Lantibiotics: Diverse activities and unique modes of action

Journal of Bioscience and Bioengineering VOL. 107 No. 5, 475 – 487, 2009 www.elsevier.com/locate/jbiosc

REVIEW

Lantibiotics: Diverse activities and unique modes of action Sikder M. Asaduzzaman,1 and Kenji Sonomoto1,2,⁎ Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 1 and Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 2 Received 3 September 2008; accepted 9 January 2009

Lantibiotics are one of the most promising alternative candidates for future antibiotics that maintain their antibacterial efficacy through many mechanisms. Of these mechanisms, some modes of activity have recently been reported, providing opportunities to show these peptides as potential candidates for forthcoming applications. Many findings providing new insight into the detailed molecular activities of numerous lantibiotics are constantly being uncovered. The combination of antibiotic mechanisms in one lantibiotic molecule shows its diverse antimicrobial usefulness as a future generation of antibiotic. Since lantibiotics do not have any known candidate resistance mechanisms, the discovered distinct modes of activity may revolutionize the design of anti-infective drugs through the knowledge provided by these super molecules. In this review, we discuss the rising assortment of lantibiotics, with special emphasis on their structure-function relationships, addressing the unique activities involved in their individual modes of action. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Lantibiotics; Structural variants; Structure-activities; Modes of action; Lipid II; Drug design]

ANTIBIOTICS AND RESISTANCE TO ANTIBIOTICS The discovery of penicillin in 1928 by Alexander Fleming was a historical milestone in human civilization; the subsequent curing of individuals with otherwise unbearable and sometimes fatal infectious diseases by antibiotics has been considered as nothing short of a medical miracle. The identification and production of a wide variety of antibiotics on a massive scale have revolutionized medical approaches. Unfortunately, the initial wide-spread use of antibiotics has generated a strong evolutionary pressure for the emergence of resistant bacteria. The exclusive reliance on broad-spectrum antibiotics has further intensified the problem by inducing the development of multi-resistant pathogens. One notorious example is that the vast majority of the clinical isolates from Staphylococcus aureus strains have been found to be resistant to methicillin (1). The devastating threats from acquired resistance to antibiotics are compounding from all regions of antibiotic end-users. Consequently, there is currently no antibiotic in clinical use to which resistance has not developed. The World Health Organization has warned that the rapid increase in resistance among pathogens may become untreatable (WHO/41. http://www.who.int 2000). Thus, there is a pressing need to discover and/or develop new agents that are active even against the emerging resistant bacteria. The discovery of new classes of antibacterial compounds based on targets identified from bacterial genomics is historically invaluable as a source of antibacterial drugs (e.g., glycopeptides) that bacteria use as ⁎ Corresponding author. Fax: +81 92 642 3019. E-mail address: [email protected] (K. Sonomoto).

“weapons” against each other. One such glycopeptide antibiotic, vancomycin, has long been reliable in treating infections caused by bacteria resistant to several other antibiotics, and is usually reserved for the treatment of serious infections, including those caused by the “super bug” methicillin-resistant Staphylococcus aureus (MRSA). However, even vancomycin-resistant enterococci (VRE) have now become quite common (2, 3), and this is made more complex by the spread of vancomycin resistance genes throughout the pathogens. Therefore, these dramatic increases in antibiotic-resistant pathogens have stimulated efforts to identify, develop, or design antibiotics that may be active against multi-resistant pathogen-caused diseases. DO LANTIBIOTICS SUPERSEDE CONVENTIONAL ANTIBIOTICS? Some antimicrobials are now being considered as alternative antibiotics, such as bacteriocins, bacteriophages, probiotics, and antimicrobial peptides. The attractive features of some of these molecules, for example, their natural sources, wide range of activities, ease of production, and the fact that they are not prone to developing resistance, have interested researchers seeking to develop new antibiotics. Among these different sources of alternative antibiotics, lantibiotics appear to be one of the most promising candidates. Traditional antibiotics usually exert their activities via a specific mode of action; for example, penicillin interferes with the cross-linking of two linear polymers by inhibiting the transpeptidase reaction and aminoglycoside antibiotics (e.g., streptomycin) inhibit protein biosynthesis by combining with the 30S subunit ribosome, whereas tertracyclines interfere with the binding of aminoacyl-tRNA to the 30S subunit ribosome and erythromycin prevents the transpeptidation

1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.01.003

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and translocation steps as a result of binding to the 50S subunit ribosome. Bacteria tend to develop resistance to all classes of these conventional antibiotics through a relatively simple mechanism. Even the antimicrobial peptides derived from many organisms, e.g., the well-studied peptide megainin, are generally based on their single action of pore formation in the membrane. In contrast, lantibiotics have quite diverse activities; for example, nisin and many other structurally related lantibiotics (e.g., epidermin/gallidermin) use the cell wall precursor lipid II bound to the membrane as a docking molecule for pore formation and combine at least two modes of action, i.e., pore formation and inhibition of cell wall biosynthesis, for antibacterial activity at nanomolar concentrations (4–6). Hasper et al. (7) recently elucidated the sequestration mechanism resulting from lantibiotic action, which helps to explain how some small lantibiotics that cannot span the bilayer of the bacterial membrane can still maintain a high level of antibacterial activity. Many other distinctive modes of action are currently known to be unique to lantibiotics, to which there are no known natural resistance mechanisms among bacteria. Therefore, we will discuss the lantibiotics' molecular mechanisms in order to clarify how these molecules carry out their exceptional activities. THE LANTIBIOTIC NISIN, THE FOREMOST ANTIBIOTIC WITH PROMISING FUTURE POTENTIAL Surprisingly, the history of lantibiotics is older than that of conventional antibiotics and dates back to a time before the discovery of penicillin. The first lantibiotic, nisin, was discovered in the 1920s and has had widespread application as a safe alternative for food preservation chemical reagents in approximately 50 countries for over 40 years, without natural resistance development (8, 9). Research regarding lantibiotics has recently gained renewed interest due to the emergence of clinical isolates that are resistant to antibiotics such as vancomycin, the last-resort drug that has been used against infections caused by Gram-positive bacteria for almost 30 years. The N-acyl-DAla-D-Ala moiety of lipid II is involved in the binding of vancomycin, and vancomycin-resistant bacteria thus remain sensitive to nisin due to its different binding site (4). Therefore, there has been a rapid and diverse expansion of research activities towards lantibiotics. Despite being the oldest known antibacterial agent, the structure of nisin was not determined until the elegant landmark studies by Gross and Morell in 1971 (10), and the word “lantibiotic” was just recently coined in 1988 as an abbreviation for lanthionine-containing antibiotic peptides (11). Therefore, although the history of lantibiotics is very old, a new paradigm is emerging due to their potential and enormous applications to meet the future challenges of developing antibiotics that can combat emerging pathogens.

J. BIOSCI. BIOENG., FEATURES OF LANTIBIOTICS All organisms have antimicrobial peptides that act as evolutionarily ancient weapons. The diversity of these antimicrobial peptides is so great that more than 1000 peptides have been included at http://www.bbcm.univ.trieste.it/∼tossi/antimic.html (described the antimicrobial peptides). Among these organisms, bacteria are remarkable producers of antimicrobial peptides. Bacterial-derived antimicrobial peptides have a large degree of structural and chemical diversity. Polypeptide antibiotics (e.g., gramicidin and valinomycin) are synthesized by large, multi-enzyme complexes from building blocks provided by a variety of cellular processes (12). Recent advances in bacterial molecular genetics have further contributed to new insights into peptide antibiotics. Ribosomally synthesized peptide antibiotics produced by certain bacteria are termed as bacteriocins (13, 14). Bacteriocins are divided into classes; lantibiotics are class-I bacteriocins that are antimicrobial peptides containing unusual amino acids, such as thioether cross-linked amino acids in lanthionine and 3-methyllanthionine, and dehydrated amino acids in 2,3-didehydroalanine (Dha) and (Z)-2,3-didehydrobutyrine (Dhb) (15, 16). Post-translational modification renders the lantibiotics biologically active. A large variety of lantibiotic structures, biosynthetic mechanisms, and modes of action have attracted significant research interest. Lantibiotics exhibit a number of notable characteristics. They are ribosomally synthesized, and in most cases the genes involved in lantibiotic biosynthesis are clustered, designated by the generic locus symbol lan, with a more specific genotypic designation for each lantibiotic member (e.g., nis for nisin, nuk for nukacin ISK-1, gdm for gallidermin). Lantibiotics are found on conjugative transposable elements (e.g., nisin), on the chromosome of the host (e.g., subtilin) or on plasmids (e.g., nukacin ISK-1). The gene clusters for the biosynthesis of representative lantibiotics are depicted in Fig. 1. Although the gene order, complexity, and transcriptional organization of the various clusters differ, three genes (lanAMT) have been identified that are involved in the biosynthesis of all type-A(II) and type-B lantibiotics, and four genes (lanABCT) are present in all type-A (I) lantibiotic gene clusters (the grouping of lantibiotics will be explained below). These essential genes obviously include the structural genes that encode the precursor peptides for post-translational maturation (prepeptides), which have been designated lanA, except for subtilin whose structural gene is historically named spaS. The lanA genes produce prepeptides that have an extension (leader peptide) of 23–59 amino acids at their N-terminus in addition to the mature lantibiotic. The sequencing of the lanA genes indicates that the dehydro amino acids in lantibiotics are the result of the dehydration of serine and threonine residues to produce dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively. Lanthionine and 3-

FIG. 1. Biosynthetic gene clusters of some representative lantibiotics. Genes with similar proposed functions are highlighted in the same pattern. LanB and lanC genes of type-A(I) lantibiotics are substituted by the gene lanM of type-A(II) lantibiotics. Despite the differences in the gene order, complexity, and transcriptional organization of the clusters, three genes (lanAMT) are involved in all type-A(II) and type-B lantibiotics, and four genes (lanABCT) are present in all type-A(I) lantibiotic gene clusters for biosynthesis.

VOL. 107, 2009 methyllanthionine rings are then generated by intramolecular conjugate additions of cysteine to these unsaturated amino acids. Though the exact function of the leader is not yet clear, the suggested possible functions include export signaling, protection of the producing strain by keeping the peptides inactive, and providing scaffolds for the posttranslational modification machinery (17, 18). In type-A(II) lantibiotics, the bifunctional lanM is responsible for dehydration and the cyclization reactions. In contrast, in type-A(I) lantibiotics, lanB is involved in the dehydration of Ser and Thr to form Dha and Dhb, respectively, and lanC codes for the cyclase that produces lanthionine or 3-methyllanthionine (Fig. 2). The C-terminus of the lanM enzyme shows 20–27% sequence identity with the lanC enzyme, but it has no homology with the lanB enzyme. Direct evidence for the bifunctional role of the lanM enzyme in catalyzing dehydration and cyclization has been provided by in vitro reconstitution of lctM in lacticin 481 biosynthesis (19). Some lantibiotics also undergo further post-translational modifications. For example, the lanD genes encode the enzyme responsible for the formation of AviCys and AviMeCys; it is likely that the epiD gene in epidermin and mesD in mersacidin carry out the in vitro decarboxylation of a C-terminal Cys residue to form AviCys and AviMeCys, respectively (20, 21). Recently, one of the most post-translationally modified lantibiotics, paenibacillin, has been isolated and identified to show a broader range of modifications, including N-terminal acetylation (22). The N-terminal leader peptide is cleaved, and the mature lantibiotic is then translocated across the membrane. The prepeptide of type-A(I) lantibiotics is translocated via ATP binding cassette transporter LanT, and the leader peptide is catalyzed by serine protease LanP. However, recent reports of the broad substrate specificity of NisT for nisin biosynthesis have suggested the secretion of unmodified, partially modified, or fully modified cyclized nisA prepeptides and non-lantibiotic peptides fused to the leader peptide of nisA (23). In contrast to the broad specificity of NisT, the processing enzyme NisP only removes the leader peptide attached to fully modified nisin. In type-A(II) lantibiotics, LanT has two functions, to remove the leader peptide and to export the matured peptide. It has an extra N-terminal cysteine peptidase domain, as compared to LanT of type-A(I) lantibiotics (24). Some gene clusters contain a second transport system, which usually consists of three genes (lanEFG), and is concerned with the immunity of the producer strains. In addition, another gene, lanI, is

MODES OF ACTION OF LANTIBIOTICS

477

also assumed to be concerned with self-immunity to some lantibiotics (25). Additionally, two directive genes (lanKR) are often found to be involved in the regulation of lantibiotic biosynthesis, encompassing an important two-component sensory system (26). STRUCTURES AND LANTIBIOTIC GROUPING Thus far, more than 50 different lantibiotics have been isolated from Gram-positive bacteria. Lantibiotics are classified by Jung (27) as types A and B, based on the topology of their structures. Representatives of the lantibiotic structures are presented in Fig. 3. Type-A lantibiotics are further divided into two subtypes, elongated type-A(I) and tail and ring region-containing type-A(II), which have different genetic organizations (28). In type-A(I) lantibiotics, the lanthionine and 3-methyllanthionine residues are formed by the action of two distinct enzymes (LanB and LanC), whereas those that are formed by a single enzyme (LanM) are termed as type-A(II). Type-B lantibiotics, such as mersacidin, cinnamycin, duramycin, and ancovenin, are more globular and compact in structure (29). In addition, a separate subgroup is formed by the twocomponent lantibiotics consisting of two post-translationally modified peptides that individually have little to no activity but synergistically display strong antibacterial action. At the present, this emerging subgroup of two-component lantibiotics encompasses the structurally closely related lacticin 3147, plantaricin W, and staphylococcin C55, and the completely unrelated streptococcal cytolysin, which combines bacteriocin and cytolytic activity against blood cells (30). The molecular mechanisms responsible for the synergistic effect of two-peptide bacteriocins are not clear at the present. Generally, the two-peptide lantibiotics work best at equimolar concentrations (1:1 stoichiometry). However, an alternative classification of lantibiotics has been proposed by comparing the leader sequences of many lantibiotics, which reveals two different conserved motifs other than those presented above. In this organization (determined by genetics rather than activity profiles or three-dimensional structures), the class I lantibiotics all have a common “FNLD” motif between positions -20 and -15 and usually contain a Pro at position -2. The biosynthetic machinery involved in the post-translational modifications in this class consists of LanB and LanC. In contrast, class II peptides contain a characteristic “GG” or “GA” cleavage site (historically termed the

FIG. 2. An example of the post-translational maturation process of the lantibiotic nisin A. Specific serine and threonine residues (bold) in the nisin prepeptides are dehydrated by NisB. The cyclization of dehydrated amino acids with cysteine residues is catalyzed by NisC in a regio- and stereo-specific manner, and the protease NisP then proteolytically cleaves the leader peptide to render the lantibiotic active (for a review of the enzymatic processes involved, see ref. 28).

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FIG. 3. Structures of a few lantibiotics. A-S-A, lanthionine; Abu-S-A, 3-methyllanthionine; Dha, dehydroalanine; Dhb, dehydrobutyrine; D-A, D-alanine. Based on the topology of their structures, lantibiotics are classified into three major groups (27), (A) elongated type-A(I); (B) tail and ring region-containing type-A(II); and (C) globular type-B lantibiotics. In addition, (D) two-component and (E) some irregularly shaped lantibiotics have also been isolated and identified.

“double Gly motif”), contain multiple Asp and Glu residues, and are usually processed by one modification enzyme (LanM).

ENGINEERING OF LANTIBIOTICS TO DETERMINE THE FUNCTIONS OF UNUSUAL STRUCTURES Lanthionine/methyllanthionine bridges are the most notable features of lantibiotic peptides. These peptides are characterized by their high contents of unusual amino acid residues that form a thioether bridge to produce lanthionine and 3-methyllanthionine and also contain the unsaturated amino acid residues Dha and Dhb (Fig. 3), which are mostly modified forms of serine, threonine, or cysteine residues. It is now well established, from studies of different lantibiotics, that these unusual amino acids play a vital role in the

stability and activity of these antibiotic peptides. The mutagenesis of lantibiotic structural genes has shown the feasibility of changing the lantibiotic structure by genetic engineering. For example, the removal of dehydro amino acids, i.e., the replacement of dehydrated amino acid residues in lantibiotics with other amino acids, reduces their antibacterial activity. The mutant Dha5Ala has activity against vegetative cells similar to that of wild-type nisin, but the activity against spores is nearly abolished (31). The removal of Dha33 by Ala or the change of Dha5 and Dha33 with Ala leads to a remarkable decrease in activity, to about 1% of the activity of wild-type nisin (32). The replacement of Dha by Dhb and vice versa has been reported for many lantibiotics. The formation of Dhb instead of Dha in the structural region at position 5 of the nisZ gene led to the production of mature nisin Z that shows 2–10 fold lower antibacterial activity (50–90% less than that of the wild-type) against many indicator strains

VOL. 107, 2009 (33, 34). The Dhb10Dha mutant of mutacin II was reported to have similar activity (35), whereas the mutant Dhb14Dha did not show any noticeable change in gallidermin activity (34). The replacement of Dhb by Ala at positions 16 and 20 of Pep5 is found to lower the activity toward some indicator strains (36). The mutant Dha16Ile in mersacidin causes a great reduction in its activity against M. luteus and S. pyogenes (37). As with the other lantibiotics mentioned above, the two-peptide lantibiotic lacticin 3147 also showed a dramatic reduction or elimination of antimicrobial activity, due to mutagenesis in the lanthionine bridges. Cotter et al. (38) reported alanine scanning results that showed that 12 out of the 14 mutations involved in 6 out of the 7 lanthionine bridges in lacticin 3147 peptides result in elimination of bioactivity. They also found that changing five dehydrated residues resulted in a drop in activity. The introduction of a new thioether bridge in the lantibiotic Pep5 results in a dramatic decrease in antimicrobial activity (36). The replacement of amino acids in all positions is not tolerated by the biosynthetic machinery, and expression does not occur. For instance, an attempt to generate the mutant Dhb10Ala mutacin II resulted in no detectable mutacin production (35), and the change of Ser3, Ser19, or Cys22, which form the lanthionines, also results in a loss of gallidermin production (39).

MODES OF ACTION OF LANTIBIOTICS

TABLE 1. Salient features of some notable structural derivatives of lantibiotics Lantibiotic Nisin A

Nisin Z

Due to the importance of the unusual structures in lantibiotics, structure-activity relationships have been determined by numerous studies. Some important structural variants from various derivatives, which show a change in the activities and/or properties of lantibiotics, are included in Table 1. Cotter et al. (38) scanned all 59 amino acids of the two-component lantibiotic 3147 and found that at least 36 retain some bioactivity and that some of the amino acids cluster to form variable domains within the peptides. The glutamate residue in the A-ring of the lacticin 481 subgroup and in the B-ring of mersacidin is conserved and is critically important for activity, but it has been shown to be nonessential in lacticin 481 (37, 40). TARGET SELECTION AND USE OF A DOCKING MOLECULE Generally, many lantibiotics (e.g., nisin, nukacin ISK-1) bind to the membrane, leading to subsequent action. Nukacin ISK-1 binds the anionic membrane by the lysine residues in the tail region, which plays a vital role in its antibacterial activity (41). In the case of nisin, membrane permeabilization occurs after target recognition and formation of a complex with nisin and lipid II (4) for further action. Hyde et al. (42) reported that the prime target of nisin in inhibiting peptidoglycan biosynthesis is near the cell division site. Epidermin shares a recognition motif with nisin and binds to both lipid I and lipid II (5). The activity of nisin against vancomycinresistant bacteria is a result of the fact that nisin does not make contact with vancomycin's binding site (L-Lys-D-Ala-D-Ala moiety of pentapeptide) on lipid II. Instead, nisin binds the pyrophosphate moiety of lipid II, allowing this lantibiotic to be effective against vancomycin-resistant bacteria (43). The antibacterial activities of plantaricin C are similar to that of nisin; it that strongly inhibits in vitro lipid II synthesis and forms a stable complex with lipid II, indicating that both nisin and plantaricin C may target the same structures in lipid II (44). Smith et al. (45) have recently shown that mutacin 1140 causes membrane disruption in the artificial membrane and reported that, although it incorporates lipid II, it is arranged in a manner different than that of the nisin A complex. The two-peptide lantibiotic lacticin 3147 binds specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. Lacticin 3147 A1 (LtnA1) forms a lipid II:LtnA1 complex and another

Derivative

Properties

Activity

Ref.

T2S N20P/M21V/ K22T/K22S S3T

Dha instead of Dhb Change in hinge region

Increased Enhanced

34 100

Residue for A ring formation Change of Dha to A Dhb instead of Dha Reduction of positive charge Additional cysteine residue K increased solubility Improved solubility

Very low

34

No production 2–10 fold lower Similar

33 33 34

S5A S5T K12P T13C M17K N20K N20E/ N21E N20V/N20A M21K M21K/Dhb / K22G N27K/H21K

Gallidermin

STRUCTURE-ACTIVITY RELATIONSHIPS OF STRUCTURAL VARIANTS

479

Nukacin ISK-1 Fragments and chimeras

V32E A12L K1A-K2AK3A NisA1–12 NisA1–20 NisA1–29 Lact4816–27 Nis1–11Sub12–32

Negative charge in hinge region Change in hinge region Improved solubility Change in hinge region Improved solubility Influenced C-term. charge Ability to form pore disrupted Reduction of positive charge from N-terminal Rings C, D and E cleaved Rings D and E cleaved All lanthionine rings retain N-terminal 5 residues removed Chimeric peptides from nisin and subtilin

Inactive/not 101 produced Reduced 34 Active against Gram- 95 negative bacteria Inactive 102 Very low 102 Active against Gram- 95 negative bacteria Very low 102 Similar 3–5 fold lower

102 101

Similar

49

32 fold lower

41

Inactive

103

100 fold lower 10 fold lower

103 103

10 fold lower

104

Similar to nisin, 6–8 fold higher than subtilin

94

component (LtnA2) recognizes the complex, leading to a high affinity three-component complex for subsequent action (46). An exchange of the associated mutant peptide LtnA1-Leu21Ala abolished peptide production (47), and it is noteworthy that a corresponding leucine is also found in a number of other lantibiotics within the same subgroup as LtnA1, i.e., mersacidin, actagardine, and plantaricin W. The surrounding residues of this leucine are highly conserved. In the case of mersacidin, it is found to be involved in lipid II binding (48), and the residue may also be related to lipid II recognition for this peptide. TWO-PEPTIDE LANTIBIOTICS WORK SYNERGISTICALLY A number of two-peptide lantibiotics (those that synergistically function at optimal concentrations) have been identified during the last decade, of which lacticin 3147, staphylococcin C55, plantaricin W, Smb, BHT-A, and haloduracin are closely related. Lacticin 3147 (Fig. 3) is a well-studied two-peptide lantibiotic with exceptional antibiotic efficacy that is achieved when two killing mechanisms are combined. It is also effective against multidrug-resistant pathogens such as MRSA and VRE. However, some reports have indicated that its significant activities in the nanomolar concentration range are, to some extent, strain or species specific. Wiedemann et al. (46) reported that lacticin 3147 peptides (LtnA1 and LtnA2) have a very strong synergistic effect against Lactococcus lactis, but a remarkably weaker effect against Micrococcus flavus. Interestingly, the A1 peptide and mersacidin are almost equally effective against the lactococcal strain, but their activities differ by a factor of 30 against Micrococcus. The activity of lacticin 3147 involves the binding of the LtnA1 peptide to lipid II. Both activities (pore formation and inhibition of cell wall biosynthesis)

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require the presence of two peptides whose intermolecular interactions appear to be stabilized by lipid II (46). O'Connor et al. (47) determined the closeness of staphylococcin C55 to lacticin 3147 and reported that 86% (LtnA1 and C55α) and 55% (LtnA2 and C55β) of the peptides are identical at the amino acid level. They also reported that the significance of the relatedness between these two lantibiotics is so remarkable that the hybrid peptide pairs LtnA1:C55β and C55α:LtnA2 show activities in the single nanomolar range, reflecting well with the native pairings. The mutagenesis of the LtnA1 peptide with the equivalent residues in C55α does not produce the mutant LtnA1-Leu21Ala. This may be due to the positioning of this residue in a putative lipid II binding loop. MODES OF ACTION OF LANTIBIOTICS The activities of lantibiotics are mostly based on different killing mechanisms that are combined in one molecule. For example, the prototypic lantibiotic nisin inhibits peptidoglycan synthesis and forms pores through specific interactions with the cell wall precursor lipid II (6). As another example, the mutant [A12L] gallidermin has a diminished pore formation ability but is as potent as wild-type gallidermin, indicating that pore formation does not contribute to the killing of bacteria for this mutant gallidermin (49). It is now well known that the multiple activities of lantibiotics combine differently for individual target strains. However, the general steps involved in lantibiotic activities include i) binding to the bacterial membrane, followed by insertion into membrane, and ii) the use of receptor/ docking molecules to exert structure-based activity. We will focus, in detail, on these steps of lantibiotic activities, which are responsible for their potential antibacterial actions. BINDING OF LANTIBIOTICS TO MEMBRANE AND INSERTION INTO MEMBRANE Many studies have shown that membrane binding is the first step in lantibiotic modes of action. Altering the charge distributions in nisin, for example, removing positive charges from the N- or Cterminal region of nisin, hampered the initial interactions of the peptide to the membrane (50). By comparing the native nisin with its variants, it was also reported that electrostatic attractions encourage the initial association of nisin with the membrane. Breukink et al. (51) reported that the highly positive-charged C-terminus of nisin interacts primarily with the anionic surface of the bacterial cell membrane. The same study also indicated a very low association with the anionic lipids by the Val32Glu mutant of nisin Z and assumed that the introduction of a negative charge into nisin Z would result in electrostatic repulsion from the negatively charged phospholipids. The strongly reduced binding affinity of a nisin1–12 fragment to anionic phospholipids further indicates the importance of the C-terminus of nisin for binding to the membrane (52). Although the initial binding to the membrane surface seems to involve the C-terminus of nisin, studies with a variant of nisin Z, in which a short peptide is fused into its C-terminus, show that the C-terminus translocates across the membrane (53). This translocation of the C-terminus is correlated with pore-forming activity, and both the activities are dependent on anionic lipids. Once it is electrostatically bound, the peptide adopts a membrane-spanning orientation in which the C-terminus of at least part of the molecules forming the pore is located in the lumen of the vesicle. However, it is now clear that the N-terminal rings of nisin bind to the disaccharide-pyrophosphate of lipid II, and the positively charged C-terminus initially interacts with the head-groups of the lipids in the membrane bilayer. Nukacin ISK-1 (Fig. 3) also has a net positive charge and binds strongly to the anionic membrane, and its potential antibacterial activity is crucially dependent on the Nterminus positive charges (Fig. 4) (41). Demel et al. (54) reported

FIG. 4. (A) Binding affinity of a cationic lantibiotic, nukacin ISK-1, to anionic [1,2dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt] and zwitterionic (1,2dioleyol-sn-glycero-3-phosphocholine) model membranes determined by a surface plasmon resonance (SPR) biosensor. Nukacin ISK-1 bound to each of the (a) anionic and (b) zwitterionic model membranes. (B) Dose-response of nukacin ISK-1 toward the anionic model membrane. Concentrations of nukacin ISK-1 were (a) 20, (b) 15, and (c) 5 μM. RU, resonance unit (41).

that lacticin 481 (with a net charge of zero) has a higher affinity for the zwitterionic membrane than nisin, which binds to the anionic membrane. PORE FORMATION BY LANTIBIOTICS Nisin and many other cationic type-A(I) lantibiotics have been well studied in terms of their modes of action involving cytoplasmic and artificial membranes (4). Numerous studies prior to the late 1990s focused on the permeabilization of bacterial cell membranes as the primary mode of action of nisin and other type-A(I) lantibiotics, which leads to the release of ions and molecules from the bacteria, eventually resulting in cell death (55). The pores formed by lantibiotics may have lifetimes of a few to several hundred milliseconds, with diameters of up to 2 nm (56). The pores of nisin are somewhat anion selective (51) and the pores of nisin and Pep5 work only in one direction (rectifying) (57, 58), whereas gallidermin and epidermin form nonrectifying channels that are more stable (55). The model membrane systems, such as planar lipid bilayers and liposomes, have a strong influence on the efficiency of pore formation (59). It has also been shown in monolayer studies that antimicrobial activity is well correlated with the nisin-anionic lipid interaction (55, 59). The two most well-established mechanisms of pore formation are the barrel-stave and wedge models. In the barrel-stave mechanism, the cationic lantibiotic monomers bind to the membrane surface through electrostatic interactions and are assembled into a preaggregate, and the pores are formed at a certain membrane potential, where the lantibiotic is perpendicular to the membrane (56). In the

VOL. 107, 2009 case of the wedge model, surface-bound lantibiotic molecules bind parallel to the membrane surface and generate local strain, bending the membrane in such a way that the lipid molecules, together with the lantibiotic, form a pore (60). Chikindas et al. (61) proposed a model for the orientation of lantibiotics in negatively charged membranes, in which the relatively elongated nisin molecule lies parallel to the membrane surface with the positively charged sidechains of amino acids pointing out of the lipid bilayer. In contrast with this model, which demonstrates the most stable orientation, transient pore formation may result as a molecule passes through the membrane by conformational change. The well-established model for pore formation by the lantibiotic nisin has been presented in Fig. 5A. Pore formation by nisin is unique, as compared to that of vancomycin, teicoplanin, and ramoplanin, in that it subsequently binds with lipid II, using it as a docking molecule to form a pore that is stable and highly efficient (62). Figure 5B depicts the structure of lipid II, which portrays the regions involved in the binding of different antibiotics. Breukink et al. (4) reported that the presence of lipid II in the membrane increases the pore-forming efficiency of nisin 1000fold as compared to peptides that do not use lipid II. Lipid II-mediated pore formation by nisin is so dramatic that the presence of only two lipid II molecules per 105 phospholipid molecules greatly enhances the release of dyes from vesicles (4, 5). Nisin forms highly specific pores through its interaction with lipid II, and the anion selectivity of nisin in model membrane systems disappears upon the addition of lipid II (6). Lipid II changes

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the orientation of nisin from parallel to perpendicular, with respect to the membrane surface (63), and is recruited into a stable pore structure (62). The involvements of manifold molecules in the lipid II-nisin complex are subsequently sufficient to form a defined pore of uniform structure (62). Therefore, the lipid II-mediated porecomplex is highly stable and unique, as other cationic antimicrobial peptides form pores in the membrane that are unstable, transient, and non-uniform in structure (62, 64). The two-component lantibiotic lacticin 3147 has also been shown to utilize lipid II in a sequential manner to form a defined pore. However, a few lantibiotics, e.g., mersacidin, do not form pores. Many novel findings of the last few years have uncovered the structure-based activities of lantibiotics, indicating that pore formation is not the major killing mechanism of lantibiotics. LIPID-II TARGETING LANTIBIOTIC ACTIVITIES Bacteria-specific cell wall precursors, e.g., lipid I and lipid II, are essential for bacterial cell wall biosynthesis. Many antibiotics bind to these precursors to interfere with peptidoglycan biosynthesis, preventing the utilization of these molecules by transpeptidase and transglycosylase enzymes in building the cross-linked network of the bacterial cell wall. Vancomycin (a peptide antibiotic) is an example of a compound that kills bacteria by targeting lipid II and has long been reliable as an essential antibiotic. Vancomycin binds to the D-Ala-DAla moiety of lipid II, and nisin binds to the disaccharides-pyrophosphate region of lipid II, so nisin is even effective against vancomycin-

FIG. 5. Pore formation by the lantibiotic nisin using lipid II as a docking molecule. (A) Nisin binds to the cell wall precursor lipid II, using it as a docking molecule. The N-terminus of nisin binds lipid II, while the C-terminus is inserted into the bacterial membrane, subsequently forming a pore to release molecules and ions. (B) Chemical structure of lipid II. NMR analysis of the lipid II-nisin complex reveals that the N-terminal region of nisin (residues 1–12) encages the pyrophosphate moiety of lipid II with a hydrogen bond network (43).

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resistant strains, though both are confined to targeting lipid II (43). It has recently been demonstrated that lipid II is the prime target of several other classes of natural products, including lantibiotics. A growing number of lantibiotics have been shown to interfere with peptidoglycan biosynthesis by binding to lipid II, which act differently on lipid II, where different structures of these compounds are used to explain the sophisticated modes of action directed by the diverse structures of lantibiotics. The prototypic type-A(I) lantibiotic nisin is an elongated amphipathic screw-shaped structure in solution, having a net positive charge. Initially, its bactericidal action was believed to be predominantly involved in the formation of short-lived pores in bacterial cell membranes (as mentioned before). During the past few years, a unique mechanism of action has been shown to be exerted by nisin, which renders it highly potent against many Gram-positive bacteria at nanomolar concentrations (4, 6). Many ambiguities have been clarified on the modes of action of lantibiotics following the report of Breukink et al. (4), suggesting that nisin interacts in a highly specific manner with lipid II. The dissimilar sensitivities of lipid-II targeting lantibiotics to different indicator strains may be due to the presence of different lipid II contents among various microorganisms (e.g., E. coli, 2 × 103 molecules per cell; Micrococcus lysodeikticus, 105 molecules per cell) (65, 66). Many studies have subsequently shown that this inhibition is caused by binding to the lipidassociated peptidoglycan precursors lipid I and lipid II, with lipid II binding having the more predominant effect (for a review see ref. 67, 68). However, structural information on the interaction of lantibiotics with the cell wall precursor so far has been restricted to lipid II. Nuclear magnetic resonance (NMR) data reveal that the pyrophosphate moiety of lipid II interacts with the backbone amides of rings A and B of nisin via six hydrogen bonds (43). Bonev et al. (69) reported that nisin can also bind to bactoprenol pyrophosphate; however, the affinity is considerably lower than that for the complete lipid II molecule. This indicates that, for high-affinity binding of nisin, additional interactions must take place, presumably between the N-acetylmuramyl moieties, whereas the pentapeptide side chain and the isoprenoid moiety are not involved. The inference of the interaction of lantibiotics with lipid I stems mainly from the observation that lipid II biosynthesis is strongly blocked, but the structural analysis of a lantibiotic-lipid I complex has not yet been reported. The A and B ring system of nisin, which has been shown to be responsible for binding with lipid II, in particular the pyrophosphate moiety, is conserved in nisin, subtilin, epidermin, gallidermin, and plantaricin C. Bonelli et al. (49) showed that gallidermin/epidermin has a higher affinity to lipid II than nisin and suggested that the structural element may be lysine at position 4 (isoleucine in nisin), which may provide an additional positive charge to enhance binding to the pyrophosphate moiety. Mersacidin, actagardin, and cinnamycin are globular type-B lantibiotics and also bind to lipid II, but have no structural similarity with nisin and epidermin (Fig. 3). They act by disrupting the enzyme function of cell wall biosynthesis, by the formation of a complex with lipid II (48, 70). Specifically, these compounds prevent the activity of transglycosylases (70). It is important to note that mersacidin does not form pores upon binding to lipid II; this is the reason for its moderate MIC values. However, the compound is very effective in vivo against staphylococcal infections (71–73), including MRSA and vancomycin-resistant enterococci (70). In vitro peptidoglycan synthesis assays suggested that epidermin and nisin accumulate lipid I, indicating that they may also inhibit the conversion of lipid I to lipid II (5). The two-peptide lantibiotic laciticn 3147 works at nanomolar concentrations with a 1:1 stoichiometry (LtnA1:LtnA2). The LtnA1 peptide interacts specifically with lipid II, which recruits LtnA2 for the inhibition of cell wall biosynthesis and pore formation (46).

J. BIOSCI. BIOENG., CHANGES IN BACTERIAL MORPHOLOGY BY LANTIBIOTICS The peptidoglycan of bacteria is a dynamic system, which is the prime target of many lantibiotics, including nisin. Hyde et al. (42) showed the effects of nisin on B. subtilis cells, which causes rapid membrane permeabilization and subsequent changes in length, crosssection, shape, and population distributions (Figs. 6 and 7). They concluded that the lethal action of nisin is due to the concerted effects of membrane permeabilization, followed by cell wall inhibition and metabolic deregulation of bacterial division. The principal site of action for nisin is located in the region of rapid cell wall growth near the site of septal formation, where the most severe cell wall malformation occurs (42). Hasper et al. (7) illustrated an action for lantibiotics by means of a pyrophosphate-mediated mechanism, through the sequestration of lipid II from sites of bacterial cell wall synthesis. These findings are consistent in their explanations involving significant aberrations in cell wall morphogenesis, where bacterial elongation is rapid. B. subtilis cells exposed to nisin form high numbers of double septa near one another and produce a number of multiseptal bacteria. We found that the external morphological appearances of B. subtilis cells that have been exposed to nukacin ISK-1 are unaltered, whereas mersacidin-treated cells showed some changes in the overall morphology. However, cells exposed to nisin show a very different reaction, which led to a drastic reduction in cell size and abnormal morphological appearances (Asaduzzaman et al., unpublished data). The comparison of these lantibiotic-treated ultra-structures showed that the cells demonstrated large variations in their internal structures, while showing no change in the inner-structure by nukacin ISK-1. However, a clear difference was observed in the cross-sections of nukacin-ISK-1 treated B. subtilis cells, which showed a striking reduction in cell wall width after addition of nukacin ISK-1 (Asaduzzaman et al., unpublished observation). The most widely studied type-B lantibiotic, mersacidin, has been reported to cause internal changes in bacterial cells, resulting in the spreading of chromosomes in the cytoplasm and ultimately leading to cell lysis (74). In contrast, the well-known type-A(I) lantibiotic nisin is a lytic-bactericidal agent that causes multiple aberrations, including leaking of cytoplasmic contents, reduction of cell width, acceleration of cell division, minicell formation, abnormal morphogenesis of bacterial cells, and eventual cell death (7, 62). DISTINCT MODES OF LANTIBIOTIC ACTIONS We have already described much of the details of different modes of the lantibiotic actions that are combined in one molecule. For example,

FIG. 6. Transmission electron microscopy observations of bacterial cross-section projections have been elucidated by Hyde et al. (42). (A) Untreated Bacillus subtilis cells, in which the cytoplasmic osmotic pressure strengthens the adhesion of the plasma membrane to the peptidoglycan layer, resulting in circular cross-sections; and (B) cell wall detachment (indicated by the arrow) from the plasma membrane is visible after nisin exposure, which relieves the osmotic stress by pore formation, leading to an astral cross-section after contraction of the plasma membrane. Scale bar: 100 nm.

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FIG. 7. Hyde et al. (42) observed the morphogenesis of Bacillus subtilis cells. (A1 and A2) Normal progression of septal formation in untreated cells; (B–E) some evidence supporting the suggestion that the bacterial morphogenesis caused by nisin is a result of morphological aberrations during septation: (B) multiseptal divisions, (C) “corkscrew” cell wall morphologies, (D) disjointed helical septa, and (E) one example of a division “dead end”, which reduces the bacteria to producing many nonviable “minicells”. Scale bar: 200 nm.

the modes of activity of the prototypic lantibiotic nisin have been shown to be so sophisticated that its effectiveness as an antibiotic is gradually increasing upon exploration of its structure-based functions. Early findings on nisin were mainly confined to the observable phenomena of pore formation to release molecules and ions (60, 75). Up until the last decade, the advances in the molecular mechanisms of lantibiotic actions had been very poor. Breukink et al. (4) were the first to report that peptidoglycan biosynthesis is inhibited by nisin, and this led to new insights into the molecular mechanisms of lantibiotic modes of actions. In a later study, Hsu et al. (43) showed that the nisin-lipid II complex reveals a novel lipid II-binding motif where the N-terminal backbone amides of nisin coordinate the pyrophosphate moiety of lipid II. Furthermore, the sequestration mechanism evident from nisin provided insight into how short peptides (e.g., gallidermin, epidermin) that may not be capable of spanning the membrane exert their high antibacterial efficacy. Nisin segregates lipid II into nonphysiological domains in its mode of action (Figs. 8A and B) (7). On the other hand, the glycopeptide antibiotic vancomycin does not segregate lipid II from the cell and clearly produces pools of lipid II in the septum (Fig. 8C). In agreement with the above findings, Hyde et al. (42) demonstrated that, in the presence of nisin, septal formation continues but the bacterial cell displays multiple aberrations, and the

FIG. 8. Hasper et al. (7) illustrated an alternative mechanism of nisin's bactericidal action, which describes the in vivo segregation of lipid II into nonphysiological domains. (A) Bacillus megaterium cells incubated with 0.5 μg/ml fluorescein-labeled nisin. The arrow indicates that the bacterium has already divided. (B) B. subtilis cells incubated with 4 μg/ml fluorescein-labeled nisin. Fluorescence from nisin appears to be clustered in patches on the membrane. (C) B. megaterium cells after incubation with 2 μg/ml labeled vancomycin. The arrows indicate the newly formed division sites or older exemplars. (D) B. subtilis cells stained with 4 μg/ml fluorescent vancomycin. The labeled vancomycin reveals pools of lipid II in the septum and as well as lipid II in helical threads. The insets are Nomarski images.

cell envelope formation is deregulated, leading to aberrant cell morphogenesis. They also proposed that this mechanism is distinctly different from the cell wall inhibitory activity of glycopeptides and βlactam antibiotics and also from the actions of pore-forming peptide antibiotics. In addition to nisin, many other lantibiotics (e.g., gallidermin, subtillin, mersacidin) use lipid II but have distinctive structure-based activities (5, 49, 70). A notable example of the molecular modes of action has been elucidated for two-peptide lantibiotics, e.g., laciticin 3147, the well-studied two-component lantibiotic that works in a sequential manner, where the LtnA1 peptide interacts specifically with lipid II, then the LtnA2 peptide recognizes the LtnA1-lipid II complex for pore formation and peptidoglycan biosynthesis inhibition (46, 76). STRUCTURAL VARIANTS TO STUDY MODES OF ACTION The mutants and fragments generated by site-directed mutagenesis and chemical and enzymatic digestion from many works have provided enormous information regarding the modes of action of lantibiotics. The introduction of an additional positive charge in nisin by the Val32Lys variant has a relatively small effect, whereas a negative charge (Val32Glu) results in about a 4-fold decrease in activity against some indicator strains (6). Epilancin K7 shares a very similar C-terminus double-ring system with nisin, which does not show interaction with lipid II (5). This evidence supports the relatively unimportant role of the C-terminus of these lantibiotics in biological activity. However, many studies have strongly suggested that the Nterminus of nisin is essential for binding. For example, a nisin1–12 fragment has no bactericidal activity but shows antagonistic activity against nisin's bactericidal activity (31), indicating that the fragment competes with nisin for the binding site. Complete proteolytic deletion of the D and E rings of nisin leads to a 100-fold decrease (99% eliminated) in activity (31), whereas chemical disruption of Dha5, which opens the A ring, results in more than a 500-fold reduction (less than 0.2%) in antibacterial activity as compared to its native form (77). The lipid II variant containing a shorter prenyl tail (3 from 11 isoprene units) can form a complex with nisin, and the length of this isoprene tail does not affect its pore-forming activity (62). Intermolecular hydrogen bonds between the amides of Dhb2, Ala3, Ile4, Dha5, and Abu8 on nisin and the oxygens of the pyrophosphate group of lipid II maintain the pyrophosphate moiety of lipid II within the cavity. Additionally, MurNAc (N-acetylmuramic acid, a component of glycan chains) and the first isoprene unit form the binding site for the recognition of nisin (Fig. 5B). The replacement of Lan in the A ring of nisin with MeLan resulted in 50-fold reduced affinity of the peptide to lipid II (6), and it is now well established that chemical opening of the

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A ring causes a nearly complete loss of activity (77). Further information regarding the binding of nisin and epidermin to both lipid I and lipid II have been revealed by their NMR structure, in which both the peptides share a recognition motif (5). Glutamate is conserved in the A ring of the lacticin 481 subgroup and the B ring of mersacidin, but it is not required for the activity of lacticin 481 (40). This discovery indicates that, since this residue is critically important for the A ring of mersacidin (37), lacticin 481 may have a different target or may recognize lipid II in a different manner. INHIBITION OF SPORE GERMINATION Most studies have mainly focused on the antibacterial activities against vegetative cells. Nisin, subtilin, and sublancin inhibit the spores' outgrowths from Bacillus and Clostridium species (78, 79). It has been proposed that this activity is a result of covalent modification of a target on the spore coat by nucleophilic attack on Dha5, in the case of nisin and subtilin (80). The reactive thiol groups on the exterior of the spores from Bacillus cereus react with compounds such as Snitrosothiols and iodoactetate, and nisin interferes with the modification of these sulfhydryl groups (81), suggesting that the target of nisin for the inhibition of spore germination is provided by these reactive thiol groups (82). However, a covalent mechanism has not yet been established. The replacement of Dha5 by Ala via site-directed mutagenesis of both subtilin (80, 83) and nisin (31) abolished the inhibition of spore germination, which indicates it as their putative site of attack. The above studies clearly suggest that the inhibition of spore germination is a different lantibiotic activity. Therefore, the inhibition of spore outgrowth is another distinct biological activity of lantibiotics, with a different structure-function relationship. FURTHER BIOLOGICAL FUNCTIONS Many lantibiotics have interesting biological activities in addition to their antibacterial activity. The SapB peptide (Fig. 3) produced by Streptomyces coelicolor works as a morphogenic peptide, and the novel lantibiotic sublancin (Fig. 3) exhibits lipid II-independent modes of action, such as the induction of autolysis of staphylococci (79). Cinnamycin (Fig. 3) and duramycin strongly inhibit the phospholipase A2 by sequestering its phosphatidylethanolamine (PE) (for multiple activities, see review 84), in addition to their bactericidal and hemolytic activities (85, 86). Cinnamycin induces transbilayer lipid movement, seemingly in a PE-dependent fashion (87). Nisin and Pep5 also induce autolysis of certain staphylococcal strains, primarily by breaking down the cell wall at the septa of the dividing cells, in addition to their usual modes of action (88, 89). The positively charged lantibiotics associate with the negatively charged teichoic and lipoteichoic acids, which displace and activate N-acetylL-alanine amidase and N-acetylglucosaminidase enzymes (88, 89). Though most lantibiotics are reported to use lipid I or lipid II as their docking molecule, not all lantibiotics bind to these, as discussed earlier. In most cases, the molecules or mechanisms involved in the activity have not yet been identified. Pep5 and epilancin K7 have specifically been shown to not bind lipid I or lipid II (90), but these lantibiotics still show activities against some bacteria that are far greater than those of other pore-forming lantibiotics. The high activity of Pep5 at nanomolar concentrations against Staphylococcus simulans and S. carnosus signifies that it employs a different high-affinity receptor or docking molecule for its potent biological activity (5, 89). In contrast with lantibiotics, conventional antibiotics do not have multiple functions in one molecule and do not possess such unique mechanisms of action. Until now, the molecular structure-based functions of only a few lantibiotics have been well clarified. Lantibiotic research is now in an advanced stage, and it is expected that more

J. BIOSCI. BIOENG., unique modes of action will be revealed, with a new era of amazing structure-based antibacterial activities. RATIONAL AND DE NOVO DESIGN OF LANTIBIOTICS TO REVOLUTIONIZE ANTIBIOTIC REPERTOIRES The discoveries of the mechanisms involved as individual lantibiotics work as a novel antibacterial, for example, the recent discoveries of lipid II as a target for nisin and, in particular, the studies of the pivotal role played by the pyrophosphate group, have brought nisin into the forefront as a candidate capable of combating resistant human infections, as a model case for the design of new antibiotics. Furthermore, the insights regarding the segregation of lipid II into non-physiological domains (7) elucidate how small lantibiotic peptides act strongly in vivo by a sequestration mechanism. While it was previously speculated that NisBTC enzymes had limited specificity, it is now clear that NisT and NisB have a broad substrate specificity. The independent functions of NisB, NisC, NisT, and NisP (for a review, ref. 91) present possibilities for designing new lantibiotics. The design of lantibiotics with respect to modification and export may be possible, based on the findings that NisB-modified peptides can be produced via the Sec or Tat system and then cyclized by the in vitro action of NisC (92). The use of lantibiotic synthetases offers much potential for designing new peptides. For example, Levengood et al. (93) have recently demonstrated the use of LctM in making thioether-containing analogs of enkephalin, contryphan, and inhibitors of human tripeptidyl peptidase II and spider venom epimerase. The versatile catalyzing capacity of lantibiotic synthetases can thus provide an approach to prepare libraries of peptides containing thioether rings and/or dehydro amino acids to overcome the inefficiency of synthetic chemistry. In addition, the design of modified peptides combined with different lantibiotics has also been explored (94). Furthermore, the enzymatic actions of lantibiotics' immunity, processing, and transportation in combination with its structure-based modes of action [for example, (i) the presence of lysines in the hinge region of nisin, which increases nisin's activity in killing Gram-negative bacteria (95), and (ii) the importance of N-terminal lysines in nukacin ISK-1 for its membrane binding and activity (41)] may aid in the design of potential lantibiotics in the future. Moreover, in vitro reconstitutions of lantibiotics are also in progress in order to revolutionize lantibotics for enormous applications in the near future. APPLICATIONS AND FUTURE OUTLOOK The fact that nisin has no known toxicity to humans has placed it in a unique position of world-wide acceptance as a powerful and safe food additive in the control of food spoilage, with widespread application as a food preservative in almost 50 countries for over 40 years. Nisin has been added to the positive list of food additives by the European Union (EU) and has also been approved by the Food and Drug Administration (FDA) (8, 9). Though the proteolytic breakdown of nisin in the gastrointestinal tract and its low stability at physiological pH levels limits the initial applications of nisin, nisin and many other lantibiotics are now being used in agricultural, veterinary, and, more recently, personal care products. Nisin, mutacin, mersacidin, etc., are in the preclinical stages of medical application (96). However, the most significant application of lantibiotics may be in the treatment of antibiotic-resistant pathogens. Ryan et al. (97) have reviewed the potential biomedical applications of lantibiotics in clinical and veterinary therapies. Some notable points are: nisin is effective against bacterial mastitis, oral decay and enterococcal infections and is effective in peptic ulcer treatment, treatment of enterocolitis, etc.; mersacidin and actagardine show remarkable activity against Staphylococcus aureus including MRSA, bacterial mastitis, oral decay, acne, etc.; gallidermin and epidermin are effective

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against acne, eczema, follicultis, and impetigo and can also be used for personal care products; mutacin 1140 may prevent dental cavities; lacticin 3147 is reported to prevent bacterial mastitis, MRSA and enterococcal infections, prevents oral hygiene, and acne; cinnamycin may be used for inflamation, viral infection treatment, and blood pressure regulation; and duramycin and ancovenin can be used for inflamation and blood pressure regulation, respectively. Some more remarkable applications of nisin have also been reported, which include the inhibition of experimental vascular graft infection caused by methicillin-resistant Staphylococcus epidermidis (98), and more interestingly, nisin inhibits sperm motility, showing its potential as a contraceptive agent (99). As there has been a recent threat of the use of spores of Bacillus anthracis in bioterrorism, the inhibitory activities of lantibiotics, such as subtilin (80, 83) and nisin (31), against spore germination may have interesting and potential future applications. In the post-genomic era, the combined knowledge of genetics, chemistry, and other approaches can promote new innovations in lantibiotics. The systematic research of lantibiotics may further resolve the existing difficulties and demonstrate potential use in food agriculture as well as in medical fields. ACKNOWLEDGMENTS Our work is partly supported by grants from “The Japan Society for the Promotion of Science (JSPS)”. References 1. Roder, B. L., Wandall, D. A., Frimodt-Moller, N., Espersen, F., Skinhoj, P., and Rosdahl, V. T.: Clinical features of Staphylococcus aureus endocarditis: a 10-year experience in Denmark, Arch. Inter. Med., 159, 462–469 (1999). 2. Weigel, L. M., Clewell, D. B., Gill, S. R., Clark, N. C., McDougal, L. K., Flannagan, S. E., Kolonay, J. F., Shetty, J., Killgore, G. E., and Tenover, F. C.: Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus, Science, 302, 1569–1571 (2003). 3. Novak, R., Henriques, B., Charpentier, E., Normark, S., and Tuomanen, E.: Emergence of vancomycin tolerance in Streptococcus pneumoniae, Nature, 399, 590–591 (1999). 4. Breukink, E., Wiedemann, I., van Kraaij, C., Kuipers, O. P., Sahl, H. G., and de Kruijff, B.: Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic, Science, 286, 2361–2364 (1999). 5. Brötz, H., Josten, M., Wiedemann, I., Schneider, U., Götz, F., Bierbaum, G., and Sahl, H. G.: Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics, Mol. Microbiol., 30, 317–327 (1998). 6. Wiedemann, I., Breukink, E., van Kraaij, C., Kuipers, O. P., Bierbaum, G., de Kruijff, B., and Sahl, H. G.: Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity, J. Biol. Chem., 276, 1772–1779 (2001). 7. Hasper, H. E., Kramer, N. E., Smith, J. L., Hillman, J. D., Zachariah, C., Kuipers, O. P., de Kruijff, B., and Breukink, E.: An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II, Science, 313, 1636–1637 (2006). 8. Delves-Broughton, J.: Nisin and its uses as a food preservative, Food Technol., 44, 100–117 (1990). 9. Delves-Broughton, J., Blackburn, P., Evans, R. J., and Hugenholtz, J.: Applications of the bacteriocin, nisin, Antonie van Leeuwenhoek, 69, 193–202 (1990). 10. Gross, E. and Morell, J. L.: The structure of nisin, J. Am. Chem. Soc., 93, 4634–4635 (1971). 11. Schnell, N., Entian, K. D., Schneider, U., Götz, F., Zahner, H., Kellner, R., and Jung, G.: Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings, Nature, 333, 276–278 (1988). 12. Mootz, H. D. and Marahiel, M. A.: Biosynthetic systems for nonribosomal peptide antibiotic assembly, Curr. Opin. Chem. Biol., 1, 543–551 (1997). 13. Nissen-Meyer, J. and Nes, I. F.: Ribosomally synthesized antimicrobial peptides: their function, biogenesis, and mechanism of action, Arch. Microbiol., 167, 67–77 (1997). 14. Tagg, J. R., Dajani, A. S., and Wannamaker, L. W.: Bacteriocins of gram-positive bacteria, Bacteriol. Rev., 40, 722–756 (1976). 15. de Vos, W. M., Kuipers, O. P., van der Meer, J. R., and Siezen, R. J.: Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria, Mol. Microbiol., 17, 427–437 (1995).

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