Manipulation of charged residues within the two‐peptide lantibiotic lacticin 3147

Lantibiotics are antimicrobial peptides which contain a high percentage of post-translationally modified residues. While most attention has been paid to the role of these critical structural features, evidence continues to emerge that charged amino acids also play a key role in these peptides. Here 16 ‘charge’ mutants of the two-peptide lantibiotic lacticin 3147 [composed of Ltnα (2+, 2−) and Ltnβ (2+)] were constructed which, when supplemented with previously generated peptides, results in a total bank of 23 derivatives altered in one or more charged residues. When examined individually, in combination with a wild-type partner or, in some instances, in combination with one another, these mutants reveal the importance of charge at specific locations within Ltnα and Ltnβ, confirm the critical role of the negatively charged glutamate residue in Ltnα and facilitate an investigation of the contribution of positively charged residues to the cationic Ltnβ. From these investigations it is also apparent that the relative importance of the overall charge of lacticin 3147 varies depending on the target bacteria and is most evident when strains with more negatively charged cell envelopes are targeted. These studies also result in, for the first time, the creation of a derivative of a lacticin 3147 peptide (LtnβR27A) which displays enhanced specific activity.     

Saturation mutagenesis of selected residues of the α‐peptide of the lantibiotic lacticin 3147 yields a derivative with enhanced antimicrobial activity

The lantibiotic lacticin 3147 consists of two ribosomally synthesized and post‐translationally modified antimicrobial peptides, Ltnα and Ltnβ, which act synergistically against a wide range of Gram‐positive microorganisms. We performed saturation mutagenesis of specific residues of Ltnα to determine their functional importance. The results establish that Ltnα is more tolerant to change than previously suggested by alanine scanning mutagenesis. One substitution, LtnαH23S, was identified which improved the specific activity of lacticin 3147 against one pathogenic strain, Staphylococcus aureus NCDO1499. This represents the first occasion upon which the activity of a two peptide lantibiotic has been enhanced through bioengineering.     

Lantibiotic Reductase LtnJ Substrate Selectivity Assessed with a Collection of Nisin Derivatives as Substrates

Lantibiotics are potent antimicrobial peptides characterized by the presence of dehydrated amino acids, dehydroalanine and dehydrobutyrine, and (methyl)lanthionine rings. In addition to these posttranslational modifications, some lantibiotics exhibit additional modifications that usually confer increased biological activity or stability on the peptide. LtnJ is a reductase responsible for the introduction of d-alanine in the lantibiotic lacticin 3147. The conversion of l-serine into d-alanine requires dehydroalanine as the substrate, which is produced in vivo by the dehydration of serine by a lantibiotic dehydratase, i.e., LanB or LanM. In this work, we probe the substrate specificity of LtnJ using a system that combines the nisin modification machinery (dehydratase, cyclase, and transporter) and the stereospecific reductase LtnJ in Lactococcus lactis. We also describe an improvement in the production yield of this system by inserting a putative attenuator from the nisin biosynthesis gene cluster in front of the ltnJ gene. In order to clarify the sequence selectivity of LtnJ, peptides composed of truncated nisin and different mutated C-terminal tails were designed and coexpressed with LtnJ and the nisin biosynthetic machinery. In these tails, serine was flanked by diverse amino acids to determine the influence of the surrounding residues in the reaction. LtnJ successfully hydrogenated peptides when hydrophobic residues (Leu, Ile, Phe, and Ala) were flanking the intermediate dehydroalanine, while those in which dehydroalanine was flanked by one or two polar residues (Ser, Thr, Glu, Lys, and Asn) or Gly were either less prone to be modified by LtnJ or not modified at all. Moreover, our results showed that dehydrobutyrine cannot serve as a substrate for LtnJ.     

Fate of the two-component lantibiotic lacticin 3147 in the gastrointestinal tract

The component peptides of lacticin 3147 were degraded by alpha-chymotrypsin in vitro with a resultant loss of antimicrobial activity. Activity was also lost in ileum digesta. Following oral ingestion, neither of the lacticin 3147 peptides was detected in the gastric, jejunum, or ileum digesta of pigs, and no lacticin 3147 activity was found in the feces. These observations suggest that lacticin 3147 ingestion is unlikely to have adverse effects, since it is probably inactivated during intestinal transit.     

Mechanistic Dissection of the Enzyme Complexes Involved in Biosynthesis of Lacticin 3147 and Nisin

The thioether rings in the lantibiotics lacticin 3147 and nisin are posttranslationally introduced by dehydration of serines and threonines, followed by coupling of these dehydrated residues to cysteines. The prepeptides of the two-component lantibiotic lacticin 3147, LtnA1 and LtnA2, are dehydrated and cyclized by two corresponding bifunctional enzymes, LtnM1 and LtnM2, and are subsequently processed and exported via one bifunctional enzyme, LtnT. In the nisin synthetase complex, the enzymes NisB, NisC, NisT, and NisP dehydrate, cyclize, export, and process prenisin, respectively. Here, we demonstrate that the combination of LtnM2 and LtnT can modify, process, and transport peptides entirely different from LtnA2 and that LtnT can process and transport unmodified LtnA2 and unrelated peptides. Furthermore, we demonstrate a higher extent of NisB-mediated dehydration in the absence of thioether rings. Thioether rings apparently inhibited dehydration, which implies alternating actions of NisB and NisC. Furthermore, certain (but not all) NisC-cyclized peptides were exported with higher efficiency as a result of their conformation. Taken together, these data provide further insight into the applicability of Lactococcus lactis strains containing lantibiotic enzymes for the design and production of modified peptides.     

Structural characterization of lacticin 3147, a two-peptide lantibiotic with synergistic activity

Lantibiotics are antibacterial peptides isolated from bacterial sources that exhibit activity toward Gram-positive organisms and are usually several orders of magnitude more potent than traditional antibiotics such as penicillin. They contain a number of unique structural features including dehydro amino acid and lanthionine (thioether) residues. Introduced following ribosomal translation of the parent peptide, these moieties render conventional methods of peptide analysis ineffective. We report herein a new method using nickel boride (Ni(2)B), in the presence of deuterium gas, to reduce dehydro side chains and reductively desulfurize lanthionine bridges found in lantibiotics. Using this approach, it is possible to identify and distinguish the original locations of dehydro side chains and lanthionine bridges by traditional peptide sequencing (Edman degradation) followed by mass spectrometry. The strategy was initially verified using nisin A, a structurally well characterized lantibiotic, and subsequently extended to the novel two-component lantibiotic, lacticin 3147, produced by Lactococcus lactis subspecies lactis DPC3147. The primary structures of both lacticin 3147 peptides were then fully assigned by use of multidimensional NMR spectroscopy, showing that lacticin 3147 A1 has a specific lanthionine bridging pattern which resembles the globular type-B lantibiotic mersacidin, whereas the A2 peptide is a member of the elongated type-A lantibiotic class. Also obtained by NMR were solution conformations of both lacticin 3147 peptides, indicating that A1 may adopt a conformation similar to that of mersacidin and that the A2 peptide adopts alpha-helical structure. These results are the first of their kind for a synergistic lantibiotic pair (only four such pairs have been reported to date).     

Fate of the Two-Component Lantibiotic Lacticin 3147 in the Gastrointestinal Tract

The component peptides of lacticin 3147 were degraded by α-chymotrypsin in vitro with a resultant loss of antimicrobial activity. Activity was also lost in ileum digesta. Following oral ingestion, neither of the lacticin 3147 peptides was detected in the gastric, jejunum, or ileum digesta of pigs, and no lacticin 3147 activity was found in the feces. These observations suggest that lacticin 3147 ingestion is unlikely to have adverse effects, since it is probably inactivated during intestinal transit.     

A system for the random mutagenesis of the two-peptide lantibiotic lacticin 3147: analysis of mutants producing reduced antibacterial activities

Lantibiotics are antimicrobial peptides that contain several unusual amino acids resulting from a series of enzyme-mediated posttranslational modifications. As a consequence of being gene-encoded, the implementation of peptide bioengineering systems has the potential to yield lantibiotic variants with enhanced chemical and physical properties. Here we describe a functional two-plasmid expression system which has been developed to allow random mutagenesis of the two-component lantibiotic, lacticin 3147. One of these plasmids contains a randomly mutated version of the two structural genes, ltnA1 and ltnA2, and the associated promoter, Pbac, while the other encodes the remainder of the proteins required for the biosynthesis of, and immunity to, lacticin 3147. To test this system, a bank of approximately 1,500 mutant strains was generated and screened to identify mutations that have a detrimental impact on the bioactivity of lacticin 3147. This strategy established/confirmed the importance of specific residues in the structural peptides and their associated leaders and revealed that a number of alterations which mapped to the -10 or -35 regions of Pbac abolished promoter activity.     

Investigating the importance of charged residues in lantibiotics

Lantibiotics are antimicrobial peptides which can have a broad spectrum activity against many Gram positive pathogens. Many of these peptides contain charged amino acids which may be of critical importance with respect to antimicrobial activity. We have recently carried out an in-depth bioengineering based investigation of the importance of charged residues in a representative two peptide lantibiotic, lacticin 3147, and here we discuss the significance of these findings in the context of other lantibiotics and cationic antimicrobial peptides.     

Cross-immunity and immune mimicry as mechanisms of resistance to the lantibiotic lacticin 3147

Lantibiotics are antimicrobial peptides that possess great potential as clinical therapeutic agents. These peptides exhibit many beneficial traits and in many cases the emergence of resistance is extremely rare. In contrast, producers of lantibiotics synthesize dedicated immunity proteins to provide self-protection. These proteins have very specific activities and cross-immunity is rare. However, producers of two peptide lantibiotics, such as lacticin 3147, face the unusual challenge of exposure to two active peptides (α and β). Here, in addition to establishing the contribution of LtnI and LtnFE to lacticin 3147 immunity, investigations were carried out to determine if production of a closely related lantibiotic (i.e. staphylococcin C55) or possession of LtnI/LtnFE homologues could provide protection. Here we establish that not only are staphylococcin C55 producers cross-immune to lacticin 3147, and therefore represent a natural repository of Staphylococcus aureus strains that are protected against lacticin 3147, but that functional immunity homologues are also produced by strains of Bacillus licheniformis and Enterococcus faecium . This result raises the spectre of resistance through immune mimicry, i.e. the emergence of lantibiotic-resistant strains from the environment resulting from the possession/acquisition of immunity gene homologues. These phenomena will have to be considered carefully when developing lantibiotics for clinical application.     

Extensive post-translational modification, including serine to D-alanine conversion, in the two-component lantibiotic, lacticin 3147

Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147. In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity. The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da. Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides. Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product. Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Gram-positive bacteria.     

An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.

Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147. The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials. Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products.     

A lacticin 3147 enriched food ingredient reduces Streptococcus mutans isolated from the human oral cavity in saliva

AIMS: To isolate and characterise Streptococcus mutans from Irish saliva samples and to assess their sensitivity to a food-grade preparation of the lantibiotic, lacticin 3147, produced by Lactococcus lactis DPC3147. METHODS AND RESULTS: Saliva samples collected from children with varying oral health status were screened on Mitis Salivarius agar for the presence of pathogenic streptococci. Following selective plating, 16S rDNA sequencing and Pulsed Field Gel Electrophoresis (PFGE), 15 distinct strains of Strep. mutans were identified. These were grouped according to their relative sensitivity to lacticin 3147 which ranged from 0.78 to 6.25%; relative to a sensitive indicator strain, Lactococcus lactis ssp. lactis HP. Inhibition of indicator Strep. mutans strains from sensitive, intermediate and tolerant groupings were assessed in microtitre plate assays with increasing concentrations of lacticin 3147. The concentration of lacticin 3147 required to give 50% growth inhibition correlated with their relative sensitivities (as assayed by well diffusion methodology) and ranged from 1280 to 5120 AU ml(-1). Concentrated preparations of lacticin 3147 caused a rapid killing of Strep. mutans strains in broth. Moreover, in human saliva deliberately spiked with Strep. mutans, the pathogen was eliminated (initial inoculum of 10(5)) in the presence of 40,000 AU ml(-1) of lacticin 3147. Furthermore, a food-grade lacticin 3147 spray dried powder ingredient was assessed for the inhibition of Strep. mutans in human saliva, spiked with a strain of intermediate sensitivity, resulting in up to a 4-log reduction in counts after 20 min. CONCLUSION: A food grade preparation of lacticin 3147 was effective in the inhibition of oral Strep. mutans. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of oral streptococci by food grade preparations of lacticin 3147 may offer novel opportunities for the development of lacticin 3147 as an anti-cariogenic agent particularly in the area of functional foods for the improvement of oral health.     

Combination of hydrostatic pressure and lacticin 3147 causes increased killing of Staphylococcus and Listeria

The use of hydrostatic pressure and lacticin 3147 treatments were evaluated in milk and whey with a view to combining both treatments for improving the quality of minimally processed dairy foods. The system was evaluated using two foodborne pathogens: Staphylococcus aureus ATCC6538 and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 were performed using concentrated lacticin 3147 prepared from culture supernatant. The results demonstrated a more than additive effect when both treatments were used in combination. For example, the combination of 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction) resulted in more than 6 logs of kill. Similar results were obtained when a foodgrade powdered form of lacticin 3147 (developed from a spray dried fermentatation of reconstituted demineralized whey powder) was evaluated for the inactivation of L. innocua DPC1770. Furthermore, it was observed that treatment of lacticin 3147 preparations with pressures greater than 400 MPa yielded an increase in bacteriocin activity (equivalent to a doubling of activity). These results indicate that a combination of high pressure and lacticin 3147 may be suitable for improving the quality of minimally processed foods at lower hydrostatic pressure levels.     

Comparison of the Potency of the Lipid II Targeting Antimicrobials Nisin, Lacticin 3147 and Vancomycin Against Gram-Positive Bacteria

While nisin (lantibiotic), lacticin 3147 (lantibiotic) and vancomycin (glycopeptides) are among the best studied lipid II-binding antimicrobials, their relative activities have never been compared. Nisin and lacticin 3147 have been employed/investigated primarily as food preservatives, although they do have potential in terms of veterinary and clinical applications. Vancomycin is used exclusively in clinical therapy. We reveal a higher potency for lacticin 3147 (MIC 0.95?C3.8???g/ml) and vancomycin (MIC 0.78?C1.56???g/ml) relative to that of nisin (MIC 6.28?C25.14???g/ml) against the food-borne pathogen Listeria monocytogenes. A comparison of the activity of the three antimicrobials against nisin resistance mutants of L. monocytogenes also reveals that their susceptibility to vancomycin and lacticin 3147 changed only slightly or not at all. A further assessment of relative activity against a selection of Bacillus cereus, Enterococcus and Staphylococcus aureus targets revealed that vancomycin MICs consistently ranged between 0.78 and 1.56???g/ml against all but one strain. Lacticin 3147 was found to be more effective than nisin against B. cereus (lacticin 3147 MIC 1.9?C3.8???g/ml; nisin MIC 4.1?C16.7???g/ml) and E. faecium and E. faecalis targets (lacticin 3147 MIC from 1.9 to 3.8???g/ml; nisin MIC ??8.3???g/ml). The greater effectiveness of lacticin 3147 is even more impressive when expressed as molar values. However, in agreement with the previous reports, nisin was the more effective of the two lantibiotics against S. aureus strains. This study highlights that in many instances the antimicrobial activity of these leading lantibiotics are comparable with that of vancomycin and emphasizes their particular value with respect to use in situations including foods and veterinary medicine, where the use of vancomycin is not permitted.     

Evaluation of a spray-dried lacticin 3147 powder for the control of Listeria monocytogenes and Bacillus cereus in a range of food systems

AIMS: The potential of a powdered preparation of the bacteriocin, lacticin 3147, was investigated for the inhibition of Listeria monocytogenes and Bacillus cereus. METHODS AND RESULTS: A 10% solution of reconstituted demineralized whey powder was fermented with Lactococcus lactis DPC3147 for the generation of a lacticin 3147 containing powdered product. A 99.9% reduction in L. monocytogenes numbers occurred in the presence of the lacticin 3147 powder within 2 h in natural yogurt, and an 85% reduction was observed in cottage cheese within the same time frame. Counts of B. cereus were reduced by 80% in soup, in the presence of 1% (w/w) lacticin 3147 powder, within 3 h. CONCLUSIONS: A powdered preparation of lacticin 3147 was effective for the control of Listeria and Bacillus in natural yogurt, cottage cheese and soup. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioactive lacticin 3147 powder may find broad applications for control of Gram-positive pathogens/spoilage bacteria in a range of foods.     

Effect of bacteriocin-induced cell damage on the branched-chain amino acid transamination by Lactococcus lactis

The effect of the bacteriocin lacticin 3147 on the branched-chain amino acid transamination by Lactococcus lactis IFPL359 was investigated. The bacteriocin provokes membrane permeabilisation of the cells, rendering them non-viable but metabolically active. Free diffusion of amino acids into the cell was facilitated. In addition, membrane permeabilisation promotes further cell lysis. Both facts render the enzymes more accessible to their substrates and hence increase branched-chain amino acid transamination. This research broadens the spectrum of technological applications of lacticin 3147 in the development of cheese flavour.     

Strategy for manipulation of cheese flora using combinations of lacticin 3147-producing and -resistant cultures

The aim of the present study was to develop adjunct strains which can grow in the presence of bacteriocin produced by lacticin 3147-producing starters in fermented products such as cheese. A Lactobacillus paracasei subsp. paracasei strain (DPC5336) was isolated from a well-flavored, commercial cheddar cheese and exposed to increasing concentrations (up to 4,100 arbitrary units [AU]/ml) of lantibiotic lacticin 3147. This approach generated a stable, more-resistant variant of the isolate (DPC5337), which was 32 times less sensitive to lacticin 3147 than DPC5336. The performance of DPC5336 was compared to that of DPC5337 as adjunct cultures in two separate trials using either Lactococcus lactis DPC3147 (a natural producer) or L. lactis DPC4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious nonstarter lactic acid bacteria in cheddar cheese. Lacticin 3147 was produced and remained stable during ripening, with levels of either 1,280 or 640 AU/g detected after 6 months of ripening. The more-resistant adjunct culture survived and grew in the presence of the bacteriocin in each trial, reaching levels of 10(7) CFU/g during ripening, in contrast to the sensitive strain, which was present at levels 100- to 1,000-fold lower. Furthermore, randomly amplified polymorphic DNA-PCR was employed to demonstrate that the resistant adjunct strain comprised the dominant microflora in the test cheeses during ripening.     

Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus

Background

Two component lantibiotics, such as the plasmid-encoded lacticin 3147 produced by Lactococcus lactis DPC3147 and staphylococcin C55 produced by Staphylococcus aureus C55, represent an emerging subgroup of bacteriocins. These two bacteriocins are particularly closely related, exhibiting 86% (LtnA1 and C55α) and 55% (LtnA2 and C55β) identity in their component peptides. The aim of this study was to investigate, for the first time for any two component bacteriocins, the significance of the relatedness between these two systems.

Results

So close is this relatedness that the hybrid peptide pairs LtnA1:C55β and C55α:LtnA2 were found to have activities in the single nanomolar range, comparing well with the native pairings. To determine whether this flexibility extended to the associated post-translational modification/processing machinery, the staphylococcin C55 structural genes were directly substituted for their lacticin 3147 counterparts in the ltn operon on the large conjugative lactococcal plasmid pMRC01. It was established that the lacticin LtnA1 post-translational and processing machinery could produce functionally active C55α, but not C55β. In order to investigate in closer detail the significance of the differences between LtnA1 and C55α, three residues in LtnA1 were replaced with the equivalent residues in C55α. Surprisingly, one such mutant LtnA1-Leu21Ala was not produced. This may be significant given the positioning of this residue in a putative lipid II binding loop.

Conclusion

It is apparent, despite sharing striking similarities in terms of structure and activity, that these two complex bacteriocins display some highly dedicated features particular to either system.     

Heterologous expression of lacticin 3147 in Enterococcus faecalis: comparison of biological activity with cytolysin

Lacticin 3147 is a broad-spectrum, two-component, lanthionine-containing bacteriocin produced by Lactococcus lactis DPC3147 which has widespread food and biomedical applications as a natural antimicrobial. Other two-component lantibiotics described to date include cytolysin and staphylococcin C55. Interestingly, cytolysin, produced by Enterococcus faecalis, has an associated haemolytic activity. The objective of this study was to compare the biological activity of lacticin 3147 with cytolysin. The lacticin 3147-encoding determinants were heterologously expressed in Ent. faecalis FA2-2, a plasmid-free strain, to generate Ent. faecalis pOM02, thereby facilitating a direct comparison with Ent. faecalis FA2-2.pAD1, a cytolysin producer. Both heterologously expressed lacticin 3147 and cytolysin exhibited a broad spectrum of activity against bacterial targets. Furthermore, enterococci expressing active lacticin 3147 did not exhibit a haemolytic activity against equine blood cells. The results thus indicate that the lacticin 3147 biosynthetic machinery can be heterologously expressed in an enterococcal background resulting in the production of the bacteriocin with no detectable haemolytic activity.