Novel lantibiotics and their pre-peptides

In recent years there has been a considerable increase in studies of bactericidal peptides produced by Grampositive bacteria, with particular emphasis upon their potential application as food preservatives. A number of these peptides contain lanthionine and other post-translationally modified amino acid residues. The lanthionine-containing molecules (lantibiotic) appear to have evolved in two quite different lineages, type A and type B. This mini-review introduces the reader to several of the more recently described type A lantibiotics for which relatively detailed biochemical and/or genetic data has already been gathered. A wider diversity of compounds of type A lantibiotics has been described in the recent years. Novel features of some of the more recently described type A lantibiotics to be reported in this review include: a) New modifications such as D-Ala and 2-hydroxypropionyl residues, both derived from serine. b) Different types of pre-lantibiotic leader sequences. c) The apparent requirement for different numbers and types of genes for synthesis of some active type A lantibiotics. d) Cytolysin functions as both a hemolysin and a bacteriocin. e) One of the newly-described lantibiotics (lactocin S) does not have any net charge at neutral pH another (carnocin UI49) is the largest of the lantibiotics discovered and the killing action of another (cytolysin) has been shown to be depend on the interaction of two peptides.     

The dawning of a 'Golden era' in lantibiotic bioengineering

There are many examples of highly modified antimicrobial peptides in nature, many of which are non-ribosomally synthesized. However, the bacterial lantibiotics are produced as gene-encoded pre-peptides that are subsequently modified by dedicated enzyme systems to form extraordinarily potent inhibitors. Consequently, they are much more amenable to bioengineering which could lead to the generation of a new arsenal of potent antimicrobials. However, although bioengineering of these compounds has been underway for at least two decades, significant progress has only been reported in recent years. This review charts these recent developments which suggest that we are entering a 'Golden era' of lantibiotic bioengineering.     

The genetics of lantibiotic biosynthesis

The lantibiotics are a rapidly expanding group of biologically active peptides produced by a variety of Gram-positive bacteria, and are so-called because of their content of the thioether amino acids lanthionine and β-methyllanthionine. These amino acids, and indeed a number of other unusual amino acids found in the lantibiotics, arise following post-translational modification of a ribosomally synthesised precursor peptide. A number of genes involved in the biosynthesis of these highly modified peptides have been identified, including genes encoding the precursor peptide, enzymes responsible for specific amino acid modifications, proteases able to remove the leader peptide, ABC-superfamily transport proteins involved in lantibiotic translocation, regulatory proteins controlling lantibiotic biosynthesis and proteins that protect the producing strain from the action of its own lantibiotic. Analysis of these genes and their products is allowing greater understanding of the complex mechanism(s) of the biosynthesis of these unique peptides.     

Bovicin HJ50-Like Lantibiotics,a Novel Subgroup of Lantibiotics Featured by an Indispensable Disulfide Bridge

Lantibiotics are ribosomally-synthesized and posttranslationally modified peptides with potent antimicrobial activities. Discovery of novel lantibiotics has been greatly accelerated with the soaring release of genomic information of microorganisms. As a unique class II lantibiotic, bovicin HJ50 is produced by Streptococcus bovis HJ50 and contains one rare disulfide bridge. By using its precursor BovA as a drive sequence, 16 BovA-like peptides were revealed in a wide variety of species. From them, three representative novel lan loci from Clostridium perfringens D str. JGS1721, Bacillus cereus As 1.348 and B. thuringiensis As 1.013 were identified by PCR screening. The corresponding mature lantibiotics designated perecin, cerecin and thuricin were obtained and structurally elucidated to be bovicin HJ50-like lantibiotics especially by containing a conserved disulfide bridge. The disulfide bridge was substantiated to be essential for the function of bovicin HJ50-like lantibiotics as its disruption eliminated their antimicrobial activities. Further analysis indicated that the disulfide bridge played a crucial role in maintaining the hydrophobicity of bovicin HJ50, which might facilitate it to exert antimicrobial function. This study unveiled a novel subgroup of disulfide-containing lantibiotics from bacteria of different niches and further demonstrated the indispensable role of disulfide bridge in these novel bovicin HJ50-like lantibiotics.     

Mode of action of modified and unmodified bacteriocins from Gram-positive bacteria

The antibiotic activity of bacteriocins from Gram-positive bacteria, whether they are modified (class I bacteriocins, lantibiotics) or unmodified (class II), is based on interaction with the bacterial membrane. However, recent work has demonstrated that for many bacteriocins, generalised membrane disruption models as elaborated for amphiphilic peptides (e.g. tyriodal pore or carpet model) cannot adequately describe the bactericidal action. Rather, specific targets seem to be involved in pore formation and other activities. For the nisin and epidermin family of lantibiotics, the membrane-bound cell wall precursor lipid II has recently been identified as target. The duramycin family of lantibiotics binds specifically to phosphoethanolamine which results in inhibition of phospholipase A2 and various other cellular functions. Most of the class II bacteriocins dissipate the proton motive force (PMF) of the target cell, via pore formation. The subclass IIa bacteriocin activity likely depends on a mannose permease of the phosphotransferase system (PTS) as specific target. The subclass IIb bacteriocins (two-component) also induce dissipation of the PMF by forming cation- or anion-specific pores; specific targets have not yet been identified. Finally, the subclass IIc comprises miscellaneous peptides with various modes of action such as membrane permeabilization, specific inhibition of septum formation and pheromone 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.     

N-terminal acetylation in paenibacillin, a novel lantibiotic

N-terminal acetylation was uncovered in paenibacillin, a novel lantibiotic recently reported as a product of Paenibacillus polymyxa OSY-DF. This N-terminal modification is unprecedented among bacteria-derived antimicrobial peptides and further illustrates the broad range of modifications that can occur in lantibiotics. Additionally, the primary structure of paenibacillin has been finally determined unequivocally by the extensive NMR analysis taken together with previous MS/MS results. These analyses revealed the structure of paenibacillin as one of the most post-translationally modified lantibiotics.     

Complete alanine scanning of the two-component lantibiotic lacticin 3147: generating a blueprint for rational drug design

Lantibiotics are post-translationally modified antimicrobial peptides which are active at nanomolar concentrations. Some lantibiotics have been shown to function by targeting lipid II, the essential precursor of cell wall biosynthesis. Given that lantibiotics are ribosomally synthesized and amenable to site-directed mutagenesis, they have the potential to serve as biological templates for the production of novel peptides with improved functionalities. However, if a rational approach to novel lantibiotic design is to be adopted, an appreciation of the roles of each individual amino acid (and each domain) is required. To date no lantibiotic has been subjected to such rigorous analysis. To address this issue we have carried out complete scanning mutagenesis of each of the 59 amino acids in lacticin 3147, a two-component lantibiotic which acts through the synergistic activity of the peptides LtnA1 (30 amino acids) and LtnA2 (29 amino acids). All mutations were performed in situ in the native 60 kb plasmid, pMRC01. A number of mutations resulted in the elimination of detectable bioactivity and seem to represent an invariable core within these and related peptides. Significantly however, of the 59 amino acids, at least 36 can be changed without resulting in a complete loss of activity. Many of these are clustered to form variable domains within the peptides. The information generated in this study represents a blue-print that will be critical for the rational design of lantibiotic-based antimicrobial compounds.     

Posttranslationally modified bacteriocins--the lantibiotics

Lantibiotics are a subgroup of bacteriocins that are characterized by the presence of the unusual thioether amino acids lanthionine and 3-methyllanthionine generated through posttranslational modification. The biosynthesis of lantibiotics follows a defined pathway comprising modifications of the prepeptide, proteolytic activation, and export. The genes encoding the biosynthesis apparatus and the lantibiotic prepeptide are organized in clusters, which also include information for proteins involved in regulation and producer self-protection. The elongated cationic lantibiotics primarily act through the formation of pores and recent progress with nisin and epidermin has shown that specific docking molecules such as lipid II play an essential role in this mechanism. Mersacidin and actagardine inhibit cell wall biosynthesis by complexing the precursor lipid II, whereas the cinnamycin-like peptides bind to phosphoethanolamine thus inhibiting phospholipase A2.     

Lantibiotics produced by lactic acid bacteria: structure,function and applications

Lantibiotics are a diverse group of heavily modified antimicrobial and/or signalling peptides produced by a wide range of bacteria, including a variety of lactic acid bacteria. Based on their diverse structures and mode of action, at least six separate lantibiotic subgroups can be suggested, but all subgroups are characterized by significant post-translational modifications, which include the formation of (-methyl)lanthionines, among other unusual alterations. These small peptides are produced, modified, exported, sensed and combated by a complex set of proteins encoded by (usually) co-ordinately regulated operons. In some instances, the production and immunity have been shown to be auto-regulated by the mature lantibiotic. Since their discovery, interest in lantibiotics has been fuelled by their obvious potential as food-grade antimicrobials to improve food safety and quality; a potential which, to date, has been realised only by the longest characterised molecule, nisin. In addition, these peptides are often mooted as alternatives to antibiotics for some biomedical applications. The purpose of this paper is to review recent developments in our understanding of lantibiotic structure, molecular genetics and applications for this unusual class of bacteriocins.     

Lichenicidin rational site-directed mutagenesis library: A tool to generate bioengineered lantibiotics

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that arise as an alternative to the traditional antibiotics. Lichenicidin is active against clinically relevant bacteria and it was the first lantibiotic to be fully produced in vivo in the Gram-negative host Escherichia coli. Here, we present the results of a library of lichenicidin mutants, in which the mutations were generated based on the extensive bibliographical search available for other lantibiotics. The antibacterial activity of two-peptide lantibiotics, as is lichenicidin, requires the synergistic activity of two peptides. We established a method that allows screening for bioactivity which does not require the purification of the complementary peptide. It is an inexpensive, fast and user-friendly method that can be scaled up to screen large libraries of bioengineered two-peptide lantibiotics. The applied system is reliable and robust because, in general, the results obtained corroborate structure–activity relationship studies carried out for other lantibiotics.     

Lantibiotics and microcins: polypeptides with unusual chemical diversity

Bacterial-derived antimicrobial polypeptides enjoy a large degree of structural and chemical diversity. Two well-studied examples of such polypeptides are the lanthionine-containing lantibiotics produced by a variety of Gram-positive bacteria, and their Gram-negative counterparts, the microcins. Both groups are produced as gene-encoded precursor peptides and undergo post-translational modification to generate the active moieties. Structure elucidation of novel lantibiotics and microcins has recently uncovered further novel structural and chemical features and, combined with the generation of analogue peptides by genetic manipulation, new insights into structure-function relationships have been gained. Furthermore, study of the mode of action of the lantibiotics nisin and mersacidin has revealed their use of a 'docking molecule' in the target cell to facilitate their biological activities. Meanwhile, in vitro studies with microcin B17 have helped to uncover the molecular mechanisms by which post-translational modification results in the formation of heterocyclic oxazole and thiazole rings. From a practical standpoint, both groups of polypeptides represent new lead structures for future development of antimicrobial agents, whilst the identification of the 'docking molecules' represents a step forward in the search for novel targets for future antibiosis.     

Microbial engineering of dehydro-amino acids and lanthionines in non-lantibiotic peptides

This minireview focusses on the use of bacteria to introduce dehydroresidues and (methyl)lanthionines in (poly)peptides. It mainly describes the broad exploitation of bacteria containing lantibiotic enzymes for the engineering of these residues in a wide variety of peptides in particular in peptides unrelated to lantibiotics. Lantibiotic dehydratases dehydrate serines and threonines present in peptides preceded by a lantibiotic leader peptide thus forming dehydroalanine and dehydrobutyrine, respectively. These dehydroresidues can be coupled to cysteines thus forming (methyl)lanthionines. This coupling is catalysed by lantibiotic cyclases. The design, synthesis, and export of microbially engineered dehydroresidue and or lanthionine-containing peptides in non-lantibiotic peptides are reviewed, illustrated by some examples which demonstrate the high relevance of these special residues. This minireview is the first with special focus on the microbial engineering of nonlantibiotic peptides by exploiting lantibiotic enzymes.     

Protein engineering of lantibiotics

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibiotics were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.     

Bacillus subtilis antibiotics: structures, syntheses and specific functions

The endospore-forming rhizobacterium Bacillus subtilis- the model system for Gram-positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. The produced anti-microbial active compounds include predominantly peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally generated, as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospholipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well-studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the "pure" anti-microbial action: Non-ribosomally produced lipopeptides are involved in biofilm and swarming development, lantibiotics function as pheromones in quorum-sensing, and a "killing factor" effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given.     

Contribution of the Actinobacteria to the growing diversity of lantibiotics

Currently, 76 lantibiotics have been described; the vast majority being produced by members of the Firmicute phylum of bacteria. There is a growing number being identified from the Actinobacteria phylum and some of these exhibit novel modifications leading to an increased functional diversity among lantibiotics. In this review, we discuss the currently characterized lantibiotics highlighting the expanding diversity provided by those from the Actinobacteria. This increased diversity has the potential to expand lantibiotic applications as antimicrobials in foods and pharmaceuticals. In addition, a phylogenetic classification system based on the full prepropeptide sequences showed remarkable consistency with current classification systems and may provide a more rapid and convenient means for classifying lantibiotics.