Abstract
Caseicins A and B are low-molecular-weight antimicrobial peptides which are released by proteolytic digestion of sodium caseinate. Caseicin A (IKHQGLPQE) is a nine-amino-acid cationic peptide, and caseicin B (VLNENLLR) is a neutral eight-amino-acid peptide; both have previously been shown to exhibit antibacterial activity against a number of pathogens, including Cronobacter sakazakii. Previously, four variants of each caseicin which differed subtly from their natural counterparts were generated by peptide synthesis. Antimicrobial activity assays revealed that the importance of a number of the residues within the peptides was dependent on the strain being targeted. In this study, this engineering-based approach was expanded through the creation of a larger collection of 26 peptides which are altered in a variety of ways. The investigation highlights the generally greater tolerance of caseicin B to change, the fact that changes have a more detrimental impact on anti-Gram-negative activity, and the surprising number of variants which exhibit enhanced activity against Staphylococcus aureus.
INTRODUCTION
Antimicrobial peptides (AMPs) are low-molecular-weight proteins which exhibit antimicrobial activity against bacteria, fungi, and/or viruses. AMPs have been the focus of significant interest in recent years (4, 7, 15, 19). In particular, many AMPs are viewed as natural preservatives and are attractive alternatives with respect to controlling spoilage and pathogenic bacteria in foods (3, 18, 25, 27). Milk-derived AMPs are of particular interest and have been investigated since the 1930s (11), resulting in the identification of a number of peptides which demonstrate activity against Gram-positive (14, 19, 29) or Gram-negative (9, 17) pathogens. Two of the most noteworthy of the milk-derived AMPs are caseicins A (IKHQGLPQE) and B (VLNENLLR), which were identified following a bacterial fermentation of sodium caseinate (9) and have previously been found to be active against a range of pathogenic bacteria, such as Cronobacter sakazakii (9), Cronobacter muytjensii, Staphylococcus aureus, and Salmonella enterica serovar Typhimurium (22). The precise mechanism by which caseicins A and B act remains unclear, and thus, to gain more insight into the importance of specific residues within these peptides, eight synthetic variants were generated which differed subtly from their natural equivalents (22). That study revealed that the replacement of the C-terminal arginine of caseicin B with alanine resulted in a generally greatly reduced activity against Gram-negative targets, whereas activity against S. aureus was not affected (22). Indeed, the results indicated that the anti-Gram-positive activity of the peptides was less sensitive to change than their anti-Gram-negative properties (22). Notably, the previous study focused on only a selection of the residues within these peptides, and these variants differed from the wild-type peptides by no more than one amino acid. In this study, we created a larger, “second-generation” collection of 26 caseicin A and B variants with which the importance of each individual residue and of overall charge and hydrophobicity can be tested. In addition to confirming that the anti-Gram-negative activity of these peptides is less tolerant to change, the investigation of this expanded group of variants highlighted the generally greater tolerance of caseicin B to change and identified a surprising number of variants which exhibit enhanced activity against S. aureus.
MATERIALS AND METHODS
Bacterial strains and culture media.
Bacterial strains used in this study are listed in Table 1. All microbes were cultured aerobically at 37°C in LB medium (Oxoid; Basingstoke, United Kingdom).
Table 1.
Strains used in this studya
| Strain | Origin or description | Source or reference |
|---|---|---|
| Gram-negative strains | ||
| C. muytjensii ATCC 51329 | Unknown source (20) | ATCC collection |
| C. sakazakii BAA-894 | Infant isolate | ATCC collection |
| C. sakazakii DSM 4485 (ATCC 2954) | Child's throat isolate | ATCC collection |
| C. sakazakii DPC6440 (NCTC08155) | Tin of dried milk | NCTC collection |
| E. coli NCIMB 11843 | Laboratory strain | NCIMB collection |
| Salmonella Typhimurium LT2 | Laboratory strain | UCC collection |
| K. pneumoniae NCIMB 13218 | Laboratory strain | NCIMB collection |
| P. fluorescens NCIMB 9046 | Laboratory strain | NCIMB collection |
| Gram-positive strains | ||
| S. aureus SA113 (ATCC 35556) | Derivative of strain NCTC 8325 | 10 |
| S. aureus RN4220 | Defective derivative of NCTC 8325-4 | 13 |
| S. aureus Newman | Osteomyelitis isolate | 6 |
| S. aureus 8325-4 | Laboratory strain | 23 |
| S. aureus RF122 (ET3-1) | Bovine mastitis-causing isolate | 8 |
ATCC, American Type Culture Collection; DSM, German Collection of Microorganisms and Cell Cultures; NCTC, National Collection of Type Cultures; NCIMB, National Collection of Industrial, Food and Marine Bacteria; UCC, University College Cork.
Peptide synthesis.
Caseicin peptides were chemically synthesized on preloaded Wang resins (Merck, Nottingham, United Kingdom) by use of microwave-assisted solid-phase peptide synthesis (MW-SPPS) performed on a Liberty CEM microwave peptide synthesizer. Caseicin A was made on an H-Glu(OtBu)-HMPB-ChemMatrix resin, and caseicin B was made on an H-Arg(Pbf)-HMPB-ChemMatrix resin (PCAS Biomatrix Inc., Quebec, Canada). Synthetic peptides were purified using reversed-phase high-performance liquid chromatography (RP-HPLC) on a preparative Vydac C18 (10 μm by 300 Å) column (Grace Davison Discovery Sciences, CA) developed in a gradient of 10 to 20% acetonitrile–0.1% trifluoroacetic acid (TFA) over 25 min for caseicin A and 15 to 35% acetonitrile–0.1% TFA over 25 min for caseicin B. Fractions containing peptides of the correct molecular mass were identified using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry, pooled, and lyophilized on a Genevac HT 4X (Genevac Ltd., Ipswich, United Kingdom) lyophilizer. The peptides were stored at −20°C. In advance of use, the peptides were resuspended in LB broth and stored at 4°C.
MICs.
MICs were determined as described previously (28). Briefly, serial 2-fold dilutions of the antimicrobial peptides were made in LB broth and added to the wells of a 96-well plate. All strains were grown in LB broth to an optical density at 600 nm (OD600) of 0.5. Bacteria were added to give a final inoculum of 105 CFU per milliliter in a final volume of 200 μl. After incubation for 16 h at 37°C, the MIC was read as the lowest peptide concentration that prevented visible growth. Results are given as mean values for three independent determinations.
Assignment of arbitrary activity units.
As a means of assessing the overall impact of variant changes on the antimicrobial activity of the caseicin peptides against Gram-negative targets and S. aureus, each peptide was assigned an arbitrary activity value which reflected its MIC relative to that of the corresponding wild-type peptide. These values were as follows: 0, no activity detected; 1, 4-fold reduced activity; 2, 2-fold reduced activity; 3, wild-type level of activity; 4, 2-fold increased activity; 5, 4-fold increased activity; and 6, 8-fold increased activity. The average arbitrary activity value corresponds to the total arbitrary activity value divided by the number of Gram-negative or S. aureus strains tested (see Tables S1 and S2 in the supplemental material).
Isoelectric point and hydrophobicity of the peptides.
The isoelectric point (pI) of each caseicin variant was calculated using the following website: http://www.innovagen.se/custom-peptide-synthesis/peptide-property-calculator/peptide-property-calculator.asp. The isoelectric point of a peptide is the recorded pH value at which the peptide possesses no electrical charge, and it has a direct effect on peptide solubility (Table 2).
Table 2.
Caseicin variant characteristics
| Caseicin sequence | Predicted mass (kDa) | Actual mass (kDa) | Amino acid change(s) | pI | Hydrophobicity | % hydrophobic residues |
|---|---|---|---|---|---|---|
| IKHQGLPQE | 1,049 | 1,049.57 | Caseicin A wild type | 7.8 | 0.3 | 44 |
| IAAAGLPQE | 869 | 869.5 | K2A, H3A, Q4A | 3.3 | −0.2 | 22 |
| AKHQGLPQE | 1,007 | 1,007.5 | I1A | 7.8 | 0.4 | 44 |
| IKHQGLPQA | 991 | 991.5 | E9A | 10.1 | −0.1 | 33 |
| IKHAGLPQE | 992 | 992.5 | Q4A | 7.8 | 0.2 | 33 |
| IKHRGLPQE | 1,077 | 1,077.4 | Q4R | 10.1 | 0.6 | 44 |
| IKHQGRPQE | 1,092 | 1,092.4 | L6R | 10.1 | 0.8 | 56 |
| IRHQGLPQE | 1,077 | 1,077.6 | K2R | 7.9 | 0.3 | 44 |
| IRRQGLPE | 968 | 968.5 | K2R, H3R, minus-Q8 | 10.9 | 0.7 | 50 |
| IHHQGLPQE | 1,058 | 1,058.5 | K2H | 6.0 | −0.1 | 33 |
| IKQGLPQE | 912 | 912.5 | Minus-H3 | 6.9 | 0.4 | 50 |
| IHQGLPQE | 921 | 921.6 | Minus-K2 | 5.1 | −0.1 | 38 |
| IKHQGLPQEE | 1,178 | 1,178.6 | Plus-E10 | 5.3 | 0.5 | 50 |
| IKHQGLPQED | 1,164 | 1,164.7 | Plus-D10 | 6.2 | 0.5 | 50 |
| VLNENLLR | 970 | 970.5 | Caseicin B wild type | 7.0 | −0.1 | 50 |
| VLNENLAA | 843 | 843.3 | L7A, R8A | 3.3 | −0.3 | 38 |
| VLNENLRR | 1,013 | 1,013.6 | L7R | 10.9 | 0.5 | 63 |
| VLNENAAR | 886 | 886.5 | L6A, L7A | 7.0 | 0.3 | 50 |
| VINENLLR | 970 | 970.6 | L2I | 7.0 | −0.1 | 50 |
| VLNANLLR | 912 | 912.7 | E4A | 11.0 | −0.5 | 38 |
| VLNEALLR | 927 | 927.5 | N5A | 7.0 | −0.2 | 38 |
| VLNENLLH | 951 | 951.5 | R8H | 5.1 | −0.5 | 38 |
| VLNENARL | 928 | 928.5 | L7R, L6A, R8L | 7.0 | 0.1 | 50 |
| VALNENLLR | 1,041 | 1,041.6 | Plus-A2 | 7.0 | −0.1 | 44 |
| VLNDNLLR | 956 | 956.5 | E4D | 6.8 | −0.1 | 50 |
| VLNENLLK | 942 | 942.7 | R8K | 6.9 | −0.1 | 50 |
| VLNNLLR | 841 | 841.4 | Minus-E4 | 11.0 | −0.5 | 43 |
| VANENLLR | 928 | 928.4 | L2A | 7.0 | 0.1 | 50 |
The hydrophobicity and percentage of hydrophobic residues were calculated using the following website: http://www.innovagen.se/custom-peptide-synthesis/peptide-property-calculator/peptide-property-calculator.asp (Table 2).
RESULTS
Design and creation of a second generation of caseicin A and B variants.
The creation of a relatively small collection of caseicin A and B variants (4 of each) has previously generated valuable information with respect to the importance of specific residues and domains within these peptides (22). To build on this knowledge, a larger collection of derivatives was designed and synthesized. In the case of caseicin A, this included peptides in which individual or multiple residues were converted to alanine (i.e., individual I1A, E8A, and Q4A changes and a combined K2A-H3A-Q4A change), charged residues were introduced (Q4R, L6R, E10, and D10) or removed (K2 or H3 removed), or positively charged residues were exchanged (K2H, K2R, and K2R-H3R-minus-Q8) (Table 2). We refer to histidine as a positively charged residue but acknowledge that its pKa is 6.0. In the case of caseicin B, alanine replacements and introductions were targeted (L2A, E4A, N5A, L6A-L7A, L7A-R8A, and plus-A2) and charged residues were exchanged (R8H, R8K, and E4D), introduced (L7R), or removed (minus-E4). Two peptides in which hydrophobic amino acids were exchanged (L2I) or the C terminus was altered significantly (L6A-L7R-R8L) were also designed (Table 2). All peptides were synthesized and purified, and mass spectrometry demonstrated that peptides of the correct masses were generated (Table 2). The antimicrobial activities of the peptides against a selection of Gram-negative targets and strains of the Gram-positive pathogen Staphylococcus aureus were tested.
Activity of caseicin A variants against Gram-negative strains.
MIC-based tests were carried out to assess the activity of the synthesized caseicin variants. The Gram-negative targets tested were Cronobacter sakazakii strains DPC6440, BAA-894, and DSM 4485, Cronobacter muytjensii ATCC 51329, Salmonella Typhimurium LT2, Escherichia coli NCIMB 11843, Klebsiella pneumoniae NCIMB13218, and Pseudomonas fluorescens NCIMB 9046 (Table 3).
Table 3.
MICs of caseicin A variants against Gram-negative strains
| Strain | MIC (mM) of variant: |
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | K2A-H3A-Q4A | I1A | E9A | Q4A | Q4R | L6R | K2R | K2R-H3R-ΔQ8 | K2H | ΔH3 | ΔK2 | Plus-E10 | Plus-D10 | |
| C. sakazakii DPC6440 | 0.625 | 0.625 | 1.25 | 0.625 | >2.5 | 0.312 | >2.5 | 1.25 | 1.25 | 1.25 | >2.5 | >2.5 | >2.5 | 0.625 |
| C. sakazakii DSM 4485 | 0.625 | 1.25 | 1.25 | 2.5 | >2.5 | >2.5 | >2.5 | 1.25 | 1.25 | >2.5 | >2.5 | >2.5 | >2.5 | 0.625 |
| C. sakazakii BAA-894 | 0.625 | 1.25 | 1.25 | 1.25 | >2.5 | >2.5 | >2.5 | 0.625 | 1.25 | 1.25 | >2.5 | >2.5 | >2.5 | 0.625 |
| C. muytjensii ATCC 51329 | 0.625 | 1.25 | 1.25 | 2.5 | >2.5 | 1.25 | >2.5 | 1.25 | 1.25 | >2.5 | >2.5 | >2.5 | >2.5 | 2.5 |
| S. Typhimurium LT2 | 1.25 | 2.5 | 2.5 | 2.5 | >2.5 | 1.25 | >2.5 | 2.5 | 2.5 | >2.5 | >2.5 | >2.5 | >2.5 | >2.5 |
| E. coli NCIMB 11843 | 1.25 | 2.5 | >2.5 | 2.5 | >2.5 | 1.25 | >2.5 | 1.25 | >2.5 | 2.5 | >2.5 | >2.5 | >2.5 | 1.25 |
| K. pneumoniae NCIMB 13218 | 1.25 | 1.25 | 2.5 | 2.5 | >2.5 | 1.25 | >2.5 | 2.5 | 2.5 | >2.5 | >2.5 | >2.5 | >2.5 | 1.25 |
| P. fluorescens NCIMB 9046 | 1.25 | 2.5 | >2.5 | >2.5 | >2.5 | >2.5 | >2.5 | 1.25 | 1.25 | 2.5 | >2.5 | >2.5 | >2.5 | 1.25 |
The caseicin L6R variant was notable because it possessed reduced activity (MIC, >2.5 mM), relative to that of wild-type caseicin A, against all Gram-negative strains tested. Three of the changes made which resulted in variants having an overall neutral charge, minus-H3, minus-K2, and plus-E10, had equally negative impacts. This contrasted with the plus-D10 variant, which retained wild-type-like levels of activity against all except 2 targets. The Q4A variation had a very detrimental impact on activity. In this case, it is apparent that it is the nature of the newly introduced residues rather than the replacement per se which is important, as the quite similar Q4R peptide retained wild-type-like levels of activity against 3 targets and, notably, displayed enhanced activity against C. sakazakii DPC6440 (0.312 mM). As indicated above, one other variant, the L6R variant, was devoid of detectable activity.
As with the Q4A variant, the replacement of other existing residues with alanine (I1A, E9A, and K2A-H3A-Q4A) had an overall negative impact, although in these instances activity was reduced, in general, rather than eliminated. The importance of the nature of the newly introduced amino acid was also apparent through alteration of the residue at position 2 of caseicin A. Although arginine and histidine are both positively charged amino acids, the presence of histidine in the K2H variant had a more detrimental impact on antimicrobial activity than did the introduction of an arginine at this position. Notably, despite differing significantly from wild-type caseicin A, the K2R-H3R-minus-Q8 variant retained at least some activity against all strains except for E. coli NCIMB 11843.
Activity of caseicin A variants against S. aureus.
The impacts of the specific amino acid changes in the caseicin A variants on antimicrobial activity against S. aureus strains were comparable, in general, with those observed when Gram-negative targets were employed, with some notable differences (Table 4). The K2H, minus-H3, minus-K2, and plus-E10 variants again displayed a reduced activity or lack of activity. In the case of the K2H variant, activity was reduced more dramatically against S. aureus targets than against their Gram-negative counterparts, but in the case of the minus-K2 and plus-E10 variants, the impact was less considerable. Yet again, the introduction of an additional aspartate at the C terminus (plus-D10) had less detrimental consequences than the introduction of a glutamate (plus-E10). Indeed, the plus-D10 variant retained wild-type-like levels of activity against all strains except strain SA113, against which it possessed enhanced activity.
Table 4.
MICs of caseicin A variants against Gram-positive S. aureus strains
| Strain | MIC (mM) of variant: |
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | K2A-H3A-Q4A | I1A | E9A | Q4A | Q4R | L6R | K2R | K2R-H3R-ΔQ8 | K2H | ΔH3 | ΔK2 | Plus-E10 | Plus-D10 | |
| 8325-4 | 1.25 | 1.25 | 1.25 | 2.5 | >2.5 | 1.25 | >2.5 | 2.5 | 0.312 | >2.5 | >2.5 | >2.5 | 2.5 | 1.25 |
| SA113 | 1.25 | 1.25 | >2.5 | 2.5 | >2.5 | 1.25 | >2.5 | 2.5 | 0.625 | >2.5 | >2.5 | >2.5 | 2.5 | 0.625 |
| RF122 | 1.25 | 0.312 | >2.5 | 0.625 | 1.25 | 0.625 | >2.5 | 0.625 | 1.25 | >2.5 | >2.5 | 1.25 | 2.5 | 1.25 |
| Newman | 1.25 | 1.25 | >2.5 | 2.5 | >2.5 | 1.25 | >2.5 | 2.5 | 1.25 | >2.5 | >2.5 | >2.5 | 2.5 | 1.25 |
| RN4220 | 1.25 | 0.312 | >2.5 | 0.625 | 0.625 | 1.25 | >2.5 | 2.5 | 1.25 | >2.5 | >2.5 | 1.25 | 2.5 | 1.25 |
The other peptides which possessed little or no activity were the L6R variant and, in slight contrast to the Gram-negative studies, the I1A variant. Although the Q4A variant also lacked activity against 3 S. aureus strains, it retained wild-type levels of activity against RF122 and, surprisingly in light of other results, enhanced activity against RN4220. However, the Q4R variant was more active in general than the Q4A variant and, indeed, exhibited enhanced activity against S. aureus RF122. Another peptide in which an arginine substitution had been made, the K2R variant, also exhibited enhanced activity against RF122, but its activity against all other S. aureus strains was reduced.
The remaining peptides were all notable as a consequence of retaining a larger proportion of wild-type activity against S. aureus than was the case when Gram-negative strains were targeted. The activity of the E9A variant was diminished against two targets but enhanced against strains RF122 and RN4220. Even more impressively, the K2A-H3A-Q4A and K2R-H3R-minus-Q8 variants retained wild-type levels of activity against three targets and exhibited enhanced activity against two others (Table 4).
Activity of caseicin B variants against Gram-negative strains.
Of the caseicin B variants synthesized, the E4D variant was by far the most dramatically affected one, resulting in the elimination of activity against all eight Gram-negative targets. Of the remaining peptides, the E4A variant was notable by virtue of being the only other peptide to lack activity against at least two targets. Despite the apparent importance of E4 on the basis of these results, the minus-E4 variant, from which the E4 residue had been omitted, retained at least some activity against all targets and, curiously, exhibited enhanced activity against C. sakazakii DPC6440. Changes at position 2 of the peptide produced much more consistent results in that the L2I and L2A variants had identical impacts, with reduced activity against 7 of 8 targets, while the plus-A2 variant differed slightly in that it resulted in enhanced activity against DPC6440. This pattern of inhibition was similar to that observed when the N5A variant was tested (Table 5).
Table 5.
MICs of caseicin B variants against Gram-negative strains
| Strain | MIC (mM) of variant: |
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | L7A-R8A | L7R | L6A-L7A | L2I | E4A | N5A | R8H | L6A-L7R-R8L | Plus-A2 | E4D | R8K | ΔE4 | L2A | |
| C. sakazakii DPC6440 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 0.625 | 1.25 | 1.25 | 0.625 | >2.5 | 1.25 | 0.625 | 1.25 |
| C. sakazakii DSM4485 | 0.625 | 0.312 | 1.25 | 0.625 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 2.5 | >2.5 | 1.25 | 2.5 | 1.25 |
| C. sakazakii BAA-894 | 0.625 | 1.25 | 1.25 | 1.25 | 1.25 | 2.5 | 1.25 | 1.25 | 1.25 | 1.25 | >2.5 | 2.5 | 1.25 | 2.5 |
| C. muytjensii ATCC 51329 | 0.625 | 2.5 | 0.625 | 1.25 | 1.25 | 2.5 | 0.625 | 1.25 | 1.25 | 0.625 | >2.5 | 1.25 | 1.25 | 1.25 |
| S. Typhimurium LT2 | 1.25 | 2.5 | 2.5 | 2.5 | 2.5 | >2.5 | 1.25 | 2.5 | 1.25 | 2.5 | >2.5 | 2.5 | 1.25 | 2.5 |
| E. coli NCIMB 11843 | 1.25 | 2.5 | 2.5 | 2.5 | 2.5 | >2.5 | >2.5 | >2.5 | 1.25 | 2.5 | >2.5 | 2.5 | 2.5 | 2.5 |
| K. pneumoniae NCIMB 13218 | 1.25 | 2.5 | 1.25 | 2.5 | 2.5 | 2.5 | 2.5 | 1.25 | 1.25 | 2.5 | >2.5 | 2.5 | 1.25 | 2.5 |
| P. fluorescens NCIMB 9046 | 1.25 | 2.5 | 1.25 | 1.25 | 2.5 | 1.25 | 2.5 | 1.25 | 1.25 | 2.5 | >2.5 | 0.625 | 2.5 | 2.5 |
Regarding the C terminus, the only peptide in which L7 alone was altered, the L7R variant, retained wild-type levels of activity against 4 targets, with activity being reduced against another 4 targets. The specific targeting of residue 8 most frequently resulted in reduced activities, but the R8K variant was exceptional by virtue of enhanced activity against P. fluorescens NCIMB 9046, while the R8H variant was notable because it lacked activity against E. coli NCIMB 11843. In the case of peptides in which multiple C-terminal residues were altered, the L6A-L7R-R8L variant retained wild-type activity against 5 targets, and the activities of the L7A-R8A and L6A-L7A variants were reduced in most cases but were enhanced against C. sakazakii DM4485 (Table 5).
Activity of caseicin B variants against S. aureus.
The most remarkable feature with respect to the activity of the caseicin B variant peptides against S. aureus was the frequent retention of wild-type-like levels of activity against all strains (Table 6). However, yet again, the E4D variant was distinctive in that its activity was reduced against all targets. Among the others, in this instance, the activity of the L2A variant was reduced most consistently (against 4 of 5 targets). The reduced activity of the L7A-R8A variant against strain 8325-4 was the only other example of a variant peptide exhibiting less-than-wild-type activity against an S. aureus target, but this contrasted with an impressive 8-fold increased activity, relative to that of caseicin B, against RF122 and a 4-fold increased activity against the RN4220 derivative of 8325-4. The activities of the L7R, L6A, L7A, L2I, L6A-L7R-R8L, and plus-A2 variants were all enhanced against one S. aureus strain, while the remaining peptides exhibited wild-type-like activities against all of these targets.
Table 6.
MICs of caseicin B variants against Gram-positive S. aureus strains
| Strain | MIC (mM) of variant: |
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | L7A-R8A | L7R | L6A-L7A | L2I | E4A | N5A | R8H | L6A-L7R-R8L | Plus-A2 | E4D | R8K | ΔE4 | L2A | |
| 8325-4 | 1.25 | 2.5 | 0.625 | 0.625 | 0.625 | 1.25 | 1.25 | 1.25 | 1.25 | 0.625 | >2.5 | 1.25 | 0.312 | 1.25 |
| SA113 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 0.625 | 1.25 | >2.5 | 1.25 | 0.312 | 2.5 |
| RF122 | 1.25 | 0.156 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | >2.5 | 1.25 | 1.25 | 2.5 |
| Newman | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | >2.5 | 1.25 | 1.25 | 2.5 |
| RN4220 | 1.25 | 0.312 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | >2.5 | 1.25 | 1.25 | 2.5 |
DISCUSSION
There has been considerable interest in the antimicrobial activity of milk-derived peptides in recent years. A number of milk proteins have been studied extensively, which led to the discovery of a range of AMPs, including caseicins (9), kappacin (19), isracidin (14), lactoferricin (1), and casocidin-I (30). This study represents a continuation of our efforts to gain a better understanding of the importance of specific regions within two caseicin peptides, caseicins A and B, through the generation of synthetic variants in which specific changes have been made and by examining the consequences of these changes. Thus, the previous modest collection of 4 caseicin A and 4 caseicin B peptides has been expanded on greatly to include a further 13 variants of each peptide. It is apparent that the impact of the changes made with respect to the MIC of the peptide varies from one species to the next and, as is evident from studies with C. sakazakii and S. aureus, from one strain to another. Nonetheless, when the data are analyzed in combination (through the assignment of arbitrary activity units and the calculation of average arbitrary activity units [AAAU] as a means of providing an overview) (Fig. 1), obvious patterns emerge. These include the generally greater tolerance of caseicin B to change; the fact that, on average, the changes made have a more detrimental impact on Gram-negative activity; and the surprising number of variants which exhibit enhanced activity against S. aureus. The obvious differences with respect to the impact on anti-Gram-negative and anti-S. aureus activities suggest differences in the mechanisms by which the peptides inhibit these two groups of targets. The fact that it was more difficult to increase activity against any of the Gram-negative strains and that none of the peptides exhibited increased AAAU values against the Gram-negative targets as a whole suggests that these milk-derived peptides are already at or close to their optimal capacity with respect to inhibiting Gram-negative organisms. It is important with respect to the differences observed in caseicin variant activity against the Gram-negative strains tested that the strains included in this study represent a variety of Gram-negative strains; therefore, one would expect the results to vary as they did. Also, it is well documented in the literature that Cronobacter strains vary considerably in their responses to a number of stresses (5, 12), which may account for the variability between strains in this study. In contrast, many changes resulted in enhanced anti-S. aureus activity, and thus the mechanism by which they act is easily improved upon.
Fig 1.
AAAU of caseicin A (IKHQGLPQE) and B (VLNENLLR) variants against Gram-negative (A) and S. aureus (B) strains. Colored residues represent those altered relative to the corresponding wild-type peptide, with the color in each case corresponding to the AAAU of the peptide in question. *, variants previously assessed by Norberg et al. (22).
Examination of the extended collection of caseicin variants also indicates the absence of specific motifs within the peptides which are of great importance with respect to antimicrobial activity. This contrasts with the outcomes observed when similar strategies were employed to identify important domains in other antimicrobial peptides, such as the bacterially produced bacteriocins (26). In these other cases, key residues were identified on the basis of their complete intolerance to change. In the case of the caseicins, while there are many instances of changes which have a negative impact, these negative impacts relate to the nature of the newly incorporated residue, and other, less detrimental changes are frequently noted. Indeed, while E4A and E4D changes both resulted in peptides with greatly reduced activity, the complete removal of this glutamate had a much less significant consequence. This was despite the fact that the removal of residues has the potential to alter the conformation of the peptide and the surface distribution of the remaining residues. These results indicate the absence of any specific receptor via which these peptides act and indicate a more generalized mechanism of action.
The hydrophobicity of the caseicin variants was also analyzed, as a number of studies have discovered a correlation between hydrophobicity and antimicrobial activity (2, 16, 21). Unfortunately, we could not link antimicrobial activity with the percent hydrophobicity of the variant caseicins. The isoelectric point of peptides is another property which has been investigated and has previously been shown to be correlated with the level of antimicrobial activity (24). Again, we did not find a significant link between the isoelectric points of the variant caseicins and the levels of activity observed.
Thus, through the generation and investigation of a series of caseicin A and B variants, we have gained further knowledge with respect to these antimicrobials, but further investigations are required to determine the precise mechanism(s) of action of these peptides and if it differs depending on whether the target is Gram positive or negative in nature.
Supplementary Material
ACKNOWLEDGMENT
This project was funded by an Enterprise Ireland Technology Development grant as part of the SAFEFORMULA project.
Footnotes
Published ahead of print 13 January 2012
Supplemental material for this article may be found at http://aem.asm.org/.
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