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REVIEWS BACTERIOCINS: DEVELOPING INNATE IMMUNITY FOR FOOD Paul D. Cotter*, Colin Hill* and R. Paul Ross‡ Abstract | Bacteriocins are bacterially produced antimicrobial peptides with narrow or broad host ranges. Many bacteriocins are produced by food-grade lactic acid bacteria, a phenomenon which offers food scientists the possibility of directing or preventing the development of specific bacterial species in food. This can be particularly useful in preservation or food safety applications, but also has implications for the development of desirable flora in fermented food. In this sense, bacteriocins can be used to confer a rudimentary form of innate immunity to foodstuffs, helping processors extend their control over the food flora long after manufacture. F O O D M I C RO B I O LO G Y BACTERIOCINS Bacterially produced, small, heat-stable peptides that are active against other bacteria and to which the producer has a specific immunity mechanism. Bacteriocins can have a narrow or broad target spectrum. Alimentary Pharmabiotic Centre, *Microbiology Department, University College Cork, Cork, Ireland. ‡ Moorepark Biotechnology Centre, Teagasc, Cork, Ireland. Correspondence to C.H. e-mail: [email protected] doi:10.1038/nrmicro1240 Perhaps the oldest and most widespread antimicrobial strategy in living systems is the use of antimicrobial peptides by the innate immune systems of many forms of life, from insects to plants to humans. Antimicrobial peptides can be fast acting and have broad-spectrum activity, which diminishes the possibility of resistance developing in target species. One example of antimicrobial peptides are the defensins, such as the α-defensins, which are produced by neutrophils in the small intestine and which act to reinforce the physical barriers of the mucosal surface. However, the production of antimicrobial peptides is not confined to multicellular organisms. BACTERIOCINS are ribosomally synthesized antimicrobial peptides produced by one bacterium that are active against other bacteria, either in the same species (narrow spectrum), or across genera (broad spectrum) and, as with host defence peptides1, cell signalling mechanisms can also be involved. Producer organisms are immune to their own bacteriocin(s), a property that is mediated by specific immunity proteins. It has been suggested that between 30–99% of the Bacteria and Archaea make at least one bacteriocin2,3, although genome analyses should ultimately provide a more definitive figure4,5. Many bacteriocins are produced by food-grade lactic acid bacteria (LAB), and this offers the possibility of manipulating food microbial ecosystems in a deliberate fashion — for NATURE REVIEWS | MICROBIOLOGY example, by using bacteriocins to protect food against contamination with, or prevent the growth of, specific pathogenic bacteria. Alternatively, narrow-host-range bacteriocins can be used to influence a microbial population in a specific ecosystem towards a particular outcome in which a desirable strain predominates (competitive exclusion). In this sense, bacteriocins can potentially be used as a form of innate immunity in food to augment physical, physiological (for example, pH) and chemical preservatives, and to influence the probable final population in complex food systems. The first description of bacteriocin-mediated inhibition was reported 80 years ago, when antagonism between strains of Escherichia coli was first discovered6. Originally called ‘colicins’, to reflect the original producer organism, gene-encoded antimicrobial peptides produced by bacteria are now referred to as ‘bacteriocins’. Although the deliberate use of bacteriocins as preservatives in food was formally proposed in 1951 REF. 7, it is likely that mankind has benefited from the serendipitous production of bacteriocins in food in the 8,000 years since cheese and other fermented foods were first manufactured. Many studies have revealed that the LAB used in cheese manufacture to convert lactose to lactic acid also produce bacteriocins that can influence the composition of the complex cheese microflora and potentially inhibit adventitious spoilage or pathogenic bacteria. Importantly, in 1928 VOLUME 3 | O CTOBER 2005 | 777 © 2005 Nature Publishing Group REVIEWS Timeline | Milestones in the commercial development of nisin Colicins first described6. 1925 1928 LAB bacteriocins first described8. Nisin discovered9. 1933 Nisin named10. LANTIBIOTIC Lanthionine-containing bacteriocins. Lanthionines are formed when a dehydrated serine or threonine is covalently bridged (through the sulphur atom) with a cysteine. BACTERIOLYSINS Large, heat-labile, lytic proteins produced by bacteria. LIPID II Lipid II (Undecaprenylpyrophosphate– MurNAc(pentapeptide)– GlcNAc) has an essential role in peptidoglycan synthesis by translocating the peptidoglycan subunits across the cell membrane. Lipid II is also the target of vancomycin, the drug of last resort in the treatment of MRSA. 778 | O CTOBER 2005 1947 Nisin first marketed in England. European Union approval11. 1953 1983 1969 Food and Agriculture Organization/ World Health Organization approval. 1988 US Food and Drug Agency approval. it was observed that certain lactococcal strains had an inhibitory effect on the growth of other LAB8. A proteinaceous antimicrobial was also described in 1933 in New Zealand9 and, in 1947, the responsible bacteriocin was named nisin (or group N inhibitory substance)10 TIMELINE. Nisin was first marketed in England in 1953 and has since been approved for use in over 48 countries. Notably, nisin was assessed to be safe for food use by the Joint Food and Agriculture Organization/ World Heath Organization Expert Committee on Food Additives in 1969. In 1983, this bacteriocin was added to the European food additive list as number E234 REF. 11 and, in 1988, it was approved by the US Food and Drug Agency (FDA) for use in pasteurized, processed cheese spreads. The successful development of nisin from an initial biological observation through regulatory approval to commercial application is a model that has stimulated a marked resurgence in bacteriocin research in recent years, but similar success with other bacteriocins has yet to be repeated on the same scale. In spite of this sobering fact, we remain convinced that bacteriocins can be exploited in food in various imaginative and commercially important applications. However, to fully realize this potential, it is first necessary to understand the biology of bacteriocins, and, in particular, to elucidate their structure–function relationships, production, immunity, regulation and mode of action. Biology of bacteriocins Bacteriocins can be defined as bacterially produced, small, heat-stable peptides that are active against other bacteria and to which the producer has a specific immunity mechanism. The bacteriocins that will probably have the most immediate potential in food applications will be those produced by food-grade LAB, as they are more likely to meet with regulatory approval owing to their origin, and they can be readily introduced into fermented foods without any concentration or purification. Bacteriocins that are produced by LAB can be broad or narrow spectrum, but in general, activity is directed against low-GC Gram-positive species. Activity against Gram-negative bacteria has been shown, but usually only in situations where the integrity of the outer membrane has been compromised. | VOLUME 3 Bacteriocins are a heterogeneous group of peptides and proteins and as many as five main classes of LAB bacteriocins have been mooted12–14. We propose that this classification scheme should be revised and suggest such a revision in TABLE 1. The proposed revised classification scheme divides the bacteriocins into two distinct categories: the lanthionine-containing LANTI BIOTICS (class I) and the non-lanthionine-containing bacteriocins (class II), while moving the large, heatlabile murein hydrolases (formerly class III bacteriocins) to a separate designation called ‘BACTERIOLYSINS’. It has recently been proposed that circular LAB bacteriocins should be regarded as class V bacteriocins14. We suggest that these should be included in the nonlanthionine-containing class II category. Class IV was a classification reserved for bacteriocins that require non-proteinaceous moieties for activity, but no members of this class have been convincingly demonstrated as yet, and so we have not included this class in the new proposal. Class I bacteriocins (lantibiotics). The lantibiotics (lanthionine-containing antibiotics) are small peptides (19–38 amino acids in length) that possess the eponymous lanthionine or β-methyllanthionine residues (FIG. 1). These unusual residues form covalent bridges between amino acids, which results in internal ‘rings’ and gives lantibiotics their characteristic structural features. Furthermore, lantibiotics can contain other unusual residues that result from post-translational modification, including the substitution of d-alanines for l-serines15–17. The lantibiotics can be divided on the basis of their structure and mode of action. In general, the elongated amphiphilic cationic lantibiotics (for example, nisin) are active through the formation of pores, leading to the dissipation of membrane potential and the efflux of small metabolites from sensitive cells. By contrast, the globular lantibiotics (for example, mersacidin) were originally defined as those lantibiotics that act through enzyme inhibition18. However, it has now been established that nisin possesses both mechanisms of action and that others, such as lacticin 3147, a two-peptide lantibiotic, function through the cooperative activity of two lanthioninecontaining peptides. Their complex structures make the lantibiotics a difficult group to subclassify; this is illustrated by a recent suggestion that they can be subdivided into 11 subgroups based on the sequences of the unmodified pro-peptides19. Some lantibiotics are active in the single nanomolar range; that is, their activity is several orders of magnitude higher than that displayed by the antimicrobial peptides encountered in the innate immune system. Although a precise mechanism of action has not been elucidated for every lantibiotic, it is apparent that a docking molecule or target is involved, which has been established as LIPID II in several cases20–24. The binding of nisin to lipid II facilitates a dual mechanism of action involving the prevention of peptidoglycan synthesis and pore formation20,21. Nisin seems to have www.nature.com/reviews/micro © 2005 Nature Publishing Group REVIEWS Table 1 | Suggested classification scheme for bacteriocins Classification* Remarks/suggestions Examples Includes both single- and two-peptide lantibiotics; up to 11 subclasses have been proposed19 Single-peptide: nisin, mersacidin, lacticin 481; two-peptide: lacticin 3147, cytolysin Heterogeneous class of small peptides; includes pediocin-like (subclass a bacteriocins), two-peptide (subclass b bacteriocins), cyclic (subclass c; formerly class V), non-pediocin single linear peptides (subclass d) Class IIa: pediocin PA1, leucocin A; class IIb: lactacin F; class IIc: enterocin AS48, reuterin 6; class IId: lactococcin A, divergicin A Large, heat-labile proteins, often murein hydrolases Lysostaphin, enterolysin A Class I Lanthionine-containing bacteriocins/lantibiotics Class II Non-lanthionine-containing bacteriocins Bacteriolysins Non-bacteriocin lytic proteins‡ * Class IV bacteriocins (bacteriocins with non-proteinaceious moieties) are not included as no members have been demonstrated. ‡ Suggested that these are no longer considered bacteriocins (see main text). a modular structure in that several N-terminally located residues, which are also present in other nisinlike, epidermin-like and streptin-like lantibiotics, have been shown to form a binding cage that is essential for the binding of nisin to the pyrophosphate of lipid II REF. 22 . The C-terminus and intervening hinge region, which is responsible for the movement of the two termini relative to one another, are essential for pore-formation21,25. It is also thought that the individual peptides of some two-peptide lantibiotics might represent discrete receptor-binding and pore-forming modules26. The combined activity of these modules helps to explain the extraordinarily high activity of at least some lantibiotics (FIG. 2). The cinnamycin-like lantibiotics represent the only other class I bacteriocins for which a binding site — the membrane phospholipid phosphatidylethanolamine — has been identified27,28. It is pertinent to note that there are examples of bacteriocins of Gram-positive bacteria that also act as virulence factors. The enterococcal two-peptide lantibiotic cytolysin exerts activity against a broad spectrum of cell types, including a wide range of Gram-positive bacteria, eukaryotic cells such as human, bovine and horse erythrocytes, retinal cells, polymorphonuclear leukocytes and human intestinal epithelial cells, and is associated with acute, terminal outcomes in human infection29. Furthermore, another non-lanthionine-containing peptide haemolysin, streptolysin S, is involved in invasive infection by some group A streptococci30. Class II bacteriocins (non-lanthionine-containing bacteriocins). The more common non-lanthioninecontaining bacteriocins are also small (<10 kDa) heat-stable peptides but, unlike lantibiotics, they are not subject to extensive post-translational modification. The majority of class II bacteriocins are active (also in the nanomolar range) by inducing membrane permeabilization and the subsequent leakage of molecules from target bacteria (FIG. 2). Several different groupings have been suggested13,31,32, but their heterogeneous nature makes rational classification difficult. NATURE REVIEWS | MICROBIOLOGY Two types are common to all classification systems and are retained in our proposed classification scheme: the class IIa pediocin-like or Listeria-active and the class IIb two-peptide bacteriocins. Pediocin-like bacteriocins have a narrow spectrum of activity but display a high specific activity against the food pathogen Listeria monocytogenes33,34. These bacteriocins range from 37 (leucocin A and mesentericin Y105 REFS 35,36 to 48 residues (carnobacteriocin B2; REF. 37) and possess one or two disulphide bridges. Conservation between peptides is most evident in the hydrophilic, cationic N-terminal ‘pediocin box’ region, which contains the amino-acid sequence motif YGNGVXCXXXXVXV, (in which X is any amino acid)38,39, and is thought to facilitate nonspecific binding to the target surface40,41. The C-terminal domains, which are located after a hinge region, are less conserved and are thought to determine the non-listerial antimicrobial spectrum. The C-terminal domains have been used as a basis for the formation of three further subdivisions42. The indications that these termini represent distinct modules has been confirmed by the generation of hybrid class IIa bacteriocins, which confirmed that the target specificity is determined by the C-terminal domain42–44. A link has been established between the expression of the mannose permease of the phosphotransferase system EIItMan in L. monocytogenes and sensitivity to class IIa bacteriocins. Mesentericin Y105- and leucocin A-resistant mutants of L. monocytogenes display reduced expression of the mptACD genes that encode this permease45–48. Although a physical interaction between a class IIa peptide and this mannose permease has not been established, the sensitisation of Lactococcus lactis strains as a consequence of the introduction of the mpt operon49 is consistent with the theory that this permease functions as a receptor for class IIa bacteriocins. The two-peptide bacteriocins require the combined activity of both peptides with a mechanism of action that again involves the dissipation of membrane potential, the leakage of ions and/or a decrease in intracellular ATP concentrations50. These peptides display very low, if any, VOLUME 3 | O CTOBER 2005 | 779 © 2005 Nature Publishing Group REVIEWS a Ser Pro Arg Ile Thr Ser Leader Ile Ser Leu Cys OH OH Thr Pro Gly SH OH Lys Cys Gly 23 amino acids SH OH Thr Dehydration of selected serines and threonines Ser Pro Arg Ile Dhb Dha Ile Dha Leu Cys Pro Dhb Gly Lys Cys Dhb Gly SH SH Lanthionine formation Dha Cleavage Ser Pro Arg Ile Ile Dhb Leu S Ala Pro S Abu Ala Gly Ala Lys Abu β-methyllanthionine Lanthionine Gly S Leu b Nisin Ala Met Dha Ile Ile Dhb Ala Leu S Ala Abu S Gly Gly Pro Gly S Ala Lys Abu S Ala Abu Ala Asn Met Lys Abu S Ala Ser Ile His Val Dha Lys Ala His Asn Gly c Lacticin 3147 Lys Asn Ala Lacticin A1 Trp Gly Tyr Ala Asp Trp Ala Ala Dhb Asn Dhb Phe D-Ala Leu Ala S S Trp Abu His Ala Abu Ala Glu Leu S Met Ala S Lacticin A2 Thr 2-ob Dhb Pro Ala Dhb Pro Ala Ile D-Ala Ile Leu D-Ala Ala Tyr Ile Ala Asn S Thr Thr Lys Arg Ala S S Ala Pro Abu Ala Abu Ala Figure 1 | Lanthionine synthesis and lantibiotic structure. As shown in a, lanthionine residues are formed when an enzymatically dehydrated serine (dehydroalanine, Dha) condenses with the sulphydryl group of a neighbouring cysteine (Cys). This forms a bridge between the two residues, thereby creating a ring within the modified peptide or lantibiotic. When the partners are threonine (Thr) and cysteine, the novel residue is a β-methyllanthionine. The resulting lanthionine and β-methyllanthionine bridges are indicated in pink as Ala–S–Ala (alanine–S–alanine) and Abu–S–Ala (aminobutyrate–S–alanine), respectively. Many lantibiotics also contain dehydrated serines (Ser) and threonines (dehydrobutyrine, Dhb). Lantibiotics can be composed of a single peptide (nisin, panel b) or two peptides acting in synergy (lacticin 3147, panel c). The dehydration and ring formation reaction can be catalysed by two enzymes (NisB and NisC in the case of nisin) or a single enzyme (LcnM in the case of lacticin 481). Several additional modified residues can be found in lantibiotics, including lysinoalanine, lanthionine sulphoxide, D-alanine, allo-isoleucine, erythro-3-hydroxyaspartate, 2-oxopyruvate, 2-oxobutyrate, hydroxypyruvate, S-(2-aminovinyl)-D-cysteine and S-(2-aminovinyl)-3-methyl-D-cysteine. bacteriocin activity when tested individually. Although members of this subgroup are relatively heterogeneous, it has been proposed that they could be subdivided into type E (enhanced) and type S (synergistic) peptides51. The class IIc (formerly class V) bacteriocins are grouped on the basis that their N- and C-termini are covalently linked, resulting in a cyclic structure52,53. Although relatively few class IIc bacteriocins have been identified, we propose two subdivisions designated 780 | O CTOBER 2005 | VOLUME 3 subclass c(i) (comprising enterocin AS48 and the non-LAB circularin A) and subclass c(ii) (comprising gassericin A, reutericin 6, the non-LAB butyrivibriocin AR10 and, although a circular structure has not yet been established, acidocin B) on the basis of percentage amino-acid sequence identity52. The class IIc bacteriocins gassericin A and reutericin 6 are the only examples of non-lantibiotic LAB bacteriocins that contain d-amino acids54. www.nature.com/reviews/micro © 2005 Nature Publishing Group REVIEWS Class II (Sakacin) Class I (Nisin) Bacteriolysins (Lysostaphin) the N-terminus that shows homology to endopeptidases, and a C-terminus that probably represents the target recognition site62,63. Unlike the ‘true’ bacteriocins, they do not always have specific immunity genes that accompany bacteriocin structural genes, but might rely on modifications of the producer cell wall to impart resistance (see below). Molecular biology of bacteriocins Cell wall Lipid II Cell membrane Peptidoglycan subunit Figure 2 | Mode of action of lactic acid bacteria bacteriocins. Lactic acid bacteria (LAB) bacteriocins can be grouped on the basis of structure, but also on the basis of mode of action. Some members of the class I (or lantibiotic) bacteriocins, such as nisin, have been shown to have a dual mode of action. They can bind to lipid II, the main transporter of peptidoglycan subunits from the cytoplasm to the cell wall, and therefore prevent correct cell wall synthesis, leading to cell death. Furthermore, they can use lipid II as a docking molecule to initiate a process of membrane insertion and pore formation that leads to rapid cell death. A two-peptide lantibiotic, such as lacticin 3147, can have these dual activities distributed across two peptides, whereas mersacidin has only the lipid-II-binding activity, but does not form pores. In general, the class II peptides have an amphiphilic helical structure, which allows them to insert into the membrane of the target cell, leading to depolarisation and death. Large bacteriolytic proteins (here called bacteriolysins, formerly class III bacteriocins), such as lysostaphin, can function directly on the cell wall of Gram-positive targets, leading to death and lysis of the target cell. The remaining bacteriocins are usually combined in a ‘miscellaneous’ or ‘one-peptide non-pediocin linear’ group (class IId). In some instances, these have been further subdivided on the basis of leader sequences31. BACTERIOCINOGENIC Bacteriocin-producing strains are said to be bacteriocinogenic. Non-bacteriocin lytic proteins (bacteriolysins). Bacteriolysins (formerly class III bacteriocins) are large, heat-labile antimicrobial proteins. They have a domain-type structure, in which different domains have functions for translocation, receptor binding, and lethal activity. Only four LAB bacteriolysins55–59 have been genetically characterized so far, although other nonLAB bacteriolysins that are of interest to food microbiologists have been identified51,60,61. Their mechanism of action is distinct from that of bacteriocins as they function through the lysis of sensitive cells by catalysing cell-wall hydrolysis (FIG. 2). These proteins are also modular in structure and have a catalytic domain at NATURE REVIEWS | MICROBIOLOGY The widespread phenomenon of bacteriocin production among LAB is undoubtedly partly due to the fact that the relevant genes are often associated with transferable elements such as conjugative transposons or plasmids. This natural association can be exploited to facilitate heterologous bacteriocin production. For example, the 10 genes that are required for the production of, and immunity to, the two-peptide lantibiotic lacticin 3147 are located on a 60-kb conjugative plasmid, which has allowed this trait to be transferred in a non-recombinant manner from the parent strain L. lactis DPC3147 to a wide variety of commercial starter strains, by simply selecting for bacteriocin immunity as a marker for plasmid transfer64. Bacteriocins are usually synthesized as an inactive pre-peptide that includes an N-terminal leader sequence. The leader sequence presumably maintains the bacteriocin in an inactive form within the producer cell, facilitates interaction with the transporter and, probably in the case of lantibiotics, has a role in recognition by the modification machinery65–68. This leader is usually cleaved during export by a dedicated bacteriocin-transport system or, less frequently, by the general secretion (Sec) pathway of the cell. It is notable that a few bacteriocins that seem to lack leader sequences have also been identified31. The genes that encode the structural pre-peptides are usually closely associated with genes that encode products involved in regulation, export, self-immunity and — in the case of the lantibiotics — modification. The export of bacteriocins is usually achieved by a dedicated membrane-associated ATP-binding cassette (ABC) transporter that can also contain a proteolytic N-terminal domain belonging to the family of cysteine proteases that is responsible for cleavage of the leader peptide69,70. In the case of some lantibiotics, cleavage might be due to a specific serine protease71. For class II peptides, accessory proteins are thought to facilitate membrane translocation and/or leader peptide cleavage, but their specific role has yet to be established. It should be noted that there are several variations to this general scheme, which involve both N- and C-terminal72 or extracellular cleavage73,74. Regulation of bacteriocin production and immunity is most frequently mediated through two-component signal-transduction systems, often as part of a quorumsensing mechanism (FIG. 3). In certain instances, more than one response regulator can be involved75,76. Other types of regulators, such as repressors of the Xre family, have also been described77,78. BACTERIOCINOGENIC bacteria also have genes that encode immunity mechanisms. These systems enable a distinction between ‘self ’ (producer) and ‘non-self ’ VOLUME 3 | O CTOBER 2005 | 781 © 2005 Nature Publishing Group REVIEWS Enterocin A Resistance to bacteriocins Enterocin F EntD EntK EntT EntR + Pent 0 ent 1 A I F 2 K 3 R 5 6 T 7 8 kb D Figure 3 | Regulation by quorum sensing. In several instances it has been established that bacteriocin production is regulated by quorum sensing. In the case of the lantibiotics nisin and subtilin, the structural peptide itself functions as a pheromone to induce its own production to high levels once a cell-density-dependent autoinduction loop is activated163–165. Although there is an example of a class II bacteriocin acting as a pheromone144, the presence of another induction peptide, which often shows many of the physico-chemical properties of bacteriocins and can function at concentrations at low as 10–17 M REF. 166, is more common. As illustrated in the figure, the regulation of enterocin A production involves one such three-component system. The genes that encode the structural protein enterocin A (EntA) and the induction factor enterocin F (EntF) are co-transcribed from the Pent promoter and these proteins are transported by a putative ATP-binding cassette transporter (EntT) through a mechanism that might involve the accessory protein (EntD). As with other bacteriocins, the other twocomponent system involved is a membrane-bound histidine kinase (EntK), which detects extracellular EntF, and a cytoplasmically located response regulator (EntR) that mediates a response — induction of transcription from the Pent promoter, which leads to increased bacteriocin production. In some cases, additional factors such as co-culture with potential target cells167–169 or even gastric transit170 can be required for successful induction. An understanding of quorum sensing and inducible bacteriocin production has facilitated the development of systems that permit inducible overexpression of desirable proteins164,171,172. (for example, food spoilage or pathogenic target bacteria). Two distinct systems are involved in class I and class II immunity, although there are some exceptions79. Protection can be provided by a dedicated immunity protein and/or a specialised ABC-transporter system involving two or three subunits that probably pumps the bacteriocin from the producer membrane76,80–83. For class I bacteriocins, immunity is provided by one or both of these systems82, for class IIa, IIb and IId bacteriocins, immunity is provided by an immunity protein alone, and class IIc bacteriocins rely on an ABC-transporter system84. Although immunity proteins rarely resemble one another, the general mechanism through which they function is probably similar, involving either the sequestration of the structural protein or an antagonistic competition for a receptor85,86. Such immunity mechanisms are highly specific and usually do not provide protection against other bacteriocins82,87,88, although again, there are some exceptions39,89,90. It has been shown that immunity to bacteriolysins is brought about through the alteration of interpeptide cross-bridges in peptidoglycan91. 782 | O CTOBER 2005 | VOLUME 3 The potential for the development of resistance among target species is an obvious cause for concern if bacterio cins are to be used extensively in food preservation strategies. Most research in this area has focused on specific bacteriocins, such as nisin and some of the class IIa peptides. The frequency of spontaneous resistance to nisin in L. monocytogenes varies from 10–2 to 10–7 in a strain-dependent manner92, whereas stable, nisin-resistant mutants of Streptococcus pneumoniae can arise (MIC increases from 0.4 to 6.4 mg l–1) following serial exposure to the lantibiotic93. In these spontaneous mutants, resistance correlates with cell envelope changes such as alterations in membrane charge and fluidity94,95, cell wall thickness96, cell wall charge97–99 and combinations thereof 100, which arise after direct exposure to a low level of lantibiotic or as part of an adaptive response to another stress101. The specific mechanism(s) by which cells become resistant to nisin is not well understood although it is apparent that variations in the lipid II content are not responsible102. Genetic loci that are associated with the development of enhanced nisin resistance103–105, or an innate tolerance of nisin, have been identified98,106,107. In the latter example, cell envelope charge seems to be the most important consideration. The most well-studied mechanism of resistance to class II bacteriocins is that observed in class IIaresistant L. monocytogenes, in which resistance seems to be linked to reduced expression of a mannose permease of the phosphotransferase system. Similar to nisin-resistant mutants, other factors such as cell membrane fluidity108 and cell surface charge109 impact on resistance. With further research, additional mechanisms will undoubtedly become apparent. The production of a bacteriocin by a Streptococcus mutans strain is inhibited by Streptococcus gordonii through the inactivation of the corresponding induction peptide, which provides an interesting example of bacteriocin-mediated communication that involves competing species110. Further studies will be required to determine the frequency with which resistance to other bacteriocins occurs. The risk of the development of large-scale, industrially significant resistance might best be limited by the intelligent use of hurdle technology, that is, the concept of combining several factors (including pH, osmolarity and temperature) to preserve food. Although the combined use of different bacteriocins can be successful, cross-resistance to class I and class II bacteriocins has been found in some instances100,104,111. It is worth noting that bacteriocins used correctly in a hurdle approach should only have to control low levels of contaminating organisms, as they should be used only to augment, and not replace, good food manufacturing practice. This creates a different situation to the use of antibiotics in clinical settings, where these inhibitors are used to eliminate established infections and usually high numbers of target bacteria, thereby greatly increasing the probability of resistance development. www.nature.com/reviews/micro © 2005 Nature Publishing Group REVIEWS Recent developments in bacteriocin biology Many exciting developments have contributed to our understanding of bacteriocin biology and have highlighted innovative ways in which these peptides can be used; for example, cell-signalling systems involved in the induction of bacteriocin production have been investigated and used as biological tools (FIG. 3). One of the most significant developments has been the discovery that the activity of nisin is mediated by the binding of lipid II REFS 20,21. Whereas lipid II is also the target for therapeutic antibiotics such as vancomycin, it has been established that a set of core amino acids at the N-terminal domain of nisin bind to an invariable region of lipid II REFS 22,112, thereby militating against resistance like that observed in vancomycin-resistant enterococci (VRE). These results indicate that nisin could act as a template for the design of novel drugs that bind an essential, invariable antimicrobial target. Although the definitive role of EIItMan as the target or docking molecule for class IIa bacteriocins has yet to be established, an ever-improving appreciation of the modular nature of these bacteriocins will facilitate the creation of hybrid proteins with different antimicrobial spectra as a consequence of C-terminal variation42. Attempts to create bioengineered derivatives of bacteriocins have met with some rare, but notable, successes113–116. The absence of post-translational modifications in class II bacteriocins makes them more amenable to change, a fact that is best illustrated by the creation of hybrid bacteriocins43,44. Whereas bioengineered peptides are usually expressed from a derivative of the original bacteriocin-producing strain, it has been shown that class II peptides can be produced heterologously by Gram-negative bacteria or yeast117,118. Although the heterologous production of a fully modified lantibiotic by non-Gram-positive microorganisms has yet to be achieved, it has recently been shown that purified modification enzymes can be used to introduce lanthionines into synthetic peptide substrates68. We predict that by combining novel insights into structure–function relationships with enhanced mutagenesis and production technologies, the bioengineering of bacteriocins will enter a new era in which the identification of derivatives with enhanced activity will be a more frequent event. One should note, however, that although some of this newly generated knowledge will undoubtedly result in novel and exciting applications, a significant suite of applications have already been demonstrated, even if, in many instances, only at the laboratory scale. Industrial applications GRAS An acronym for substances that are ‘generally recognized as safe’. This group includes several hundred substances that experts consider safe for use in food on the basis of either a history of safe use before 1958 or on published scientific evidence. Food systems. Food processors face a major challenge in an environment in which consumers demand safe foods with a long shelf-life, but also express a preference for minimally processed products that do not contain chemical preservatives. Bacteriocins are an attractive option that could provide at least part of the solution. They are produced by food-grade organisms, they are usually heat stable and they can inhibit many NATURE REVIEWS | MICROBIOLOGY of the primary pathogenic and spoilage organisms that cause problems in minimally processed foodstuffs. However, at present, only nisin and pediocin PA1/AcH have found widespread use in food. The form of nisin used most widely in food is Nisaplin (Danisco), which is a preparation that contains 2.5% nisin with NaCl (77.5%) and non-fat dried milk (12% protein and 6% carbohydrate). The use of pediocin PA1 for food biopreservation has also been commercially exploited in the form of ALTA 2431 (Quest), which is based on LAB fermentates generated from a pediocin PA1-producing strain of Pediococcus acidilactici119. Its use is covered by several US and European patents119,120. When screening for a bacteriocin with a food application in mind, there are several important criteria: first, the producing strain should preferably have ‘generally recognized as safe’ (GRAS) status; and second, the bacteriocin should have a broad spectrum of inhibition that includes pathogens, or have activity against a particular pathogen. Third, the bacteriocin should be heat stable; fourth, have no associated health risks; fifth, its inclusion in products should lead to beneficial effects such as improved safety, quality and flavour; and sixth, it should have high specific activity121. Bacteriocins have been shown to have potential in the biopreservation of meat, dairy products, canned food, fish, alcoholic beverages, salads, egg products, high-moisture bakery products, and fermented vegetables, either alone, in combination with other methods of preservation, or through their incorporation into packaging film/food surfaces (for comprehensive reviews, see REFS 122124). Bacteriocins can be introduced into food in at least three different ways: in fermented food, bacteriocins can be produced in situ by bacterial cultures that substitute for all or part of the starter culture; purified or semi-purified bacteriocins (for example, Nisaplin) can be added directly to food; or an ingredient based on a fermentate of a bacteriocin-producing strain can be used (for example, ALTA 2431). Although bacteriocins with a wide spectrum of activity are usually the most sought after, other factors including pH optima, solubility and stability are as important and are major considerations in choosing a particular inhibitor for a particular food or target bacterium. Furthermore, the antimicrobial spectra of a variety of LAB bacteriocins can be extended to encompass Gram-negative bacteria through their use in combination with measures that affect the integrity of the outer membrane, such as temperature shock, high pressure, chelators and eukaryotic antimicrobial peptides125–133. There are also rare natural (for example, AS48 REF. 134) and bioengineered bacteriocins114 that possess inherent activity against Gram-negative microorganisms. There is also a niche for narrow-spectrum bacteriocins. This is true of fermented foods, in which contamination with L. monocytogenes is a problem. L. monocytogenes is the cause of approximately 25% of deaths caused by food-borne pathogens in the US annually135 and, as a consequence of the zero-tolerance standards for the organism in ready-to-eat (RTE) food, was responsible for 71% of all recalls of food products VOLUME 3 | O CTOBER 2005 | 783 © 2005 Nature Publishing Group REVIEWS owing to bacterial contamination in the US between 1993 and 1998 REF. 136. In mid-2003, the Food Safety and Inspection Service (FSIS) announced a ruling that required the manufacturers of RTE food to take further steps to address the problem posed by the presence of L. monocytogenes in consumables. This ruling encourages the implementation of technologies that can kill the bacteria or prevent its growth after cooking or packaging. The Listeria-active class IIa bacteriocins seem to provide an ideal solution for such applications, especially with respect to fermented foods, as any potential L. monocytogenes contamination can be negated without impacting on the success of the fermentation itself owing to the low activity of class IIa bacteriocins against lactococci39. Bacteriocins can also be used to promote quality, rather than simply to prevent spoilage or safety problems. For example, bacteriocins can be used to control adventitious non-starter flora such as non-starter lactic acid bacteria (NSLAB) in cheese and wine. The uncontrolled growth of NSLAB can cause major economic losses owing to calcium-d-lactate formation137 and slit defects in cheeses, and the production of detrimental compounds in wine. Bacteriocinproducing starters and adjuncts (one- or two-strain strategies) have been found to significantly reduce these problems138–142. However, as some NSLAB such as lactobacilli and other starter adjuncts in cheese, and Leuconostoc oenos and Pediococcus damnosus in some red wines can improve flavour, the complete elimination of NSLAB is not always desirable143. This problem has been overcome through the use of a three-strain system in which an adjunct strain with reduced bacteriocin sensitivity (obtained on repeated exposure to increasing concentrations of the bacteriocin) is used with a bacteriocin-producing starter144 (FIG. 4). Bacteriocins can also be applied in other ways to enhance food fermentation. This has been shown during semi-hard and hard cheese manufacture in which bacteriocin production brings about the controlled lysis of starter LAB, which results in the release of intracellular enzymes and ultimately accelerated ripening and even improved flavour139,140,145,146. Although traditionally, the use of bacteriocins is associated with the preservation of food, in the near future food might merely act as a vehicle for the delivery of bacteriocin-producing probiotic bacteria. The production of antimicrobials by a probiotic culture is a desirable trait as they are thought to contribute to the inhibition of pathogenic bacteria in the gut147–149. Whereas bacteriocins in food are degraded by the proteolytic enzymes of the stomach, probiotic bacteria might be ingested in a form that facilitates gastric transit, allowing the in vivo production of the bacteriocin in the small or large intestine. It has also been speculated that recombinant probiotic strains that can be induced to produce bacteriolysin could be developed to facilitate the in vivo delivery of bioactive compounds that are produced intracellularly150. Unlike most other preservation methods such as heat or low pH, which are essentially indiscriminate in 784 | O CTOBER 2005 | VOLUME 3 their antimicrobial effect, it is this ability to precisely influence the developing flora in an otherwise perishable food that led us to describe the use of bacteriocins as a form of ‘innate immunity’ for food. As already described, the inclusion of Listeria-active class IIa bacteriocins can specifically prevent the growth of this pathogen, without affecting harmless LAB, or bacteriocin-tolerant strains can be introduced into an otherwise hostile food environment. It is unlikely that the use of bacteriocins in food will negatively impact on the natural flora of either the human (or animal) host, or on the environment. The low level of bacteriocins required to eliminate or reduce small numbers of pathogenic or spoilage organisms in food are unlikely to have an impact on more microorganism-rich environments. In any event, bacteriocins are unlikely to survive gastric transit, as they are sensitive to proteolytic degradation. Clinical applications. Although this review has emphasized the role (existing and potential) of LAB bacteriocins in food, the non-toxicity of lantibiotics and their activity against Gram-positive human and animal pathogens has led to research investigating their potential clinical application. In particular, the elucidation of the precise mechanism of action of some lantibiotics and their activity against multidrugresistant pathogens by a novel mechanism makes them an attractive option as possible therapeutic agents. The broad-spectrum lantibiotics could theoretically be of use against any clinical Gram-positive human or animal pathogen. For example, the two-peptide lantibiotic lacticin 3147 has in vitro activity against Staphylococcus aureus (including methicillin-resistant S. aureus (MRSA)), enterococci (including VRE), streptococci (S. pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus mutans), Clostridium botulinum, and Propionibacterium acnes151. Initial in vivo trials with animal models have demonstrated the success of lantibiotics in treating infections caused by S. pneumoniae152, and MRSA153,154, and in preventing tooth decay and gingivitis155–159. The use of nisin for human clinical applications has been licensed to Biosynexus Incorporated by Nutrition 21 and ImmuCell Corporation has licensed the use of the anti-mastitic nisin-containing product Mast Out to Pfizer Animal Health. Bovine mastitis is defined as an inflammation of the udder and is the most persistent disease in dairy cows. Nisin is also used as an active agent in WipeOut (a teat wipe), and lacticin-3147-containing Teat Seals (Cross Vetpharm Group Ltd) have been shown to prevent deliberate infection by mastitic staphylococci and streptococci in animal challenge trials159 (FIG. 4). A strain that produces the lantibiotic mutacin 1140 is entering Phase I clinical trials in the US with a view to replacement therapy, and the dietary supplement BLIS K12 throat guard, which contains a Streptococcus salivarius that produces two lantibiotics salivaricin A2 and B, is sold in New Zealand as an inhibitor of the bacteria responsible for bad breath160. From a nonantimicrobial medical perspective, the cinnamycin-like www.nature.com/reviews/micro © 2005 Nature Publishing Group REVIEWS Bac– a Food quality Bac+ c Veterinary medicine b Food safety 80,000 Bac– S. aureus L. monocytogenes 104 103 102 Bacteriocins 20,000 10,000 Bac+ nd 1 2 3 4 5 Teat seal Number of days d Human medicine Phe Lys Ala Leu S S Asn Ala Dha Trp Ala Abu Pro Phe Gly S Ala Ala Arg Dhb Gly Ala Teat seal and lacticin 3147 CH Tyr S CH Ala N H Figure 4 | Selected applications of bacteriocins. a | Food quality. A cheese made with a commercial starter culture (Bac–) will develop an undefined flora called non-starter lactic acid bacteria (typified by different fingerprints generated by random amplified polymorphic DNA patterns). However, a cheese inoculated with the same commercial strain that can produce a bacteriocin (Bac+) (lacticin 3147, in this example) and a resistant adjunct strain of Lactobacillus, chosen for a flavour attribute, will develop a single defined culture once the starter culture has died off, offering the cheese manufacturer control over previously adventitious flora development. b | Food safety. A simple example of the role of bacteriocins in food safety is the production of cottage cheese with a starter culture that produces a bacteriocin with activity against Listeria monocytogenes, which results in a cheese that is inherently anti-Listeria. c | Veterinary medicine. A teat seal is a physical barrier against infection. Here, a bacteriocin was incorporated into the teat seal and the teat was challenged with Staphylococcus aureus. The number of staphylococci recovered from 14 teats with or without bacteriocin is shown. d | Human medicine. A Streptococcus mutans strain that cannot produce acid, but that produces the lantibiotic mutacin (shown), can competitively exclude acidogenic S. mutans, thereby offering protection against tooth decay173. lantibiotics have attracted interest owing to their novel activities against the functions of medically important specific human enzymes, such as phospholipase A2 and angiotensin-converting enzyme, and nisin has also been found to have contraceptive efficacy161,162. Conclusions For the past 50 years, bacteriocins have frequently been proposed as the solution to a myriad of foodcontamination problems, but nisin and, to a lesser extent, pediocin PA1, remain the only examples of the large-scale deliberate use of these peptides in food applications. The under-utilization of bacteriocins in food can perhaps be ascribed to a combined lack of awareness of what bacteriocins can achieve in food systems and a lack of enthusiasm to move away from existing food-preservation techniques. However, although there have been several ‘false dawns’ heralding the bacteriocin revolution, modern consumer and regulatory demands associated with minimal processing might now provide an opportunity for their more widespread application. Bacteriocin screening NATURE REVIEWS | MICROBIOLOGY programmes have yielded a large arsenal of bacteriocins with different properties, target species and producer organisms. Furthermore, if even the most conservative of estimates concerning the percentage of bacteria that produce bacteriocins are confirmed, then many other bacteriocins remain to be identified and exploited. Whether deliberately added or produced in situ by food bacteria, bacteriocins can play a beneficial role in the control of undesirable flora and in the establishment of desirable microbial populations. It is this ability of bacteriocins to direct microbial flora that can be described as a type of programmable innate immunity for food. Genomic advances will also contribute to the identification of novel bacteriocins, and enhanced understanding of regulatory mechanisms will allow us to determine how to switch on production when desired and even how to generate overproducing strains. By determining the specific activity of bacteriocins against particular spoilage/pathogenic bacteria, ensuring stability and assessing the likelihood of emerging resistance, it should be possible to pre-determine VOLUME 3 | O CTOBER 2005 | 785 © 2005 Nature Publishing Group REVIEWS which bacteriocin is best suited to a particular food application. Such an application has been exemplified by the tailored use of class IIa bacteriocins in the treatment of L. monocytogenes. From a clinical perspective, the emergence of drug-resistant pathogens makes the identification of novel antimicrobials even more important. 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Online links DATABASES The following terms in this article are linked online to: Entrez: http://www.ncbi.nlm.nih.gov/Entrez Escherichia coli | Lactococcus lactis | Leuconostoc oenos | Listeria monocytogenes | Staphylococcus aureus | Streptococcus gordonii | Streptococcus mutans | Streptococcus pneumoniae FURTHER INFORMATION Alimentary Pharmabiotic Centre: http://apc.ucc.ie/content/bacteriocins.htm Gateway to Government Food Safety Information: http://www.foodsafety.gov/~dms/lmr2-toc.html Access to this interactive links box is free online. www.nature.com/reviews/micro © 2005 Nature Publishing Group