Lantibiotic structures as guidelines for the design of peptides that can be modified by lantibiotic enzymes

Lantibiotics are (methyl)lanthionine-containing bacterial peptides. (Methyl)lanthionines are posttranslationally introduced into the prepropeptides by biosynthetic enzymes that dehydrate serines and threonines and couple these dehydrated residues to cysteine residues. Thirty seven lantibiotic primary structures have been proposed to date, but little is known about the substrate specificity of the lantibiotic modifying enzymes. To define rules for the rational design of modified peptides, we compared all known lantibiotic structures by in silico analysis. Although no strict sequence motifs can be defined that govern the modification, statistical analysis demonstrates that dehydratable serines and threonines are more often flanked by hydrophobic than by hydrophilic amino acids. Serine residues escape dehydration more often than threonines. With these rules, novel hexapeptides were designed that either were predicted to become modified or will escape modification. The hexapeptides were fused to the nisin leader and expressed in a Lactococcus lactis strain containing the nisin modifying and export enzymes. The excreted peptides were analyzed by mass spectrometry. All designed fusion peptides were produced, and the presence or absence of modifications was found to be in full agreement with the predictions based on the statistical analysis. These findings demonstrate the feasibility of the rational design of a wide range of novel peptides with dehydrated amino acid residues.     

Sec-mediated transport of posttranslationally dehydrated peptides in Lactococcus lactis

Nisin is a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis. Its (methyl)lanthionines are introduced by two posttranslational enzymatic steps involving the dehydratase NisB, which dehydrates serine and threonine residues, and the cyclase NisC, which couples these dehydrated residues to cysteines, yielding thioether-bridged amino acids called lanthionines. The prenisin is subsequently exported by the ABC transporter NisT and extracellularly processed by the peptidase NisP. L. lactis expressing the nisBTC genes can modify and secrete a wide range of nonlantibiotic peptides. Here we demonstrate that in the absence of NisT and NisC, the Sec pathway of L. lactis can be exploited for the secretion of dehydrated variants of therapeutic peptides. Furthermore, posttranslational modifications by NisB and NisC still occur even when the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively. However, transport of fully modified prenisin via the Sec pathway is impaired. The extent of NisB-mediated dehydration could be improved by raising the intracellular concentration NisB or by modulating the export efficiency through altering the signal sequence. These data demonstrate that besides the traditional lantibiotic transporter NisT, the Sec pathway with an established broad substrate range can be utilized for the improved export of lantibiotic enzyme-modified (poly)peptides.     

Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis

Nisin is a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis. Its (methyl)lanthionines are introduced by two posttranslational enzymatic steps involving the dehydratase NisB, which dehydrates serine and threonine residues, and the cyclase NisC, which couples these dehydrated residues to cysteines, yielding thioether-bridged amino acids called lanthionines. The prenisin is subsequently exported by the ABC transporter NisT and extracellularly processed by the peptidase NisP. L. lactis expressing the nisBTC genes can modify and secrete a wide range of nonlantibiotic peptides. Here we demonstrate that in the absence of NisT and NisC, the Sec pathway of L. lactis can be exploited for the secretion of dehydrated variants of therapeutic peptides. Furthermore, posttranslational modifications by NisB and NisC still occur even when the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively. However, transport of fully modified prenisin via the Sec pathway is impaired. The extent of NisB-mediated dehydration could be improved by raising the intracellular concentration NisB or by modulating the export efficiency through altering the signal sequence. These data demonstrate that besides the traditional lantibiotic transporter NisT, the Sec pathway with an established broad substrate range can be utilized for the improved export of lantibiotic enzyme-modified (poly)peptides.     

Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by Gram-positive bacteria

Lantibiotics form a family of highly modified peptides which are secreted by several Gram-positive bacteria. They exhibit antimicrobial activity, mainly against other Gram-positive bacteria, by forming pores in the cellular membrane. These antimicrobial peptides are ribosomally synthesized and contain leader peptides which do not show the characteristics of signal sequences. Several amino acid residues of the precursor lantibiotic are enzymatically modified, whereafter secretion and processing of the leader peptide takes place, yielding the active antimicrobial substance. For several lantibiotics the gene clusters encoding biosynthetic enzymes, translocator proteins, self-protection proteins, processing enzymes and regulatory proteins have been identified. This MicroReview describes the current knowledge about the biosynthetic, immunity and regulatory processes leading to lantibiotic production. Most of the attention is focused on the lantibiotic nisin, which is produced by the food-grade bacterium Lactococcus lactis and is widely used as a preservative in the food industry.     

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.     

Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.     

Production of dehydroamino acid-containing peptides by Lactococcus lactis

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.     

Bacteriocins: mechanism of membrane insertion and pore formation

Lactic acid bacteria produce several types of pore forming peptides. Class I bacteriocins are lantibiotics that contain (methyl)lanthionine residues that may form intramolecular thioether rings. These peptides generally have a broad spectrum of activity and form unstable pores. Class II bacteriocins are small, heat stable peptides mostly with a narrow spectrum of activity. Most bacteriocins interact with anionic lipids that are abundantly present in the membranes of Gram-positive bacteria.'Docking molecules' may enhance the conductivity and stability of lantibiotic pores, while'receptors' in the target membrane may determine specificity of class II bacteriocins. Insertion into the membrane of many bacteriocins is proton motive force driven. Lantibiotics may form pores according to a'wedge-like' model, while class II bacteriocins may enhance membrane permeability either by the formation of a'barrel stave' pore or by a'carpet' mechanism.     

Production of Dehydroamino Acid-Containing Peptides by Lactococcus lactis

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.     

NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides

Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.     

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.     

Lantibiotic engineering: molecular characterization and exploitation of lantibiotic-synthesizing enzymes for peptide engineering

Lanthionine-containing peptide antibiotics called lantibiotics are produced by a large number of Gram-positive bacteria. Nukacin ISK-1 produced by Staphylococcus warneri ISK-1 is type-A(II) lantibiotic. Ribosomally synthesized nukacin ISK-1 prepeptide (NukA) consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events including unusual amino acid formation by the modification enzyme NukM, cleavage of leader peptide and export by the dual functional ABC transporter NukT, finally yielding a biologically active peptide. Unusual amino acids in lantibiotics contribute to biological activity and also structural stability against proteases. Thus, lantibiotic-synthesizing enzymes have a high potentiality for peptide engineering by introduction of unusual amino acids into desired peptides with altering biological and physicochemical properties, e.g., activity and stability, termed lantibiotic engineering. We report the establishment of a heterologous expression of nukacin ISK-1 biosynthetic gene cluster by the nisin-controlled expression system and discuss our recent progress in understanding of the biosynthetic enzymes for nukacin ISK-1 such as localization, molecular interaction in biophysical and biochemical aspects. Substrate specificity of the lantibiotic-synthesizing enzymes was evaluated by complementation of the biosynthetic enzymes (LctM and LctT) of closely related lantibiotic lacticin 481 for nukacin ISK-1 biosynthesis. We further explored a rapid and powerful tool for introduction of unusual amino acids by co-expression of hexa-histidine-tagged NukA and NukM in Escherichia coli.     

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.     

Discovery of Unique Lanthionine Synthetases Reveals New Mechanistic and Evolutionary Insights

Lantibiotic synthetases are remarkable biocatalysts generating conformationally constrained peptides with a variety of biological activities by repeatedly utilizing two simple posttranslational modification reactions: dehydration of Ser/Thr residues and intramolecular addition of Cys thiols to the resulting dehydro amino acids. Since previously reported lantibiotic synthetases show no apparent homology with any other known protein families, the molecular mechanisms and evolutionary origin of these enzymes are unknown. In this study, we present a novel class of lanthionine synthetases, termed LanL, that consist of three distinct catalytic domains and demonstrate in vitro enzyme activity of a family member from Streptomyces venezuelae. Analysis of individually expressed and purified domains shows that LanL enzymes install dehydroamino acids via phosphorylation of Ser/Thr residues by a protein kinase domain and subsequent elimination of the phosphate by a phosphoSer/Thr lyase domain. The latter has sequence homology with the phosphothreonine lyases found in various pathogenic bacteria that inactivate host mitogen activated protein kinases. A LanC-like cyclase domain then catalyzes the addition of Cys residues to the dehydro amino acids to form the characteristic thioether rings. We propose that LanL enzymes have evolved from stand-alone protein Ser/Thr kinases, phosphoSer/Thr lyases, and enzymes catalyzing thiol alkylation. We also demonstrate that the genes for all three pathways to lanthionine-containing peptides are widespread in Nature. Given the remarkable efficiency of formation of lanthionine-containing polycyclic peptides and the latter''s high degree of specificity for their cognate cellular targets, it is perhaps not surprising that (at least) three distinct families of polypeptide sequences have evolved to access this structurally and functionally diverse class of compounds.     

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.     

Roseocin,a novel two-component lantibiotic from an actinomycete

Lantibiotics are lanthionine ring containing natural products that belong to the class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Recent expansion in the availability of microbial genome data and in silico analysis tools have accelerated the discovery of these promising alternatives to antibiotics. Following the genome-mining approach, a biosynthetic gene cluster for a putative two-component lantibiotic, roseocin, was identified in the genome of an Actinomycete, Streptomyces roseosporus NRRL 11379. Posttranslationally modified lanthipeptides of this cluster were obtained by heterologous expression of the genes in Escherichia coli, and were in vitro reconstituted to their bioactive form by exploiting commercial proteases like endoproteinase GluC, and proteinase K. The two peptides displayed synergistic antimicrobial activity against Gram-positive bacteria including the WHO high-priority pathogens, MRSA and VRE. Structural characterization confirmed the installation of four (methyl)lanthionine rings with an indispensable disulfide bond in the α-peptide, and six (methyl)lanthionine rings in the β-peptide, by a single promiscuous lanthionine synthetase, RosM. Roseocin is the first two-component lantibiotic from a non-Firmicute, with extensive lanthionine bridging.     

Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5.

Pep5 is a 34-amino-acid antimicrobial peptide, produced by Staphylococcus epidermidis 5, that contains the thioether amino acids lanthionine and methyllanthionine, which form three intramolecular ring structures. In addition, two didehydrobutyrines are present in the central part of the lantibiotic and an oxobutyryl residue is located at the N terminus. All rare amino acids are introduced by posttranslational modifications of a ribosomally made precursor peptide. To elucidate the function of the modified residues for the antimicrobial action of Pep5, mutant peptides, in which single modified residues had been eliminated, were produced by site-directed mutagenesis. All of these peptides showed a reduced antimicrobial activity. In addition, those peptides from which the ring structures had been deleted became susceptible to proteolytic digest. This demonstrates that the ring structures serve as stabilizers of conformations essential for activity, e.g., amphiphilicity, as well as for protecting Pep5 against proteases of the producing strains. In addition, residues that could serve as precursors of new modified amino acids in lantibiotics were introduced into the Pep5 precursor peptide. This way, a novel methyllanthionine and a didehydroalanine were inserted into the flexible central part of Pep5, demonstrating that biosynthesis of modified amino acids is feasible by protein engineering and use of the lantibiotic modification system.     

The generation of nisin variants with enhanced activity against specific gram-positive pathogens

Nisin is the prototype of the lantibiotic group of antimicrobial peptides. It exhibits broad spectrum inhibition of Gram-positive bacteria including important food pathogens and clinically relevant antibiotic-resistant bacteria. Significantly, the gene-encoded nature of nisin means that it can be subjected to gene-based bioengineering to generate novel derivatives. Here, we take advantage of this to generate the largest bank of randomly mutated nisin derivatives reported to date, with the ultimate aim of identifying variants with enhanced bioactivity. This approach led to the identification of a nisin-producing strain with enhanced bioactivity against the mastitic pathogen Streptococcus agalactiae resulting from an amino acid change in the hinge region of the peptide (K22T). Prompted by this discovery, site-directed and site-saturation mutagenesis of the hinge region residues was employed, resulting in the identification of additional derivatives, most notably N20P, M21V and K22S, with enhanced bioactivity and specific activity against Gram-positive pathogens including Listeria monocytogenes and/or Staphylococcus aureus . The identification of these derivatives represents a major step forward in the bioengineering of nisin, and lantibiotics in general, and confirms that peptide engineering can deliver derivatives with enhanced antimicrobial activity against specific problematic spoilage and pathogenic microbes or against Gram-positive bacteria in general.     

Homologues and bioengineered derivatives of LtnJ vary in ability to form D-alanine in the lantibiotic lacticin 3147

Ltnα and Ltnβ are individual components of the two-peptide lantibiotic lacticin 3147 and are unusual in that, although ribosomally synthesized, they contain d-amino acids. These result from the dehydration of l-serine to dehydroalanine by LtnM and subsequent stereospecific hydrogenation to d-alanine by LtnJ. Homologues of LtnJ are rare but have been identified in silico in Staphylococcus aureus C55 (SacJ), Pediococcus pentosaceus FBB61 (PenN), and Nostoc punctiforme PCC73102 (NpnJ, previously called NpunJ [P. D. Cotter et al., Proc. Natl. Acad. Sci. U. S. A. 102:18584-18589, 2005]). Here, the ability of these enzymes to catalyze d-alanine formation in the lacticin 3147 system was assessed through heterologous enzyme production in a ΔltnJ mutant. PenN successfully incorporated d-alanines in both peptides, and SacJ modified Ltnα only, while NpnJ was unable to modify either peptide. Site-directed mutagenesis was also employed to identify residues of key importance in LtnJ. The most surprising outcome from these investigations was the generation of peptides by specific LtnJ mutants which exhibited less bioactivity than those generated by the ΔltnJ strain. We have established that the reduced activity of these peptides is due to the inability of the associated LtnJ enzymes to generate d-alanine residues in a stereospecific manner, resulting in the presence of both d- and l-alanines at the relevant locations in the lacticin 3147 peptides.