Simultaneous Production of Formylated and Nonformylated

Article pubs.acs.org/JAFC

Simultaneous Production of Formylated and Nonformylated Enterocins L50A and L50B as well as 61A, a New Glycosylated Durancin, by Enterococcus durans 61A, a Strain Isolated from Artisanal Fermented Milk in Tunisia Hasna Hanchi,†,§ Riadh Hammami,*,† Benoit Fernandez,† Rim Kourda,§ Jeannette Ben Hamida,§ and Ismail Fliss*,† †

STELA Dairy Research Centre, Institute of Nutrition and Functional Foods, Université Laval, Québec, QC, Canada G1K 7P4 Unité de Protéomie Fonctionnelle & Biopréservation Alimentaire, Institut Supérieur des Sciences Biologiques Appliquées de Tunis, Université Tunis El Manar, Tunis, Tunisia

§

S Supporting Information *

ABSTRACT: Enterococcus durans 61A, a broad-spectrum strain, was isolated from artisanal fermented dairy products. The strain is a multibacteriocin producer, free from virulence genes, and could be considered a good candidate for application in food preservation. In the present study, E. durans 61A was shown to produce simultaneously formylated and nonformylated forms of leaderless enterocins L50A and L50B as well as 61A, a new glycosylated durancin. Bacteriocins were characterized using mass spectrometry. Formylation was found to increase enterocin antimicrobial activity of enterocin L50A (8×) and, to a lesser extent, the activity of L50B (2×). Durancin 61A was found glycosylated by two hexoses (glucose and arabinose) and exhibited broadspectrum inhibition against Gram-positive and Gram-negative bacteria and fungal spores. Durancin 61A was highly bactericidal at 15.6 μg/mL (10× the MIC) on Listeria innocua HPB13 and seems to target bacterial membrane as shown by ion efflux and transmission electron microscopy. KEYWORDS: Enterococcus durans 61A, glycosylated bacteriocin, durancin 61A, enterocin L50A, enterocin L50B

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INTRODUCTION Enterococci are a widespread class of lactic acid bacteria in many traditional dairy and meat products. Several enterococci were found to produce broad-spectrum bacteriocins often termed enterocins, reviewed by Franz et al.1 Bacteriocins are defined as bacteriostatic or bactericidal proteinaceous molecules that have a relatively narrow killing spectrum, being toxic primarily to bacteria closely related to the producing strain.2 Bacteriocins belong to a highly diverse family of peptides in terms of size, structure, microbial target, mode of action, and release. Bacteriocins generally exert their killing action by interfering with the cell wall or membrane of target organisms, by either inhibiting cell wall biosynthesis or causing pore formation, subsequently resulting in death.2 Nowadays, an increasing number of multibacteriocin producer enterococci are reported.3−6 For instance, Enterococcus faecium L50 was shown to produce enterocins L50A and L50B at low temperature range (16−25 °C) and, alternatively, enterocins P and Q at higher temperatures (37−47 °C).3 Previously, we have isolated five antilisterial enterococci strains from artisanal fermented dairy products.7 Among the five strains, the Enterococcus durans 61A was the most potent, free of virulence genes and sensitive to vancomycin. Beside its safety, the strain was able to survive under simulated gastrointestinal conditions and reached the effluent with high survival.7 In the present study, we report that E. durans 61A produces simultaneously formylated and nonformylated forms of leaderless enterocins L50A and L50B as well as 61A, a new glycosylated durancin. The physicochemical characteristics, the mode of action, and © 2016 American Chemical Society

safety of durancin 61A were investigated. In addition, the effects of formylation on the antimicrobial activity of EntL50A and EntL50B were also reported.

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MATERIALS AND METHODS

Microorganisms and Culture Conditions. E. durans 61A previously isolated from spontaneous fermented milk was grown aerobically in M17 broth supplemented with 5 g of glucose at 30 °C.7 Indicator strains used in this study, the culture media, and their origins are listed in Table 1. All strains were maintained in 50% glycerol at −80 °C until use. Antimicrobial Activity and MIC Determination. The minimal inhibitory concentrations (MIC) were determined using polystyrene microassay plates (96-well Microtest, Becton Dickinson Labware, Sparks, MD, USA) as described by Hammami et al.8 Briefly, microplates loaded with 2-fold serial dilutions of durancin 61A (starting at 200 μg/mL) in tryptic soy broth (TSB) were seeded with log-phase culture of target strain diluted in TSB to (0.5−1.0) × 106 cfu/mL (approximately 1 × 104 cfu/well). Microplates were then incubated at 37 °C for 24 h, and absorbance at 595 nm was measured hourly using an Infinite F200 PRO photometer (Tecan US Inc., Durham, NC, USA). Supernatant activity was expressed in arbitrary units per milliliter (AU/mL) and calculated as AU/mL = 2n × (1000/125),8 where n is the number of wells at different concentrations in the serial dilution showing inhibition of the indicator strain. MIC values of pure Received: Revised: Accepted: Published: 3584

February April 20, April 25, April 25,

13, 2016 2016 2016 2016 DOI: 10.1021/acs.jafc.6b00700 J. Agric. Food Chem. 2016, 64, 3584−3590

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Journal of Agricultural and Food Chemistry

detected at 214 nm and manually collected. Acetonitrile solvent was then evaporated before antimicrobial activity assays using the critical dilution micromethod. Protein concentrations of the pure peptides were determined spectrophotometrically at 280 nm using Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE, USA). Mass Spectrometry and Peptide Identification. The molecular weight of the purified fractions was measured by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) on an AB SCIEX 4800 Plus MALDI-TOF/TOF instrument using α-cyano-4-hydroxycinnamic acid (Aldrich Chemical, Mississauga, ON, Canada) as matrix. For peptide identification, peptides were analyzed using ESI-MS/MS on a Thermo Fisher Orbitrap XL mass spectrometer. The mass spectrometer was operated in a data-dependent mode with full scan (m/z 200−2000) MS spectra acquired at 60000 resolution in the Orbitrap. The most abundant precursor ions were selected for fragmentation in the LTQ using collision-induced dissociation at a CE setting of 35. Fragment ions were analyzed in the ion trap. The raw centroid data were searched using Mascot search engine v2.10 (Matrix Science) against a custom database (E. faecium, 267,757 entries) with deamidation of asparagine and glutamine, and oxidation or formylation of methionine was specified in Mascot as variable modification. Searches were performed with a precursor mass tolerance of 15 ppm and fragment ion tolerances of 0.6 Da. Identification of Carbohydrates Moieties in Durancin 61A. Sensitivity to α-Amylase. To test enzymatic inactivation of antimicrobial substance, the pure peptide was treated with the α-amylase (sigma) at a final concentration of 1 mg/mL. Colorimetric Method. For the preliminary detection of sugar, the phenol−sulfuric acid method was used;10 0−1.5 mL of 5% phenol was added to 1.2 mL of the sample and immediately vortexed after mixing with 2.5 mL of concentrated sulfuric acid. After the mixture had stood for 20 min at room temperature, the absorbance of the sample solution was measured at 490 nm against the blank.11 Sugar Identification Using GC-MS. The presence of the sugar moiety was confirmed by gas chromatography−mass spectrometry (GC-MS) analysis and library matching of the sugar, according to protocol.12 The purified durancin 61A was subjected to acidic hydrolysis to release the sugar followed by derivatization. N-Methyl-N(trimethylsilyl) trifluoroacetamide (Tri-Syl PIERCE Thermo Scientific) was used as derivatizating agent and pyridine as a solvent. Derivative molecules were analyzed by GC-MS [Hewlett-Packard model 6890 series II gas chromatograph attached to an Agilent (Palo Alto, CA) model 5973N selective quadrupole mass detector] under an ionization voltage of 70 eV at 230 °C and connected to a computer with a Hewlett-Packard ChemStation. Hexose sugar standards including glucose, mannose, fructose, galactose, and arabinose taken as control were treated and derivatized using the conditions described above. Finally, mass spectra were compared with those of the base NIST 11 Mass Spectral Library http://www.sisweb.com/software/ms/nist.htm (National Institute of Standards and Technology) standards. Mode of Action of Durancin 61A. Bactericidal Effect. The purified durancin 61A at 0×, 5×, or 10× the MIC was added to 10 mL culture of L. innocua HPB13 in early exponential phase and incubated at 30 °C. The number of viable cells was determined by plate counting on TSB agar hourly during 8 h and expressed as log10 (CFU/mL). Hemolytic Activity. Bacteriocin hemolytic activity was tested as previously described.13 Briefly, horse defibrinated blood (Quelab, Montreal, QC, Canada) was centrifuged at 5000g at 4 °C for 10 min, and the pellet was washed three times with PBS buffer (100 mM, pH 7.4). Ten microliters of durancin 61A at different concentrations were added to 90 μL of erythrocytes and incubated at 37 °C for 1 h. Positive and negative controls were performed with 1% (w/v) Triton X-100 and PBS buffer. The release of hemoglobin was monitored at 405 nm. All samples were run in triplicate. Hemolysis was calculated as a percentage: hemolysis (%) = [(Abs405 of the compound treated sample) − (Abs405 of buffer treated sample)]/[(Abs405 of Triton X-100 treated sample) − Abs405 of buffer treated sample)] × 100. Determination of Ion Efflux. Efflux of K+ and Na+ from L. innocua HPB13 cells in the presence of durancin 61A (1× or 5× the MIC) was

Table 1. Inhibitory Spectra of the Enterococcus durans 61A Supernatant and Durancin 61Aa microorganism

growth conditions

supernatant (AU/mL)

durancin 61A MIC (μg/mL)

Listeria monocytogenes ATCC19113 Listeria monocytogenes LSD532 Listeria monocytogenes Scott A3 Listeria innocua HPB13 Listeria ivanovii HPB28 Listeria monocytogenes LMA-1045 Lactococcus lactis biovar diacetylactis UL719 Pediococcus acidilactici UL5 Enterococcus faecalis ATCC 27275 Staphylococcus aureus ATCC 6538 Staphylococcus aureus ATCC 25923 Streptococcus pyogenes ATCC 10389 Clostridium perf ringens ATCC 3628 Staphylococcus epidermidis HER 1241

TSYB 30 °C

16

25

TSYB 30 °C

2048

6.25

TSYB 30 °C

128

25

TSYB 30 °C TSYB 30 °C TSYB 30 °C

512 128 128

1.56 6.25 25

MRS 30 °C

65536

0.097

MRS 30 °C TSYB 30 °C

65536 128

0.19 50

TSYB 30 °C

16

6.25

TSYB 30 °C

na

100

TSYB 30 °C

na

50

BHI 37 °C, anaerobic TSYB 30 °C

na

25

na

25

Pseudomonas aeruginosa ATCC 15442 Escherichia coli ATCC 35150 Escherichia coli ATCC 35695 Salmonella choleraesuis ATCC 8387

TSYB 37 °C

na

100

TSB 37 °C

na

50

TSB 37 °C

na

50

TSB 37 °C

na

100

Aspergillus versicolor LMA-417 Penicillium commune LMA-212

YEG 26 °C

2048

3.59

YEG 26 °C

1024

7.18

a

AU, arbitrary units; MIC, minimal inhibitory concentration; na, not active; TSYB, tryptic soy broth + yeast extract; MRS, de Man, Rogosa, and Sharp; YEG, yeast extract glucose broth; BHI, brain−heart infusion medium.

bacteriocin were expressed in micrograms per milliliter and corresponded to the lowest concentration that inhibited the growth of target organism after 16−20 h. The MIC values are reported as means of two independent experiments in duplicate. Bacteriocin Purification. Bacteriocins were purified from the culture supernatant of E. durans 61A, grown overnight at 30 °C in a chemically defined medium.9 Cell-free culture supernatant was loaded into a Sep-Pak C18 cartridge column (Waters, Milford, MA, USA) previously equilibrated with 5 mM HCl in HPLC grade water and eluted with increasing concentrations of acetonitrile (0, 25, 50, and 100%). The active fraction was then subjected to rotary evaporation to remove acetonitrile. The active fraction was loaded onto a preparative C18 reverse-phase column (Luna 10 μm, 250 × 21.10 mm, Phenomenex, CA, USA) using a Beckman Gold System (Beckman Coulter Canada, Mississauga, ON, Canada) and a linear gradient from 25 to 50% of acetonitrile. The eluted peaks were collected, tested, and concentrated under azote. The active peak was then injected into an analytic C18 reverse-phase column (Aeris 3.6 μm PEPTIDE XB-C18, 250 × 4.6 mm, Phenomenex). Elution was performed at a flow rate of 1 mL/min using gradient from 70% solvent A (100% water supplemented with 5 mM HCl) and 30% solvent B (100% acetonitrile) to 50% of solvents A and B, respectively, within 50 min. Peptides were 3585

DOI: 10.1021/acs.jafc.6b00700 J. Agric. Food Chem. 2016, 64, 3584−3590

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Journal of Agricultural and Food Chemistry determined as previously described.13 Ion efflux was expressed in nanomoles per milligram of dry cells. Transmission Electronic Microscopy. Morphological structure of L. innocua HPB13 and E. coli ATCC 35695 cells after treatment with 20× the MIC of durancin 61A was monitored as previously described.13

methionine formylation on the antimicrobial activity of EntL50A and EntL50B was assessed on L. innocua HPB13. Whereas the MIC value of EntL50A was 625 nM against L. innocua HPB13, N-formylation increased the peptide antilisterial action 8 times (MIC = 78.1 nM). To a lesser extent, N-formylation increased the inhibitor activity of EntL50B 2 times, with MIC values being lowered from 1250 to 625 nM. Biochemical and Functional Characterization of Durancin 61A. Identification of Carbohydrate Moieties. The antimicrobial activity of durancin 61A decreased from 2048 to 1024 AU/mL after treatment with α-amylase, indicating the presence of carbohydrate moieties. In addition, sugar quantification using the Dubois method revealed that durancin 61A (100 μg/mL) contains 22.5 μg/mL sugar. To determine the identity of the hexose, durancin 61A was subjected to acidic hydrolysis to release the sugar followed by derivatization. Analysis by GC-MS and comparison of mass spectra with those of the base NIST 11 Mass Spectral Library http://www.sisweb. com/software/ms/nist.htm (National Institute of Standards and Technology) standards demonstrated the presence of both glucose and galactose in durancin 61A (Figure S3). Thus, durancin 61A was identified as a glycosylated peptide. Spectrum of Activity and Cytotoxicity. The antimicrobial spectrum of durancin 61A is summarized in Table 1. The glycosylated bacteriocin exhibited a broad-spectrum inhibition against Gram-positive foodborne pathogens including Listeria sp. (MIC range of 1.56−25 μg/mL), C. perfringens (MIC = 25 μg/mL), S. aureus (MIC range of 6.25−100 μg/mL), and S. pyogenes (MIC = 50 μg/mL). Although E. durans 61 supernatant was not active against Gram-negative strains, durancin 61A was found active against E. coli (MIC = 50 μg/mL), S. choleraesuis (MIC = 100 μg/mL), and P. aeruginosa (MIC = 100 μg/mL). Durancin 61A was also highly antifungal against spores of A. versicolor and P. commune with MIC values of 3.59 and 7.18 μg/mL, respectively. In addition, the cytotoxicity of glycosylated durancin 61A against horse erythrocytes was assessed

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RESULTS Antimicrobial Spectrum of E. durans 61A. The antimicrobial activity of E. durans 61A was assessed against various food-borne Gram-positive and Gram-negative bacteria and fungi (Table 1). Strain 61A was active against a wide range of Gram-positive pathogens such as Listeria sp. The inhibition was significant against related bacteria such as Lactococcus lactis, Pediococcus acidilactici, and E. faecalis. None of the tested Gramnegative indicator strains was sensitive to E. durans 61A supernatant. Besides, E. durans 61A was active against Aspergillus versicolor and Penicillium commune spores. Purification and Identification of E. durans 61A Bacteriocins. The supernatant of E. durans 61A was desalted and fractionated using a C18 Sep-Pak column. The active fraction eluted at 50% of acetonitrile and was evaporated and then loaded onto an RP-C18 HPLC preparative column. A subsequent separation of peptides by RP-HPLC was run using a linear gradient of 30−50% of acetonitrile. Five active peptides were isolated and are presented in Figure 1A. The MALDI-TOF spectra showed a single peak for each peptide (Figure 1B). The respective monoisotopic masses of peptides 1−5 were 5175.70, 5187.95, 5203.37, 5215.37, and 5217.34 Da. All peptides were subject to identification by LC-MS/MS. Peptides 1 and 3 were respectively identified as EntL50B and formylated EntL50B (+28 Da), as shown in Figure S1. Similarly, peptides 2 and 4 were identified as EntL50A and formylated EntL50A (Figure S2). The experimental b ions of peptides 3 (EntL50B) and 4 (EntL50B) closely match those of the theoretically N-formylated forms. However, identification attempt of HPLC peptide 5 (durancin 61A) using LC-MS/MS was unsuccessful. Besides, the impact of such

Figure 1. (A) Reverse-phase chromatography separation profile of Enterococcus durans 61A bacteriocins at 214 nm. (B) Matrix-assisted laser desorption ionization−time-of-flight mass spectrometry (MALDI-TOF MS) profile of purified peaks. 3586


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from artisanal fermented dairy products.7 Of particular interest, strain E. durans 61A was the most potent, free of virulence genes and sensitive to vancomycin. Beside its safety, the strain was able to survive under simulated gastrointestinal conditions and reached the effluent with a high survival rate of 17.21 ± 0.65%.7 In the present study, we report the identification and characterization of multiple bacteriocins from E. durans 61A supernatant. Purification of the antimicrobial substances was achieved using reverse-phase chromatography, and five active peaks were purified to homogeneity from the culture supernatant of E. durans 61A. MALDI-TOF spectra confirmed the presence of a single peak in each collected peptide. The monoisotopic masses of peaks 1 and 2 were close to those previously reported for EntL50A and EntL50B (5178 and 5190 Da).14 In addition, LC-MS/MS analyses confirmed the identity of the purified bacteriocins. Enterocins L50A and L50B are widespread bacteriocins that are produced by several E. faecium strains and by E. durans.15 Besides, peaks 3 and 4 were the most abundant (44.2 and 35.4%, respectively) and differ from peaks 1 and 2 by +28 Da. LC-MS/MS confirmed that the N-terminal methionines of EntL50A (peak 4) and EntL50B (peak 3) were formylated. To our knowledge, this is the first report of the simultaneous production of formylated and nonformylated leaderless enterocins. Previously, Dezwaan et al.16 reported the production of formylated EntL50A and EntL50B, whereas Cintas et al.14 reported the nonformylated forms of EntL50A and EntL50B. Similarly, E. faecalis 710 C was found to produce formylated enterocin 7A (MR10A) and 7B (MR10B),17 whereas E. faecalis MRR 10-3 produces the nonformylated forms.18 The strain E. durans 61A seems to produce mostly formylated isoforms of EntL50A and EntL50B, with relative abundance 79.6% compared to nonformylated forms (14%). The EntL50A was more potent than EntL50B as previously reported in the literature. The formylated forms of EntL50A and EntL50B were more potent than the nonformylated. Formylation highly increased the activity of enterocin L50A (8×) and, to a lesser extent, the activity of L50B (2×). Recently, removal of methionine formylation in bacteriocin BACsp222 by cyanogen bromide resulted in a slight decrease of bactericidal potential compared to the native form.19 Molecular determination of durancin 61A (peak 5) revealed a monoisotopic mass of 5217.34 Da. The bacteriocin was faintly produced by E. durans 61A with a relative abundance of 2.4% in the active fraction. LC-MS/MS spectra did not permit identification of durancin 61A, and the sequence remains unresolved. Nevertheless, durancin 61A peptide was shown to contain sugar moieties as revealed by GC-MS. The presence of such a compound could explain the difficulties observed in peptide identification. The glycosylated durancin 61A contained two hexoses, namely, glucose and arabinose. Similar but few bacteriocins with carbohydrate moieties were previously reported. For instance, sublancin 168 was shown to be an S-linked glycopeptide containing a glucose attached to a cysteine residue.20 Another post-translational modification was reported for glycocin F, a 43 amino acid bacteriocin from Lactobacillus plantarum, which contains two β-linked N-acetylglucosamine moieties, attached via side-chain linkages to a serine via oxygen and to a cysteine via sulfur.21 More recently, E. faecalis F4-9, isolated from Egyptian salted-fermented fish, was shown to produce a glycosylated bacteriocin.22 Enterocin F4-9 is a 5516.6 Da peptide β-O-linked to two molecules of N-acetylglucosamine at Ser37 and Thr46.

Figure 2. Hemolytic activity of durancin 61A against horse erythrocytes. Data were fit by nonlinear regression (f = y0 + a/(1 + exp(−(x − x0)/b)), SigmaPlot 12.0).

in vitro (Figure 2). The bacteriocin was not hemolytic at concentrations up to 25 μg/mL. Mode of Action. The mode of action of durancin 61A on sensitive cells was investigated using different peptide concentrations (5× and 10× the MIC) against L. innocua HPB13. As shown in Figure 3A, addition of 5× the MIC decreased Listeria counts by 1.3 log units within 1 h, and inhibition lasted for 6 h. A higher concentration of durancin 61A (10× the MIC) was more detrimental on indicator strain, with reduction of 3.2 log CFU/mL after only 1 h of contact. In addition, no cells were detected during the 8 h of experiment. The induced efflux of K+ and Na+ ions from L. innocua HPB13 cells by durancin 61A was determined after treatment of cells with 1× and 5× the MIC for 30 min (Figure 3B). Whereas a concentration of 1× the MIC induced potassium efflux of only 1.63 ± 0.78 nmol/mg, the glycosylated bacteriocin at 5× the MIC induced a total efflux of 67.60 ± 1.45 nmol/mg. Similarly, the presence of the peptide at 1× and 5× the MIC caused the release of 11.76 ± 0.65 and 61.26 ± 6.83 nmol of Na+ per milligram of cells. The transmission electron microscopy (TEM) images of untreated cells of L. innocua HPB13 revealed regular rodshaped structure with intact cell walls and well-defined membranes (Figure 4A). After 5 min of exposure to durancin 61A, listerial cells presented an altered morphology with cell wall damage and loss of cytoplasmic content (Figure 4B,C). Untreated cells of E. coli ATCC 35695 showed a normal cell shape with an undamaged structure of the inner membrane and an intact, slightly corrugated, and well-defined outer membrane (Figure 4D). Similar damage of the inner or outer membranes of E. coli was detected in the presence of durancin 61A, and cells presented blurred boundaries and highly altered cell walls (Figure 4E,F). In addition, durancin 61A induced the formation of mesosome-like structures (Figure 4E) and a very fragmented and heterogeneous electron density of the cytoplasm (Figure 4F).

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DISCUSSION Due to their ability to survive adverse environmental conditions such as extreme pH, temperatures, and salinity, enterococci are most frequently present in food products. Several enterococci were found to produce broad-spectrum bacteriocins, which makes them interesting candidates for the food industry.1 Previously, we have isolated five antilisterial enterococci strains 3587


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Figure 3. (A) Inhibitory action of durancin 61A against Listeria innocua HPB13 in the absence (circle) or presence of 5× (squares) and 10× (triangles) the MIC. (B) Ion efflux from L. innocua HPB13 in the presence of durancin 61A at 1× and 5× the MIC.

Figure 4. Transmission electron microscopy: (A) untreated Listeria innocua HPB13; (B, C) L. innocua HPB13 in the presence of durancin 61A; (D) untreated Escherichia coli ATCC 35695; (E, F) E. coli ATCC 35695 in the presence of durancin 61A.

The glycosylated durancin 61A exhibited a broad-spectrum inhibition against Gram-positive and Gram-negative foodborne pathogens including Listeria sp., C. perfringens, S. aureus, S. pyogenes, E. coli, S. choleraesuis, and P. aeruginosa. The presence

of inhibitory activity against Gram-negative bacteria is in agreement with reported data on enterococcal bacteriocins.17,22−24 In addition, durancin 61A was highly antifungal against spores of A. versicolor and P. commune. Previously, Belguesmia et al.25 3588


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Journal of Agricultural and Food Chemistry Notes

have reported antifungal activity of E. durans A5-11 bacteriocins (durancin A5-11a and A5-11b) against Fusarium culmorum, Penicillium roqueforti, and Debaryomyces hansenii. The glycosylated durancin 61A at 15.6 μg/mL (10× the MIC) was highly bactericidal against L. innocua HPB13. Whereas glycosylated glycocin F and enterocin F4-9 were bacteriostatic,21,22 durancin 61A’s mode of action is clearly bactericidal. As demonstrated by TEM, durancin 61A’s mechanism of action includes pore and vesicle formation followed by cell disruption and loss of membrane integrity and cytoplasm content. The potency of durancin 61A may result from induced efflux of K+ and Na+ ions and membrane lysis. The data presented herein would seem to indicate that durancin 61A activity is dependent on pore formation but does not exclude the presence of another (receptor-mediated) mechanism. A similar bactericidal mode of action was previously reported for enterocins L50A and L50B14 and enterocin 012.26 E. durans 61A produces simultaneously three different bacteriocins EntL50A, EntL50B, and durancin 61A. In addition, the strain produces both formylated and nonformylated forms of enterocins L50A and L50B. Multibacteriocin production by enterococci is a widely reported phenomenon.3−6 Enterococci are most frequently present in many traditional food products. Several enterococci were found to produce multiple and broad-spectrum bacteriocins. The present study provides evidence that E. durans 61A produces simultaneously formylated and nonformylated forms of leaderless enterocins L50A and L50B as well as 61A, a new glycosylated durancin. Formylation was found to increase enterocin antimicrobial activity. Durancin 61, a new glycosylated bacteriocin, exhibits broad-spectrum inhibition against Gram-positive and Gramnegative bacteria and fungal spores. Durancin 61A is bactericidal and seems to act on the bacterial membrane with a pore formation mechanism. Further investigation of durancin 61A’s structure and safety is required before possible consideration in the food and biomedical sectors. Multiple bacteriocin production by E. durans 61A is an interesting feature in preventing the growth of undesired bacteria rather than the use of a single bacteriocin producer. E. durans 61A is free from virulence genes and could be considered a good candidate for application in food preservation.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Isabelle Kelly (Proteomics Platform, CHUQ) and Pascal Dubé (INAF) for their assistance in mass spectrometry analyses.

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(1) Franz, C. M. A. P.; van Belkum, M. J.; Holzapfel, W. H.; Abriouel, H.; Galvez, A. Diversity of enterococcal bacteriocins and their grouping in a new classification scheme. FEMS Microbiol. Rev. 2007, 31, 293−310. (2) Hammami, R.; Fernandez, B.; Lacroix, C.; Fliss, I. Anti-infective properties of bacteriocins: an update. Cell. Mol. Life Sci. 2013, 70, 2947−2967. (3) Cintas, L. M.; Carmen Herranz, P. C.; Sigve Håvarstein, L.; Holo, H.; Hernández, P. E.; Nes, I. F. Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the Sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q. J. Bacteriol. 2000, 182, 6806−6814. (4) Izquierdo, E.; Marchioni, E.; Aoude-Werner, D.; Hasselmann, C.; Ennahar, S. Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer, against Listeria monocytogenes. Food Microbiol. 2009, 26, 16−20. (5) Ishibashi, N.; Himeno, K.; Fujita, K.; Masuda, Y.; Perez, R. H.; Zendo, T.; Wilaipun, P.; Leelawatcharamas, V.; Nakayama, J.; Sonomoto, K. purification and characterization of multiple bacteriocins and an inducing peptide produced by Enterococcus faecium NKR-5-3 from Thai fermented fish. Biosci., Biotechnol., Biochem. 2012, 76, 947− 953. (6) Liu, G.; Griffiths, M. W.; Wu, P.; Wang, H.; Zhang, X.; Li, P. Enterococcus Faecium LM-2, a multi-bacteriocinogenic strain naturally occurring in “byaslag”, a traditional cheese of Inner Mongolia in China. Food Control 2011, 22, 283−289. (7) Hanchi, H.; Hammami, R.; Kourda, R.; Hamida, J. B.; Fliss, I. Bacteriocinogenic properties and in vitro probiotic potential of Enterococci from Tunisian dairy products. Arch. Microbiol. 2014, 196, 331−344. (8) Hammami, R.; Zouhir, A.; Hamida, J. B.; Neffati, M.; Vergoten, G.; Naghmouchi, K.; Fliss, I. Antimicrobial properties of aqueous extracts from three medicinal plants growing wild in arid regions of Tunisia. Pharm. Biol. 2009, 47, 452−457. (9) Zhang, G.; Mills, D. A.; Block, D. E. Development of chemically defined media supporting high-cell-density growth of Lactococci, Enterococci, and Streptococci. Appl. Environ. Microbiol. 2009, 75, 1080−1087. (10) Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956, 28, 350−356. (11) Masuko, T.; Minami, A.; Iwasaki, N.; Majimab, T.; Nishimura, S.-I.; Lee, Y. C. Carbohydrate analysis by a phenol−sulfuric acid method in microplate format. Anal. Biochem. 2005, 339, 69−72. (12) Ruiz-Matute, A. I.; Hernandez-Hernandez, O.; RodríguezSánchez, S.; Sanz, M. L.; Martínez-Castro, I. Derivatization of carbohydrates for GC and GC−MS analyses. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2011, 879, 1226−1240. (13) El Arbi, M.; Théolier, J.; Pigeon, P.; Jellali, K.; Trigui, F.; Top, S.; Aifa, S.; Fliss, I.; Jaouen, G.; Hammami, R. Antibacterial properties and mode of action of new triaryl butene citrate compounds. Eur. J. Med. Chem. 2014, 76, 408−413. (14) Cintas, L. M.; Casaus, P.; Holo, H.; Hernandez, P. E.; Nes, I. F.; Havarstein, L. S. Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are related to Staphylococcal hemolysins. J. Bacteriol. 1998, 180, 1988−1994. (15) Batdorj, B.; Dalgalarrondo, M.; Choiset, Y.; Pedroche, J.; Métro, F.; Prévost, H.; Chobert, J. M.; Haertlé, T. Purification and

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00700. MS/MS spectra of nonformylatedand formylated forms of enterocin L50B and structure determination; MS/MS spectra of nonformylated and formylated forms of enterocin L50A and structure determination; GC-MS analysis of the sugar profile of durancin 61A with glucose and arabinose standards (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(I.F.) E-mail: ismail.fl[email protected]. *(R.H.) E-mail: [email protected]. Funding

This work was supported by the Fonds Québécois de Recherche sur la Nature et les Technologies (FQRNT) and the Ministry of Higher Education and Scientific Research, Republic of Tunisia. 3589


Article

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Simultaneous Production of Formylated and Nonformylated

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