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JAC Advance Access originally published online on April 21, 2007
Journal of Antimicrobial Chemotherapy 2007 59(6):1102-1108; doi:10.1093/jac/dkm096
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Short peptides derived from the NH2-terminus of subclass IIa bacteriocin enterocin CRL35 show antimicrobial activity

Emiliano Salvucci, Lucila Saavedra and Fernando Sesma*

Centro de Referencia para Lactobacilos (CERELA-CONICET), Chacabuco 145 (T4000ILC), S.M. de Tucumán, Tucumán, Argentina

* Corresponding author. Fax: +54-381-4005600; E-mail: fsesma{at}cerela.org.ar

Received 12 January 2007; returned 29 January 2007; revised 1 March 2007; accepted 5 March 2007


   Abstract
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Objectives: Subclass IIa bacteriocins are characterized by a hydrophilic N-terminal domain that shares a YGNGVxCxxxxC consensus and a variable hydrophobic C-terminus. Enterocin CRL35 is a 43-amino-acid heat stable peptide with antilisterial activity. Short synthetic peptides derived from the N-terminal half of enterocin CRL35 and other subclass IIa bacteriocins were evaluated for antimicrobial properties.

Methods: In vitro activities of synthetic peptides were evaluated in complex, chemically defined and minimal media. MIC assays were performed by the agar well-diffusion method. Fluorescence assays to evaluate the dissipation of membrane potentials in intact cells were carried out. Time–kill kinetics of Listeria innocua cells with the active peptide were performed.

Results and conclusions: A 15-mer peptide derived from enterocin CRL35 inhibited the growth of L. innocua and Listeria monocytogenes in synthetic/minimal media and dissipated the membrane potential of sensitive cells, with MICs of 10 and 50 µM, respectively. 15-mer derivatives from other class IIa bacteriocins (mesentericin Y105, pediocin PA-1 and piscicolin 126) also showed antimicrobial activities.

Keywords: enterococci , pediocin , liposomes


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Bacteriocins are antimicrobial proteinaceous compounds synthesized ribosomally by bacteria. The ecological function of these peptides is not yet fully understood.1 Most food-grade lactic acid bacteria (LAB) produce bacteriocins. These peptides permeabilize the membrane and deplete the proton motive force of sensitive cells and artificial liposomes or induce lysis through the activation of autolysins.2–4 Subclass IIa bacteriocins are heat stable short peptides with antilisterial activity.3,5,6 These compounds share a YGNGVxCxxxxC consensus in their cationic and hydrophilic N-terminal domain; however, the C-terminal domains are somewhat more diverse, which allows subclass IIa antimicrobials to be divided into three subgroups.7,8 It has been postulated that the C-terminal half is implicated in cell specificity.9

As subclass IIa bacteriocins have strong antilisterial activity, these peptides constitute a novel approach to control Listeria monocytogenes, the causative agent of listeriosis, in food and make them attractive candidates as next-generation therapeutic agents.10

The fact that they are ribosomally synthesized peptides opens the possibility of improving the characteristics of each peptide in order to enhance their spectra of activity. As a preliminary approach, numerous studies about the relationship between primary structure and function have been performed.5,9,11,12 Fleury et al.12 showed that truncated analogues of mesentericin Y105 are not active against Listeria. Fimland et al.13 have demonstrated that a peptide derived from the C-terminal domain could increase the MIC of different bacteriocins. Alternatively, Johnsen et al.9 demonstrated that hybrid bacteriocins remain active and suggested that the C-terminal hairpin domain is implicated in the specificity-determining step that seems to involve interactions with lipids and/or proteins of the cell membrane. Yan et al.14 reported that the interaction with a chiral receptor is a critical feature of the mode of action of subclass IIa bacteriocins and a non-specific mode of action at high concentrations.

Enterocin CRL35 is a subclass IIa bacteriocin with antilisterial and antiviral activities.15 It is a 43-amino-acid heat stable peptide produced by Enterococcus mundtii CRL35 isolated from artisanal cheese from Tafí del Valle (Tucumán, Argentina). It has demonstrated synergism with some antibiotics even at sublethal concentrations.8 Saavedra et al.15 showed that peptides derived from the C-terminal domain of enterocin CRL35 were able to inhibit the action of the bacteriocin and those derived from the N-terminal half were able to enhance the antimicrobial activity.

In the present work, we analyse the antimicrobial properties of short synthetic peptides derived from the N-terminal sequence of enterocin CRL35 and other subclass IIa bacteriocins in complex and chemically defined media.


   Materials and methods
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Strains and media

Complex media brain heart infusion (BHI), LAPTg (1.5% peptone, 1% tryptone, 1% glucose, 1% yeast extract, 0.1% Tween 80, pH 6.5) and Mueller–Hinton (M–H), the minimal medium HTM (Hsiang-Ning Tsai medium)16 and the chemically defined Bacto B12 vitamin assay medium (Difco; supplemented with tryptone, peptone or yeast extract at 10 g/L for each) were used for the assays. Agarized media were prepared with 1.5% agar. Susceptible and resistant target cells were Lactococcus lactis subsp. cremoris MG1363,17 Lactobacillus plantarum CRL691 (CERELA culture collection), L. lactis subsp. lactis IL140318 (INRA-Jouy-en-Josas, France), Enterococcus faecium DPC1143 (Dairy Products Research Center, Teagasc, Moorepark, Fermoy, County Cork, Ireland), Escherichia coli BL21(DE3), L. monocytogenes FBUNT (Bacteriology Department, Faculty of Biochemistry, Universidad Nacional de Tucumán) and Listeria innocua 7 (INRA-Jouy-en-Josas). All bacterial stock cultures were maintained in their appropriate broths containing 20% glycerol at –80°C. L. innocua 7 and L. monocytogenes FBUNT were grown in BHI broth, HTM broth and B12 assay bacto agar (Difco), supplemented with 24 pM lipoic acid. L. plantarum CRL961 was cultured in Lactobacillus MRS broth. L. lactis MG1363 was cultured in LAPT with 0.5% (w/v) glucose (LAPTg). All cultures were grown overnight without aeration at 30°C.

Synthetic peptides and bacteriocins

Enterocin CRL35 and short truncated peptides derived from N-terminal sequences of enterocin A, divercin V41, piscicolin, pediocin PA-1, Listeriocin 743A, sakacin P, mesentericin and enterocin CRL35 were designed and synthesized (Table 1). Freeze-dried peptides were reconstituted in sterile distilled water and stored at –20°C until used. Different parameters of the truncated peptides were calculated using the online ProtParam tool from Expasy (www.expasy.org/tools/protparam.html). Mature divercin V41 and enterocin A were purified from cell-free supernatants from producer strains Carnobacterium divergens V4119 and E. faecium CRL988,20 respectively.21 The active fractions were pooled and concentrated by N2 flux using a TurboVap evaporator (Caliper Life Sciences, Hopkinton, MA, USA) and resuspended in distilled water.


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  		Table 1.. Bacteriocins and short truncated N-terminal peptidesa used in this work

 
MIC assays

The inhibitory activity of truncated derivatives and their corresponding mature bacteriocins as models of subclass IIa was studied using the well-diffusion method described previously.12 MICs were determined by successive dilutions of antimicrobials on agar plates and defined as the lowest dilution of the bacteriocin/peptide that forms a clear inhibition zone of 1 mm, or following growth inhibition at OD560 in a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA). In this case, MIC was defined as the concentration of bacteriocin/peptide that inhibited growth of the indicator strain by 50% after 12 h.

Determination of membrane potential

The ability of the peptides to depolarize the cytoplasmic membrane of L. innocua 7 was examined using the membrane potential-sensitive fluorescent cyanine dye 3,5-dipropylthiadicarbocyanine iodide [DiSC3(5); Molecular Probes, Eugene, OR, USA].22 The dye concentrates in the cytoplasmic membrane of energized cells, resulting in the autoquenching of fluorescence. If the peptide forms a channel or disrupts the membrane, the membrane potential will be dissipated and the dye will be released into the medium, increasing the fluorescence. Mid-log phase L. innocua cells were washed twice with cold 50 mM HEPES buffer (pH 7.4) containing 12.5 mM glucose and resuspended in the same buffer up to 1 x 108 cells/mL at 4°C and immediately placed on ice for fluorescence measurements. The cell suspension was incubated with 0.5 µM DiSC3(5) in a fluorescence cuvette until the uptake was maximal, as indicated by a stable reduction in fluorescence. Different concentrations of enterocin CRL35, divercin V41 and enterocin A and their truncated peptides were added to the suspension and the fluorescence was monitored at 30ºC in a fluorescence spectrophotometer (Cary Eclipse, Varian) with the excitation monochromator set at 632 nm and the emission at 670 nm. Complete dissipation was obtained in the presence of 1 µM valinomycin.

Leakage measurement

The release of liposomal content was measured by following the fluorescence quenching of pre-encapsulated terbium-dipicolinic acid (Tb/DPA) complex upon its release into external medium containing 0.1 mM EDTA.23 To prepare vesicles containing Tb/DPA complex, a chloroformic solution of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) was mixed (9:1, final concentration 0.5 µmol) and exhaustively dried under nitrogen stream and suspended in 50 mM Tris–HCl buffer with Tb/DPA, pH 6.5. The large multilamellar vesicles formed were sonicated for 20 min with a probe-type sonicator. Non-encapsulated material was eliminated by incubating liposomes for 1 h at 4°C and by gel filtration using a Sephadex-50 column with 50 mM Tris–HCl/10 mM EDTA as the mobile phase. Liposomes were collected in the void volume. Total phospholipid concentration was determined by phosphate analysis. Excitation and emission wavelengths were set at 280 and 545 nm. The assays were carried out at 15ºC.

Viability assays

These assays were carried out as described by McAuliffe et al.24 Briefly, cells of L. innocua in the exponential growth phase were collected and resuspended in 50 mM HEPES plus 12.5 mM glucose; then, 1 x 108 cells were taken and resuspended in 1 mL of the same buffer. Different concentrations of the peptides to evaluate were added and cells were quantified (as cfu/mL) at different times.


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As shown in Table 1, 15- and 16-mer N-terminal derivative peptides of enterocin CRL35 (called S and V peptide, respectively) showed antimicrobial activity (Figure 1a). The S peptide was active against L. innocua 7 at concentrations of 10 µM, whereas L. monocytogenes FBUNT was more resistant and was inhibited with concentrations of at least 50 µM in HTM or Bacto B12 vitamin assay medium (Table 2 and Figure 1b). When assayed in complex media (M–H, BHI and LAPTg) the antimicrobial activity was lower and variable (with frequent negative well-diffusion assays) when compared with that obtained in chemically defined medium (Bacto B12 vitamin assay medium) and minimal medium (HTM) (Table 3). The increased antimicrobial activity in chemically defined medium suggests that some components of the complex media might interfere with detection or activity of antimicrobial peptides. To test this hypothesis, we added different components (one by one) to the base minimal medium and analysed the activity. After 16 h, the sensitive strain forms a lawn on agar and we found a decreased antimicrobial activity in Bacto B12 vitamin assay medium supplemented with tryptone, yeast extract or peptone when compared with the activity displayed in medium without the addition of supplements (Table 3). These results demonstrate that S and V peptides derived from the N-terminal domain of enterocin CRL35 displayed antimicrobial activity in minimal medium and confirm the importance of using chemically defined medium to study the spectrum of action of bacteriocins. Besides, these results might explain in part the apparent contradictory data observed in our previous work, because antimicrobial assays done with peptide S in complex media such as LAPTg showed negative results.15


Figure 1
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  		Figure 1.. (a) Comparative activity by the well-diffusion agar method between S peptide (10 µM) and enterocin CRL35 (10 µM) in an overlay of L.innocua 7. (b) MIC, the highest dilution of peptide that could inhibit the growth of the susceptible strain forming a halo of at least 1 mm, of S peptide on a lawn of L. innocua 7 in Bacto B12 vitamin assay medium. (c) Inhibition halos by peptide derivatives (labelled with the name of the entire bacteriocin with the number of amino acids) on an overlay of L. innocua cells in BHI (600 µM) (spot-on-lawn).

 


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  		Table 2.. MIC of S peptide in complex and chemically defined media (µM)

 


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  		Table 3.. Sensitivity of Listeria in different media to S peptide

 
In contrast, shorter (10- to 14-mer) derivatives of enterocin CRL35 did not show antimicrobial activity (Table 1) even at high concentrations (1000 µM), indicating that 15 amino acids would be the smallest size for the peptide to retain its activity.

To evaluate the effect of peptides on membrane potential, fluorescence assays were carried out with S and V derivatives. These peptides were able to dissipate the transmembrane electrical potential ({Delta}{Psi}) of sensitive cells in a buffer system (Figure 2). In agreement with the well-diffusion assays, the S derivative was more active at dissipating {Delta}{Psi} than peptide V. However, the more active peptide (S) displayed an activity 100-fold lower than parental bacteriocin. Furthermore, the dissipating action of S peptide occurred more slowly than the complete bacteriocin, suggesting that though lacking the C-terminal domain, the peptide derivatives could destabilize the cell membrane by forming pores in the membrane at concentrations in the order of micromolar. This could be due to the accumulation of major quantities of short peptides that auto-enhance the pore formation or activate another mechanism. Although the lack of the C-terminal domain causes an important loss of activity, the peptide at elevated concentrations retains the ability to form pores or to destabilize the cell membrane.


Figure 2
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  		Figure 2.. (a) Dissipation of {Delta}{Psi} induced by enterocin CRL35 15-mer peptide derivative. L. innocua cells (1 x 108 cfu/mL) were charged with 3,5-dipropylthiadicarbocyanine iodide in 50 mM HEPES, pH 7.4. (b) Comparative dissipation of {Delta}{Psi} induced by S and V peptide derivatives when compared with enterocin CRL35. Results are the mean values of three independent experiments. Standard deviation (<10%) was omitted to facilitate visualization. ent., enterocin.

 
In order to extend our knowledge, we analysed short peptide derivatives from other class IIa bacteriocins. As the length of 15 amino acids appears to be critical for the activity, we evaluated 15-mer peptides derived from the following bacteriocins: sakacin P, mesentericin Y105, piscicolin 126, pediocin PA-1 and listeriocin 743A. Besides these 15-amino-acid class IIa derivatives, we also assayed derivatives from divercin 41 (17 amino acids) and enterocin A (20 amino acids). These last two derivatives are also derived from class IIa bacteriocins but present other amino acids in the NH2 terminus: T in divercin V41 and TTHSG in enterocin A. Like peptides S and V, the derivatives from mesentericin Y105, pediocin PA-1, sakacin P, piscicolin 126 and listeriocin 743A were also able to inhibit a lawn of Listeria (Figure 1c). The peptidic derivatives from enterocin A and divercin V41 were not active against L. innocua, which is in agreement with previously reported data.11 In the case of mesentericin Y105, Fleury et al.12 have shown that the N-terminus is also essential for bacteriocin activity and that the C-terminal domain is involved in determining the target cell specificity of these bacteriocins; it could also play a role in electrostatic interactions with anionic lipids of the target membrane.

All peptides that showed antimicrobial activity were also evaluated for their ability to disrupt DMPC/DMPG liposomes (Figure 3). All of them were active at 10 µM.


Figure 3
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  		Figure 3.. Leakage of Tb+3/DPA from liposomes by the action of peptide derivatives. At concentrations of 10 µM, peptides could deplete the signal of fluorescence (arrow). Total extrusion was obtained with 0.1% Triton X-100 (arrowhead). Results are the mean values of three independent experiments. Standard deviation (<10%) was omitted to facilitate visualization.

 
S and V peptides were also capable of dissipating the membrane potential of L. plantarum CRL691 (data not shown). S peptide was used for MIC assays using L. innocua 7 as a susceptible strain in broth (Figure 4); 10 µM of the peptide was able to inhibit the growth to half of the control after 14 h. Therefore, it is necessary for a great amount of peptides to auto-enhance their affinity to the membranes and disrupt them.


Figure 4
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  		Figure 4.. Microplate MIC assays: S peptide has an MIC of 10 µM. Peptide at millimolar concentrations inhibits growth totally.

 
It is known that sublethal concentrations of bacteriocins could dissipate {Delta}{Psi} without altering the viability of Listeria cells.8 In order to evaluate the bactericidal effect of S peptide on Listeria, viability assays were carried out as described by McAuliffe et al.24 After 5 min of treatment with peptide concentrations equal to MIC, the viable cell count was 250-fold lower than the control without treatment (Figure 5). This effect was observed with the parental bacteriocin at nanomolar concentrations. Thus, the antimicrobial activity was reduced by more than 1000-fold.


Figure 5
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  		Figure 5.. Time–kill kinetics of membrane-active S peptide against L. innocua 7. Cell viability, expressed as cfu/mL, was determined at the indicated times. Results represent the average of three independent experiments using different peptide concentrations. Circles, 0 µM; squares, 2.5 µM; diamonds, 5 µM; stars, 10 µM. ent., enterocin.

 
In contrast, truncated peptides S and V were not active against E. coli BL21(DE3), L. lactis MG1363, L. lactis IL1403 or E. faecium DPC1143 (data not shown). However, the 15-mer peptide did show activity against L. plantarum CRL691, displaying a relatively narrow spectrum of action as was previously observed with the entire bacteriocin.7,12 Although S and V peptides showed a similar biological activity when compared with the entire enterocin CRL35, further studies are necessary to elucidate the mechanism of action of these peptides that lack the amphiphilic domain but remain active in synthetic or minimal media. Although the elucidation of mode of action of these active peptides will be analysed in future works, it is possible to speculate that these peptides could form pores in the membrane at elevated concentrations in order to compensate for their lower activity (and size) when compared with parental bacteriocin.

As noted earlier, we reported in a previous work that the 15-mer derivative (peptide S) of enterocin CRL35 was unable to inhibit L. innocua.15 Although that result appears to be contradictory with the present data, one important difference has been the implementation of synthetic or minimal media. In contrast, Fimland et al.13 showed that an N-terminal derivative of pediocin PA-1 did not show antimicrobial activity. However, the peptide design in that work was different as the N-terminal amino acids were modified and the Cys was replaced with {alpha}-aminobutyric acid. Probably, the Cys is needed to form intra- and intermolecular disulphide bridges that could be crucial for the antimicrobial activity of these short peptides. Furthermore, Yan et al.14 reported that a 22-mer N-terminal derivative of carnobacteriocin B2 did not show antimicrobial activity. In this experiment, the length and the sequence of the peptide (VNYGNGV in carnobacteriocin B12) were different from those of the peptides used in the present work.

It is known that many peptide derivatives from antimicrobial proteins of vertebrates have antimicrobial activity, i.e. human ß-defensins25 and mucins26 or frog skin antimicrobial peptides,27,28 and peptides derived from human proteins or antibiotics;29–31 however, to the best of our knowledge, this is the first report of antimicrobial activity of short N-terminal peptidic derivatives from LAB bacteriocins.32 It would be interesting to evaluate their use as biopreservatives in fermented food,33 or for topical use in veterinary or medical applications.


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None to declare.


   Acknowledgements
 
This work was supported by grants PIP6229 from CONICET (Buenos Aires) and PICT 9-13406 from ANPCyT (Buenos Aires).


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2 Bierbaum G, Sahl HG. Influence of cationic peptides on the activity of the autolytic endo-ß-N-acetylglucosaminidase of Staphylococcus simulans 22. FEMS Microbiol Lett (1989) 49:223–8.[CrossRef][Web of Science][Medline]

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5 Morisset D, Berjeaud JM, Marion D, et al. Mutational analysis of mesentericin Y105, an anti-Listeria bacteriocin, for determination of impact on bactericidal activity, in vitro secondary structure, and membrane interaction. Appl Environ Microbiol (2004) 70:4672–80.[Abstract/Free Full Text]

6 Papagianni M. Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications. Biotechnol Adv (2003) 21:465–99.[CrossRef][Web of Science][Medline]

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8 Minahk CJ, Dupuy F, Morero RD. Enhancement of antibiotic activity by sub-lethal concentrations of enterocin CRL35. J Antimicrob Chemother (2004) 53:240–6.[Abstract/Free Full Text]

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11 Bhugaloo-Vial P, Douliez JP, Moll D, et al. Delineation of key amino acid side chains and peptide domains for antimicrobial properties of divercin V41, a pediocin-like bacteriocin secreted by Carnobacterium divergens V41. Appl Environ Microbiol (1999) 65:2895–900.[Abstract/Free Full Text]

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16 Tsai HN, Hodgson DA. Development of a synthetic minimal medium for Listeria monocytogenes. Appl Environ Microbiol (2003) 69:6943–5.[Abstract/Free Full Text]

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