Characterization of the antimicrobial phenotype ofL. salivarius DPC6502.

The peptide responsible for the antimicrobial activity ofL. salivarius DPC6502 was purified from an overnight culture of the strain using cation exchange followed by reversed-phase chromatography. Subsequent matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis revealed an associated mass of 2,782 Da in the active fractions (Fig. 1). Edman analysis revealed the N-terminal sequence KRKXHRXRVYNNGMPTGMYRYM, where “X” at positions 4 and 7 indicates blank cycles for which no amino acid derivative was detected. A homology search did not identify any similar sequences in protein databases. The purified bacteriocin peptide inhibited closely related lactobacilli, as well asStaphylococcus aureus and, to a lesser extent,Listeria innocua (Fig. 1).

Considering the unusual nature of this bacteriocin, the genome of the producing strainL. salivarius DPC6502 was sequenced to identify the corresponding gene cluster.

Characterization of the bacteriocin locus in the genome ofL. salivarius DPC6502.

Scanning of the entire draft genome sequence for a gene corresponding to the N-terminally derived amino acid sequence revealed a chromosomally located open reading frame (ORF), DLSL_0050, present on a gene cluster which was absent from the publically availableL. salivarius genome sequences. The unidentified amino acids at positions 4 and 7 in the sequence determined by Edman degradation were, on the basis of the corresponding codons, found to be lysine and cysteine residues, respectively (Fig. 2). The bacteriocin structural gene consists of 159 nucleotides and is predicted to encode a 53-amino-acid (aa) precursor peptide comprised of a 31-aa double-glycine leader sequence and a 22-aa propeptide sequence which differed from that derived by peptide sequencing with respect to the two most C-terminally located residues (Fig. 2). The predicted mass of the mature translation product (2,785 Da) differed from that determined by MALDI-TOF MS analysis of the active peptide (2,782 Da) by 3 Da. MS analysis revealed an increase of 2 Da (2,784 Da) in the mass of the active peptide when the cysteine residues were in a reduced state (after treatment with dithreitol [DTT] and iodoacetamide), indicating that an intramolecular disulfide bond is formed between Cys7 and Cys22. A search of the BLAST database did not identify any homologues for this ORF, indicating that this peptide is unlike any previously characterized bacteriocins. However, a search of the antimicrobial peptide database (29) revealed that bactofencin A shares the greatest similarity (42%) with a plant antimicrobial peptide (AMP) isolated from extracts of the seed ofImpatiens balsamina (Ib-AMP3) which displays both antibacterial and antifungal properties (30). In consequence of the greater similarity of this bacteriocin to cationic peptides of eukaryotic origin, it was designated bactofencin A. Despite its similarity to Ib-AMP3, neitherL. salivarius DPC6502 nor the purified bactofencin A peptide exhibited antifungal activity against the indicator strains investigated (data not shown).

The ORF immediately downstream from the bacteriocin structural gene (bfnA) encodes a putative product of 396 aa which shares 74% identity with an operon-encodedd-alanyl transfer protein (DltB) ofPediococcus pentosaceus ATCC 25745 (accession no.YP_805052) and 65% identity with the operon-encoded DltB ofL. salivarius UCC118 (LSL_0891). DltB is a transmembrane protein responsible for the transfer of activatedd-alanine across the cytoplasmic membrane, which is indispensable for thed-alanyl esterification of teichoic acids (31,32). Interestingly,d-alanylation of teichoic acids has previously been found to contribute to bacterial resistance to cationic antimicrobial peptides due to the resultant reduction in the net negative charge generated in the bacterial cell wall, thereby attenuating essential electrostatic interactions (32–36). A second DltB-encoding gene (DLSL_0976), present in adlt operon (DLSL_0974 to DLSL_0978), is also located on the chromosome ofL. salivarius DPC6502, the product of which shares 99% identity with DltB of UCC118 (encoded by LSL_0891) and 65% identity with the deduced protein product of DLSL_0051. The ORFs downstream from thedltB homologue encode a putative bacteriocin ABC transporter (DLSL_0052) and a bacteriocin transport accessory protein (DLSL_0053). The deduced protein sequence of the ABC transporter contains an N-terminal peptidase C39 domain of 139 aa which contains a conserved cysteine motif and histidine motif characteristic of the putative catalytic site responsible for the cleavage of double-glycine leader sequences (37,38). Thus, these genes encode proteins which are probably responsible for the processing and secretion of mature active bactofencin A. This putative bacteriocin transport system likely completes the bacteriocin gene cluster (approximately 4 kb), as the adjacent ORFs, which are conserved in UCC118 (LSL_0035 to LSL_0042), display similarity to the WalRK (YycGF) regulon responsible for the regulation of bacterial cell wall metabolism of low-G+C Gram-positive bacteria (39).

Bactofencin A is active at micromolar concentrations.

The identification of the structural gene and deduced peptide sequence facilitated the production of synthetic bactofencin A. MS analysis revealed a mass of 2,784 Da for the synthetic form of the peptide, which displayed anti-Listeria and anti-S. aureus activity that was similar to that of the purified natural bactofencin A peptide (data not shown). This suggests that the disulfide bond of the natural form of bactofencin A is not crucial for activity. In consequence of this and of the fact that the synthetic approach provided access to larger quantities of peptide, the synthetic bactofencin A peptide was employed for further antimicrobial activity assays, which on this occasion took the form of more-sensitive broth-based MIC₅₀ assays. These investigations revealed that, although concentrations of up to 50 µM of synthetic bactofencin A did not inhibitEscherichia coli,Cronobacter sakazakii,Salmonella, or any of the fungal indicator strains employed, concentrations of 1 to 5 µM were sufficient to inhibit the growth of bothS. aureus DPC5246 andL. monocytogenes NCTC 11994 by 50% (Fig. 3). Furthermore, the bactericidal effect of bactofencin A was demonstrated by a 4-log reduction in viableS. aureus DPC5246 cells following an 8-h incubation with the bacteriocin (Fig. 4).

A DltB homologue is responsible for bactofencin A-specific immunity.

Typically, the unmodified class II bacteriocin structural genes are cotranscribed with an ORF encoding a cognate immunity protein, usually 50 to 150 aa in size and located downstream of the structural gene, which provides self-protection for the producing strain. To determine whether the DltB homologue encoded by the ORF immediately downstream ofbfnA, designatedbfnI, has a role in bactofencin A immunity, an expression plasmid (pEOS01) harboringbfnI was constructed. When introduced into the sensitive indicator strainsL. monocytogenes NCTC11994 andS. aureus DPC5246, a statistically significant increase in resistance to bactofencin A, relative to the resistance of the corresponding isogenic control strains harboring the pNZ44 plasmid, was evident for transformants of theS. aureus DPC5246 indicator strain (Fig. 5) but not for those of theL. monocytogenes NCTC11994 strain (data not shown). Moreover, introducing the pEOS01 construct into the more-similar, yet sensitive strainL. salivarius UCC118 resulted in almost-complete immunity to the bacteriocin for the corresponding transformants relative to the control (Fig. 5B). However, this resistance was specific for bactofencin A, as these clones did not exhibit enhanced resistance when exposed to a range of cationic antimicrobial peptides, suggesting that the protection provided is not due to a general impact on cell wall charge (Fig. 6).

The results of cytochromec binding assays also correlated with these results. Cytochromec is a highly positively charged protein (pI 10), readily detected at 530 nm, whose binding is dependent on the net negative cell surface charge. In a control study, following a 10-min exposure, the level of unbound cytochromec detected in the cell-free supernatant (CFS) of thedltA-deficientS. aureus Sa113 mutant was considerably lower than the level in the CFS of the wild-type strain. This indicates an increase in the net negative cell surface charge of the mutant relative to that of the wild-type strain, due to the associated decrease ind-alanylation of teichoic acids (Fig. 7). However, the affinity for cytochromec ofS. aureus DPC5246 orL. salivarius UCC118 containing pEOS01 did not differ from that of the respective control (Fig. 7).

The bactofencin A locus is a novel hypervariable gene cluster characteristic ofL. salivarius of porcine origin.

The production of similar or identical bacteriocins is frequent among genetically distinct strains or even species of lactic acid bacteria (LAB) (6,40–42). Indeed, the genetic determinants for abp118-like bacteriocins are among the relatively high content of hypervariable gene clusters ofL. salivarius (6,21,43). To investigate whether the bactofencin A locus described in this study is similarly among the hypervariable loci of the flexible gene pool ofL. salivarius, we investigated eight additionalL. salivarius isolates, four of human and four of porcine intestinal origin (Table 1), for the presence of the bactofencin A structural gene. A PCR product corresponding to this gene could not be generated from the genomic DNA of the four strains of human origin, namely, DPC6488, DPC6196, DPC6107, and DPC6095. Cross-immunity assays also revealed that each of these isolates was sensitive to the bactofencin A-producing strain. However, all four additional porcine strains (L. salivarius DPC6005, DPC6027, DPC6189, and 7.3) were immune to bactofencin A and PCR positive forbfnA. Sequencing of the PCR products generated confirmed 100% identity with the bactofencin A structural gene of DPC6502 in each case. Subsequent purification and MS analysis confirmed bactofencin A production in these strains.

Bacterial and fungal strains and culture conditions.

TheL. salivarius strains used in this study are listed inTable 1. All lactobacilli were routinely cultured under anaerobic conditions at 37°C in MRS medium (Difco Laboratories, Detroit, MI), unless otherwise stated. Anaerobic conditions were maintained with the use of anaerobic jars and Anaerocult A gas packs (Merck, Darmstadt, Germany). TheEscherichia coli,Cronobacter (Enterobacter),Listeria, and staphylococcal indicator strains employed for antimicrobial characterization (Table 1) were grown aerobically at 37°C in brain heart infusion (BHI) medium (Merck).E. coli XL-1 Blue, used as an intermediate host for DNA manipulations, was also grown aerobically at 37°C in LB medium (Merck). Chloramphenicol (Sigma, Poole, United Kingdom) was used in selective medium, being added at concentrations of 20 µg ml⁻¹ (Escherichia coli) and 5 µg ml⁻¹ (Staphylococcus aureus,L. monocytogenes, andL. salivarius strains). The indicator strains employed for antifungal characterization includedSaccharomyces cerevisiae,Aspergillus niger,Botrytis cinerea,Geotrichium candidum,Penicillium notatum, andRhizopus stolonifer. These were maintained aerobically on potato dextrose medium (Merck) at 25°C.

Purification of the hydrophilic antimicrobial peptide produced byL. salivarius DPC6502.

The antimicrobial peptide was purified from a 2-liter overnight culture ofL. salivarius DPC6502 grown in MRS medium. The cells were removed by centrifugation at 8,000 ×g for 15 min, and the supernatant applied to a column containing 90 ml SP Sepharose fast-flow cation-exchange resin (GE Healthcare, United Kingdom) previously equilibrated with 20 mM potassium phosphate buffer, pH 2.5, containing 25% acetonitrile. The column was washed with 20 mM potassium phosphate buffer, pH 2.5, containing 25% acetonitrile, 600 mM KCl, and the bioactive peptide was subsequently eluted from the column using 20 mM potassium phosphate buffer, pH 2.5, containing 25% acetonitrile, 1 M KCl. Acetonitrile was removed by rotary evaporation before the sample was applied to a 5-g Strata-E C₁₈ SPE column (Phenomenex, Cheshire, United Kingdom) preequilibrated with methanol and water. The column was washed with distilled water, and the peptide eluted with 70% (vol/vol) propan-2-ol containing 0.1% (vol/vol) trifluoroacetic acid (TFA). The propan-2-ol was removed by rotary evaporation, and 2- by 4-ml aliquots of the resultant preparation were applied to a Jupiter proteo reversed-phase high-performance liquid chromatography (RP-HPLC) column (250.0 by 10.0 mm, 4-µm particle size, 90-Å pore size; Phenomenex). The column was preequilibrated with 10% (vol/vol) acetonitrile containing 0.1% (vol/vol) TFA, followed by separation and elution of the bioactive peptide by gradient RP-HPLC using 0.1% (vol/vol) TFA and acetonitrile concentrations that ranged from 10% to 30% (vol/vol), over a period of 5 to 45 min at a flow rate of 2.5 ml min⁻¹. Absorbance was monitored at a wavelength of 214 nm.

Bacteriocin activity was monitored throughout the purification procedure by well diffusion assay using the sensitive indicator strainLactobacillus delbrueckii subsp.bulgaricus LMG6901. MALDI-TOF MS (Axima-TOF²; Shimadzu Biotech, Manchester, United Kingdom) analysis was performed on bioactive fractions as described previously (48). Fractions of interest were further purified by reapplying them to the RP-HPLC column under the conditions described above. N-terminal sequence analysis of the purified antimicrobial peptide by Edman degradation was performed by Aberdeen Proteomics (University of Aberdeen, Scotland). Reduction and alkylation of the cysteine residues were performed using DTT and iodoacetamide (Sigma).

Genome sequencing and analysis.

The sequence of the genomic DNA extracted fromL. salivarius DPC6502 was determined through random shotgun pyrosequencing (Beckman Coulter Genomics, United States). The draft genome assembly was processed using the annotation software package GAMOLA (49). The gene model was determined with Gene Locator and Interpolated Markov ModelER (GLIMMER) 3.02 (50). Sequence similarity analyses were performed using the gapped BLASTp algorithm and the nonredundant database provided by NCBI (ftp://ftp.ncbi.nih.gov/blast/db) (51). The Artemis Comparison Tool (ACT) (52) facilitated comparative analysis of the draft genome assembly ofL. salivarius DPC6502 with the complete genome sequence ofL. salivarius UCC118 (28). The genome annotation was manually verified also using the ARTEMIS package. Global whole-genome and megaplasmid alignments were performed using stretcher, available at the EMBOSS server (http://emboss.sourceforge.net/) (53).

Generation of a synthetic analogue of bactofencin A.

The bactofencin A peptide was synthesized according to the deduced amino acid sequence of the chromosomally located bacteriocin structural gene (DLSL_0050) using microwave-assisted solid-phase peptide synthesis (MW-SPPS) performed on a CEM Liberty microwave peptide synthesizer using an H-Cys-HMPB-ChemMatrix resin (PCAS Biomatrix, Inc., Quebec, Canada). The synthetic peptide was purified by RP-HPLC as described above. Fractions containing a peptide with the desired molecular mass, as identified by MALDI-TOF MS, were pooled and lyophilized using a Genevac HT 4× evaporator (Genevac Ltd., Ipswitch, United Kingdom). The peptide was dissolved in 70% (vol/vol) propan-2-ol at a concentration of 5 mg ml⁻¹ and stored at −20°C. Appropriate dilutions of the peptide in 50 mM potassium phosphate buffer, pH 6.8, were used to determine the specific activity of the synthetic analogue.

Specific activity determination.

A microtiter plate assay system was used to determine the MIC₅₀ values of the synthetic bactofencin A analogue for a range of pathogenic indicator strains (Table 1). The microtiter plate was first treated with bovine serum albumin (BSA) to prevent adherence of the peptide to the sides of the wells, as described previously (54). Each plate included triplicate assays at each concentration of synthetic bactofencin A examined. Each well contained a total volume of 200 µl, comprised of purified bactofencin A, the first component added to the well, and 150 µl of a 1-in-10 dilution of the indicator culture (A₅₉₀ of 0.1) in BHI broth. Control wells contained medium only (blanks) and untreated indicator culture. The microtiter plate cultures were incubated at 37°C for 24 h, and the optical densities at 590 nm (OD₅₉₀) recorded at 30-min intervals (GENios plus; Tecan, Switzerland). Triplicate readings were averaged, and blanks were subtracted from these readings. The amount of bacteriocin that inhibited the indicator strain by 50% was defined as 50% of the final OD₅₉₀ reading ± 0.05 of the untreated control culture.

To determine whether the antimicrobial activity of bactofencin A is bactericidal or bacteriostatic in nature, the effect of bactofencin A onS. aureus DPC5246 was further investigated. Three independent cultures ofS. aureus DPC5246 were grown overnight at 37°C. 3 replicate 1-ml volumes of sterile double-strength BHI broth medium were inoculated with the test organism to give initial cell numbers of approximately 10⁶ CFU/ml. Bactofencin A was added, and the volume made up to 2 ml with sterile distilled water. The bacteriocin was omitted from the control and its volume replaced with sterile water. Samples were removed at intervals, serially diluted, plated on BHI agar, and then incubated aerobically at 37°C.

DNA manipulations, transformation, and plasmid construction.

The DltB homologue encoded by DLSL_0051, designatedbfnI, was amplified by routine PCR using velocity DNA polymerase (Bioline, United Kingdom) and the primer pairbfnIF, 5′ CGGGGTACCCCAGGAATGTATCGTTGGTGC 3′, andbfnIR, 5′ CGAGCTCGTTACATACAATTGCAACCATAC 3′, containing the restriction sites for the KpnI and SacI enzymes (underlined). Digestion facilitated insertion of the resultant product downstream from the constitutive p44 lactococcal promoter in the expression vector pNZ44 (55) to generate pEOS01. The integrity of the insert was confirmed by DNA sequencing using the primer pair pNZ44_for, 5′ TTACAGGTACATCATTCTGTTTGT 3′, and pNZ44_rev, 5′ TGTTTTAACGATTATGCCGATAAC 3′, specific for the pNZ44 multiple cloning site (56). Restriction enzymes and T4 DNA ligase (New England Biolabs, Beverly, MA) were used according to the manufacturer’s instructions. PCR products were purified using the Isolate PCR and gel kit (Bioline), and the Qiagen plasmid minikit (Qiagen, West Sussex, United Kingdom) was used to isolate plasmid DNA fromE. coli transformants.E. coli,S. aureus,L. monocytogenes, andL. salivarius were transformed by electroporation in accordance with methods described previously (57–60). The sensitivities of the clones were compared to those of the control strains using the microtiter well-based assay system described above and also by well diffusion assay using 2-fold serial dilutions of bactofencin A prepared in 50 mM potassium phosphate buffer, pH 6.8. Similarly, the clones and their respective controls were assayed against the cationic antimicrobial peptides magainin II, polymyxin B, protegrin-1, ε-polylysine, and nisin, as well as gramicidin D, an antimicrobial of neutral charge.

Comparison of relative net cell surface charges.

The cytochromec binding affinity of thebfnI clones and their respective isogenic controls were measured as described previously (32). Briefly, mid-log-phase cells (with an OD₆₀₀ of 0.5) were harvested and washed twice with 20 mM MOPS (morpholinepropanesulfonic acid), pH 7. The cells were then resuspended in 0.5 ml 20 mM MOPS, pH 7, and cytochromec from equine heart (Sigma) was added at a final concentration of 0.5 mg ml⁻¹. Following a 10-min incubation at room temperature, the cells were pelleted and the unbound cytochromec present in the supernatant was quantified spectrophotometrically at 530 nm. A control consisting of 0.5 mg ml⁻¹ cytochromec in 20 mM MOPS, pH 7 (buffer without cells), was also included as a reference. The absorbance measurements obtained compared with that of the reference were calculated as the absorption ratios (reflecting the bacterial surface charge). AdltA-deficientS. aureus Sa113 mutant and the corresponding parent strain (32) served as experimental controls. The data are representative of at least three independent studies.

Detection ofbfnA.

The presence ofbfnA was determined by PCR using template DNA from eight additional genetically distinct intestinalL. salivarius isolates (Table 1) and the primer pairbfnAF, 5′ CAGTCGACAATGATCATGATGGAGTAGCG 3′, andbfnAR, 5′ GGAAGTAAGTAGGGTTTTGATAAGGTGTC 3′, to amplify a product of 430 nucleotides. This was performed using the Expand high-fidelity PCR system (Roche) according to the manufacturer’s instructions. The products derived from PCR were purified and sequenced (Beckman Coulter Genomics), and analysis of DNA sequence data was performed using Lasergene 8 software (DNASTAR, Inc., Madison, WI). Template DNA ofL. salivarius DPC6502 andL. salivarius UCC118 were used as positive and negative controls, respectively. Subsequent purification and MS analyses, as described above, further confirmed positive results.

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