI] [PubMed] [Google Scholar]
  • 2.Li Y, Xiang Q, Zhang Q, Huang Y, Su Z. 2012. Overview on the recent study of antimicrobial peptides: origins, functions, relative mechanisms and application. Peptides 37:207–215 [DOI] [PubMed] [Google Scholar]
  • 3.Mills S, Ross RP, Hill C, Fitzgerald GF, Stanton C. 2011. Milk intelligence: mining milk for bioactive substances associated with human health. Int. Dairy J. 21:377–401 [Google Scholar]
  • 4.Hayes A, Ross RP, Fitzgerald GF, Hill C, Stanton C. 2006. Casein-derived antimicrobial peptides generated by Lactobacillus acidophilus DPC6026. Appl. Environ. Microbiol. 72:2260–2264 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Malkoski M, Dashper SG, O'Brien-Simpson NM, Talbo GH, Macris M, Cross KJ, Reynolds EC. 2001. Kappacin, a novel antibacterial peptide from bovine milk. Antimicrob. Agents Chemother. 45:2309–2315 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Lahov E, Regelson W. 1996. Antibacterial and immunostimulating casein-derived substances from milk: casecidin, isracidin peptides. Food Chem. Toxicol. 34:131–145 [DOI] [PubMed] [Google Scholar]
  • 7.Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita M. 1992. Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J. Appl. Bacteriol. 73:472–479 [DOI] [PubMed] [Google Scholar]
  • 8.Zucht HD, Raida M, Adermann K, Magert HJ, Forssmann WG. 1995. Casocidin-I: a casein-alpha s2 derived peptide exhibits antibacterial activity. FEBS Lett. 372:185–188 [DOI] [PubMed] [Google Scholar]
  • 9.Recio I, Visser S. 1999. Identification of two distinct antibacterial domains within the sequence of bovine αs2-casein. Biochim. Biophys. Acta 1428:314–326 [DOI] [PubMed] [Google Scholar]
  • 10.López-Expósito I, Amigo L, Recio I. 2008. Identification of the initial binding sites of αs2-casein f(183–207) and effect on bacterial membranes and cell morphology. Biochim. Biophys. Acta 1778:2444–2449 [DOI] [PubMed] [Google Scholar]
  • 11.Shai Y. 1999. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta 1462:55–70 [DOI] [PubMed] [Google Scholar]
  • 12.Fjell CD, Hiss JA, Hancock RE, Schneider G. 2012. Designing antimicrobial peptides: form follows function. Nat. Rev. Drug Discov. 11:37–51 [DOI] [PubMed] [Google Scholar]
  • 13.Haversen L, Kondori N, Baltzer L, Hanson A, Dolphin GT, Dunér K, Mattsby-Baltzer I. 2010. Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial α-helix of human lactoferrin. Antimicrob. Agents Chemother. 54:418–425 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Norberg S, O'Connor PM, Stanton C, Ross RP, Hill C, Fitzgerald GF, Cotter PD. 2011. Altering the composition of caseicins A and B as a means of determining the contribution of specific residues to antimicrobial activity. Appl. Environ. Microbiol. 77:2496–2501 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Norberg S, O'Connor PM, Stanton C, Ross RP, Hill C, Fitzgerald GF, Cotter PD. 2012. Extensive manipulation of caseicins A and B highlights the tolerance of these antimicrobial peptides to change. Appl. Environ. Microbiol. 78:2353–2358 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Schneider G, Schuchhardt J, Wrede P. 1994. Artificial neural networks and simulated molecular evolution are potential tools for sequence-oriented protein design. Comput. Appl. Biosci. 10:635–645 [DOI] [PubMed] [Google Scholar]
  • 17.Norberg S, Stanton C, Ross RP, Hill C, Fitzgerald GF, Cotter PD. 2012. Cronobacter spp. in powdered infant formula. J. Food Prot. 75:607–620 [DOI] [PubMed] [Google Scholar]
  • 18.Farber JM. 2004. Enterobacter sakazakii—new foods for thought? Lancet 363:5–6 [DOI] [PubMed] [Google Scholar]
  • 19.Friedemann M. 2007. Enterobacter sakazakii in food and beverages (other than infant formula and milk powder). Int. J. Food Microbiol. 116:1–10 [DOI] [PubMed] [Google Scholar]
  • 20.Beuchat LR, Kim H, Gurtler JB, Lin LC, Ryu JH, Richards GM. 2009. Cronobacter sakazakii in foods and factors affecting its survival, growth and inactivation. Int. J. Food Microbiol. 136:204–213 [DOI] [PubMed] [Google Scholar]
  • 21.Osaili T, Forsythe S. 2009. Desiccation resistance and persistence of Cronobacter species in infant formula. Int. J. Food Microbiol. 136:214–220 [DOI] [PubMed] [Google Scholar]
  • 22.Warriner K, Namvar A. 2009. What is the hysteria with Listeria? Trends Food Sci. Tech. 20:245–254 [Google Scholar]
  • 23.Romestand B, Molina F, Richard V, Roch P, Granier C. 2003. Key role of the loop connecting the two beta strands of mussel defensin in its antimicrobial activity. Eur. J. Biochem. 270:2805–2813 [DOI] [PubMed] [Google Scholar]
  • 24.Chen Y, Guarnieri MT, Vasil AI, Vasil ML, Mant CT, Hodges RS. 2007. Role of peptide hydrophobicity in the mechanism of action of alpha-helical antimicrobial peptides. Antimicrob. Agents Chemother. 51:1398–1406 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Nan YH, Park KH, Park Y, Jeon YJ, Kim Y, Park IS, Hahm KS, Shin SY. 2009. Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin. FEMS Microbiol. Lett. 292:134–140 [DOI] [PubMed] [Google Scholar]
  • 26.Leptihn S, Har JY, Wohland T, Ding JL. 2010. Correlation of charge, hydrophobicity and structure with antimicrobial activity of S1 and MIRIAM peptides. Biochemistry 49:9161–9170 [DOI] [PubMed] [Google Scholar]
  • 27.Paulsen VS, Blencke HM, Benincasa M, Hauq T, Eksteen JJ, Styrvold OB, Scocchi M, Stensvag K. 2013. Structure-activity relationships of the antimicrobial peptide arasin1—and mode of action studies of the N-terminal, proline-rich region. PLoS One 8:e53326. 10.1371/journal.pone.0053326 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Zhang L, Benz R, Hancock REW. 1999. Influence of proline residues on the antibacterial and synergistic activities of alpha-helical peptides. Biochemistry 38:8102–8111 [DOI] [PubMed] [Google Scholar]
  • 29.Malmsten M, Kasetty G, Pasupuleti M, Alenfall J, Schmidtchen A. 2011. Highly selective end-tagged antimicrobial peptides derived from PRELP. PLoS One 6:e16400. 10.1371/journal.pone.0016400 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Schmidtchen A, Pasupuleti M, Morgelin M, Davoudi M, Alenfall J, Chalupka A, Malmsten M. 2009. Boosting antimicrobial peptides by hydrophobic oligopeptide end tags. J. Biol. Chem. 284:17584–17594 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Deslousches B, Phadke SM, Lazarevic V, Cascio M, Islam K, Montelaro RC, Mietzner TA. 2005. De novo generation of cationic antimicrobial peptides: influence of length and tryptophan substitution on antimicrobial activity. Antimicrob. Agents Chemother. 49:316–322 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Stromstedt AA, Pasupuleti M, Schmidtchen A, Malmsten M. 2009. Evaluation of strategies for improving proteolytic resistance of antimicrobial peptides by using variants of EFK17, an internal segment of LL-37. Antimicrob. Agents Chemother. 53:593–602 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Rydlo T, Rotem S, Mor A. 2006. Antibacterial properties of dermaseptin S4 derivatives under extreme incubation conditions. Antimicrob. Agents Chemother. 50:490–497 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Vogt TC, Bechinger B. 1999. The interactions of histidine-containing amphipathic helical peptide antibiotics with lipid bilayers. The effects of charges and pH. J. Biol. Chem. 274:29115–29121 [DOI] [PubMed] [Google Scholar]
  • 35.Annamalai T, Venkitanarayanan KS, Hoagland TA, Khan MI. 2001. Inactivation of Escherichia coli O157:H7 and Listeria monocytogenes by PR-26, a synthetic antibacterial peptide. J. Food Prot. 64:1929–1934 [DOI] [PubMed] [Google Scholar]
  • 36.Maisnier-Patin S, Forni E, Richard J. 1996. Purification, partial characterisation and mode of action of enterococcin EFS2, an antilisterial bacteriocin produced by a strain of Enterococcus faecalis isolated from a cheese. Int. J. Food Microbiol. 30:255–270 [DOI] [PubMed] [Google Scholar]
  • 37.Goldman MJ, Anderson GM, Stolzenberg ED, Kari UP, Zasloff M, Wilson JM. 1997. Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88:553–560 [DOI] [PubMed] [Google Scholar]
  • 38.Hancock REW, Lehrer RI. 1998. Cationic peptides: a new source of antibiotics. Trends Biotechnol. 16:82–88 [DOI] [PubMed] [Google Scholar]
  • 39.Appendini P, Hotchkiss JH. 1999. Antimicrobial activity of a 14-residue peptide against Escherichia coli O157:H7. J. Appl. Microbiol. 87:750–756 [DOI] [PubMed] [Google Scholar]
  • 40.Yaron S, Rydlo T, Shachar D, Mor A. 2003. Activity of dermaseptin K4-S4 against foodborne pathogens. Peptides 24:1815–1821 [DOI] [PubMed] [Google Scholar]

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