Class I The Lanthionine Containing Lantibiotic Bacteriocins

The term lantibiotic (Schnell et al. 1988; Jung 1991) refers to the diverse array of bacterial antibiotic peptides that contain the non-genetically encoded amino acids lanthionine (Lan) and/or 3-methyllanthionine (MeLan), as well as various other highly modified amino acids, commonly including the 2,3-unsaturated amino acids dehydroalanine (Dha) and dehydrobutyrine (Dhb). All of the lantibiotics currently described are thought to be produced as ribo-somally synthesized precursor peptides, which then undergo a series of post-translational modification reactions to produce the unusual amino acids described above as intrinsic components of the biologically active peptides. To date, lantibiotics have been found to be produced only by Gram-positive bacteria. Furthermore, they are generally considered to predominantly, if not exclusively, act on Gram-positive targets. As the family of lantibiotic molecules grew, the individual members were initially classified according to the topology of their ring structures and their biological activities (Jung and Sahl 1991), as either type A (elongated amphipathic structures) or type B (globular and more compact structures). In order to encompass the more recently described two-component varieties, the type C lantibiotics has been proposed. The type A lantibiotics are further divided into subtypes AI and AII based on the size, charge and sequence of their leader peptides (de Vos et al. 1995). It must be noted, however, that the lantibiotics are a difficult group to subdivide, and indeed it has been proposed that on the basis of similarities in their unmodified propeptide sequences, they could be split into 11 groups (Cotter et al. 2005a). The lantibiotics have been reviewed extensively over the last decade, and the reader is referred to some of these accounts for a more complete overview (McAuliffe et al. 2001b; Chatterjee et al. 2005). We propose to focus only on a selection of lantibiotics that we consider illustrate some of the significant diversity of these molecules, and to update the reader on recent developments in the field not covered by other reviews (cf. Table 4.1).

4.2.1 Type AI Lantibiotics

The prototype type AI lantibiotic, nisin, is perhaps the most extensively characterized of all bacteriocins. Produced by Lactococcus lactis, nisin has a long history of research, its discovery in 1928 (Rogers 1928) predating that of penicillin. Nisin has now been used safely in the food industry as a preservative for over 40 years without the appearance of significant bacterial resistance. Since the nisin biosynthetic pathway, requiring the coordinated expression and action of at least 11 gene products, is generally mirrored by most other lantibiotics, we will give a brief description of the processes involved. The precursor peptide, encoded by nisA, is acted upon by the proteins NisB and

Table 4.1 A selection of class I (lantibiotic) bacteriocins

Bacteriocin Producer species

Distinctive characteristics

Recommended reference(s)

Type A

Subtype AI Nisin A

Lactococcus lactis

Nisin U Streptococcus uberis

Streptin Streptococcus pyogenes

Subtype All

SA-FF22 S. pyogenes

Lacticin481 L. lactis

Salivaricin A Streptococcus salivarius

Prototype AI lantibiotic, widely used as a food preservative for over 40 years; the best-characterized of the bacteriocins of Gram-positive bacteria; induces its own expression through binding to a two-component signal transduction system

First nisin variant to be characterized from a non-lactococcal host bacterium

Utilizes a general bacterial protease (SpeB), rather than a dedicated protease (LanP) to remove the leader sequence from the prepeptide

First subtype All lantibiotic to be characterized; has a two-component sensor-kinase histidine-response regulator system for which the inducing molecule (most likely a peptide) is not SA-FF22 itself

Considered by some to be the prototype of this subtype; in contrast to SA-FF22, the lacticin 481 locus does not encode a two-component sensor-kinase histidine-response regulator

Multiple SalA variants have been characterized from different species of streptococci; all tested M serotypes of S. pyogenes (other than Mil and M37) have the SalA structural gene variant salAl; each SalA variant has been shown to effect both auto- and heterologous induction of SalA production in their respective host strains

Mattick and Hirsch (1947), Gross et al. (1971), McAuliffe et al. (2001b)

Wirawan et al. (2006) Wescombe and Tagg (2003)

van den Hooven et al. (1996)

Sublancin 168 Bacillus subtilis Type B

Mersacidin Bacillus spp.

Cinnamycin Streptomyces cinnamoneus

TypeC

Lacticin3147 L. lactis

Smb Streptococcus mutans

Cytolysin Enterococcus faecalis

The only lantibiotic shown to have a disulfide bond in Paik et al. (1998)

addition to the typical MeLan and Dha residues

Prototype type B lantibiotic, and the only lantibiotic of Chatterjee et al. (1992)

which the structure has been resolved by X-ray crystallography; effective in treating systemic staphylococcal (St. aureus) infections and nasal carriage in a mouse model system

The only characterized lantibiotic thought to be exported Kessler et al. (1987)

by the general secretory (Sec) pathway; has two MeLan rings in which the nucleophilic Cys is positioned

N-terminally to the Dhb; may have use as an anti-inflammatory and anti-allergy drug

The prototype two-component lantibiotic; structures and functions of both peptide components have been defined; killing of sensitive cells requires initial binding of LtnAl to lipid II, followed by complexing with LtnA2 to effect pore formation

Regulated in response to the external levels of CSP; a variant (BHT-A) has been identified in S. rattus that appears to be closely associated with the locus for the class II bacteriocin BHT-B

A lantibiotic that exhibits toxicity for both bacterial and eukaryotic cells; cytolysin subunits are activated by a two-step process involving transport and initial GG specific cleavage by CylB (an ABC transporter), following which the cytolysin subunits are activated via the removal of an additional six amino acids by CylA (a serine protease)

Martin et al. (2004)

Yonezawa and Kuramitsu (2005), Hyink et al. (2005)

Booth et al. (1996), Coburn and Gilmore (2003), Coburn et al. (2004)

NisC to dehydrate particular Ser/Thr residues, some of which are then used to form specific thioether bonds (i.e., Lan and MeLan) with Cys residues located (generally) further toward the C-terminus of the molecule. NisT is an ABC transporter responsible for export of the modified prepeptide, and NisP is a membrane-anchored protease able to cleave the leader peptide to release active nisin. NisI is involved in nisin immunity by an as yet ill-defined mechanism. Although the majority of NisI appears to be localized within the cytoplasmic membrane of the producer cell (Qiao et al. 1995), a significant amount is also secreted into the cytoplasm where it may bind to external nisin before it can aggregate at the cell surface (Koponen et al. 2004). Expression of nisi in nisin-sensitive L. lactis strains results in moderately decreased sensitivity to nisin (Qiao et al. 1995). However, full immunity levels are not achieved without the presence of NisFEG. NisFEG is an ABC transport protein complex, presumably contributing to nisin immunity in a manner similar to that used by multi-drug transporters, by reducing the concentration of nisin in direct contact with the cytoplasmic membrane. NisR and NisK together form a two-component sensor-kinase/response-regulator element involved in the regulation of nisin biosynthesis, which characteristically occurs late in the exponential phase of growth. Interestingly, since nisin itself is the specific ligand recognized by the sensor NisK, it up-regulates its own expression (Kuipers et al. 1995). The basic elements of the nisin biosyn-thetic pathway are conserved for all lantibiotics, with only minor variations such as the use of the LanM modification enzyme, rather than of the LanB/C complex for dehydratase and ring formation reactions and the encoding of LanD enzymes by a minority of lantibiotic loci to effect formation of the unusual amino acids S-[(Z)-2-aminovinyl]-D-cysteine (AviCys) and S-[(Z)-2-aminovinyl]-(3S)-3-methyl-D-cysteine (AviMeCys). For some lantibiotics, specific immunity appears attributable either only to LanI (e.g., Pep5; Reis et al. 1994) or only to the LanFEG system (e.g., lacticin 481; Rince et al. 1997). On the other hand, the epidermin and gallidermin gene clusters encode an additional accessory factor LanH, which enhances LanFEG-mediated immunity (Hille et al. 2001).

In addition to its widespread use as a food preservative, nisin and other members of the lantibiotic class have been investigated for their potential applications in medicine. The MICs of mutacin B-Ny266 and nisin A were shown to be comparable to those of vancomycin and oxacillin against various bacterial pathogens (Mota-Meira et al. 2000). Both lantibiotics were active against vancomycin- and oxacillin-resistant strains of Helicobacter pylori and Neisseria spp., making them potential candidates for treatment of infections caused by these bacteria (Hancock 1997; Mota-Meira et al. 2000). A novel potential application of nisin is as a spermicidal contraceptive. Studies with rabbits indicated that vaginal administration of 1 mg of nisin stopped sperm motility completely, none of the treated animals having become pregnant (Reddy et al. 2004). Complete histopathologic evaluation of the vagina indicated no adverse effects resulting from the intravaginal application of nisin, in terms of either tissue damage or subsequent reproductive performance (Aranha et al. 2004; Reddy et al. 2004). A future direction for lantibiotic application may involve the rational design of new peptides based on desirable structural features of some well-characterized biologically active peptides such as nisin. An analysis of 37 known lantibiotics indicated that although there were no hard and fast rules, Ser/Thr residues were more likely to be dehydrated when flanked by hydrophobic amino acids than by hydrophilic residues. To test the predicted dehydration sequence rules, hexapeptide-encoding sequences were fused to the nisin leader peptide, and expressed in a L. lactis strain containing the nisin modification and export enzymes. Analysis of the composition of the hexapeptide products confirmed the designers' predictions, demonstrating the feasibility of rational design of novel peptides having specific dehydrated amino acid residues (Rink et al. 2005).

As ever more lantibiotics are being detected, it has become increasingly obvious that a continuum of natural variants exists, some exhibiting only a single amino acid residue difference from previously documented lantibi-otics, but others having multiple sequence variations. A variant of nisin produced by Streptococcus uberis strain 42 has recently been identified (Wirawan et al. 2006), the first of the nisin family not produced by a Lactococcus strain. The biologically active 31-amino acid (aa) nisin U differs from the 34-aa nisin A in 12 of its amino acids (82% similarity of the propeptides; Fig. 4.2a). Nisin U is predicted to share the same bridging pattern as nisin A, and the producer strains of nisin A and nisin U are cross-immune. This apparent cross-immunity to the two nisin peptides is particularly interesting, since the putative immunity peptide for nisin U, NsuI, shares only 55% homology with NisI. By contrast, there is no indication of cross-immunity between subtilin (from Bacillus subtilis) and nisin, despite them having 60% propeptide sequence similarity (McAuliffe et al. 2001b). In addition, the antimicrobial spectrum of nisin U appears to match closely that of nisin A, although there appears to be some relative reduction in the activity of nisin U against S. pyo-genes and L. lactis strains. Significantly, nisin U and nisin A exhibited both auto-inducing and cross-inducing activity when added to cultures of the respective nisin-producing Lactococcus and Streptococcus strains, further emphasizing the close functional identity of the two peptides and justifying the classification of the S. uberis lantibiotic as a nisin variant. The nisin U genetic locus comprises 11 open reading frames, closely similar to their nisin A counterparts, but with nsuPRKFEG located upstream of nsuA rather than downstream of nsuI, as in the nisin A locus (Fig. 4.2b). The nisin U locus is flanked by transposon-related sequences, and also has a 742-bp region between nsuG and nsuA encoding remnants of a transposase (Fig. 4.2b), indicating that a rearrangement of the locus has occurred. Streptin is a 23-aa type AI lantibiotic produced by S. pyogenes strain M25 exhibiting similarity in its first two ring structures with the corresponding region in nisin (Karaya et al. 2001; Wescombe and Tagg 2003). The streptin locus appears similar to that of subtilin, in that it does not encode a specific protease (LanP) for propeptide a

Nisin A

Nisin U

SA-FF22

Mersacidin

Cinnamycin b

Nisin A

Nisin U

SA-FF22

Fig. 4.2 a The primary structures of the type A lantibiotics nisin, nisin U and SAFF-22, and the representative type B lantibiotics mersacidin and cinnamycin. Modified amino acid abbreviations: a, d-alanine; B, 2,3-dihydrobutyrine; O, 2,3-dihydroalanine; u, d-a-aminobutyric acid; a-S-A, lanthionine (Lan); u-S-A and A-S-u, methyllanthionine (MeLan); A-NH-K, lysinoala-nine; D-OH, erythro-3-hydroxyaspartic acid. All other conventional amino acids are given in one-letter code. The solid lines represent the Lan and MeLan bridges that have been confirmed experimentally, whereas those with dotted lines are predicted. b Organization of the biosyn-thetic loci of nisin A, nisin U and SA-FF22. Note the different order of the lanPRKFEG genes between the nisin A and nisin U loci. The X symbols in the nisin U locus represent remnants of mobile genetic elements (see text)

activation (Stein and Entian 2002). Rather, it appears that the producers of subtilin and streptin utilize other host cell proteases to remove the lantibiotic leader sequences, probably following prepeptide export. In the case of streptin, the S. pyogenes cysteine proteinase, SpeB, has been implicated in the prepeptide cleavage reaction, since proteinase-negative mutants of strain M25 concomitantly lose the ability to express the streptin phenotype (Hynes and Tagg 1986; S. O'Brien and J.R. Tagg, unpublished data). The utilization of host cell proteases for the processing of lantibiotics could be viewed as an efficient way to reduce the metabolic burden of lantibiotic production, although it may limit the dissemination of the locus to other species.

4.2.2 Type AII Lantibiotics

The type AII lantibiotics differ from those in type AI in that their thioether ring formation is effected by bifunctional LanM enzymes, rather than by the

Mersacidin

Cinnamycin

Fig. 4.2 a The primary structures of the type A lantibiotics nisin, nisin U and SAFF-22, and the representative type B lantibiotics mersacidin and cinnamycin. Modified amino acid abbreviations: a, d-alanine; B, 2,3-dihydrobutyrine; O, 2,3-dihydroalanine; u, d-a-aminobutyric acid; a-S-A, lanthionine (Lan); u-S-A and A-S-u, methyllanthionine (MeLan); A-NH-K, lysinoala-nine; D-OH, erythro-3-hydroxyaspartic acid. All other conventional amino acids are given in one-letter code. The solid lines represent the Lan and MeLan bridges that have been confirmed experimentally, whereas those with dotted lines are predicted. b Organization of the biosyn-thetic loci of nisin A, nisin U and SA-FF22. Note the different order of the lanPRKFEG genes between the nisin A and nisin U loci. The X symbols in the nisin U locus represent remnants of mobile genetic elements (see text)

combined action of LanB and LanC, and also because they generally have the conserved consensus sequences E(L/V)S and E(L/M) in their leader peptides. Furthermore, their leader sequences resemble more closely those of class II bacteriocins, in that they contain a "double-glycine" (GG/GA/GS) motif immediately preceding the cleavage site (McAuliffe et al. 2001a; Chatterjee et al. 2005). This group of lantibiotics also includes a most unusual member, sublancin 168 produced by a B. subtilis strain, which appears to be the first bacteriocin to contain both lanthionine ring structures and stabilizing disulfide bonds (Paik et al. 1998).

Although lacticin 481 is largely touted as the prototype of this subclass, the first of this group to be characterized was actually the 26-aa lantibiotic strep-tococcin A-FF22 (SA-FF22) produced by S. pyogenes strain FF22 (Jack and Tagg 1991, 1992). Lacticin 481, now extensively characterized, is a 27-aa lantibiotic containing two Lan, one MeLan and one Dhb residue (Piard et al.

1992). Interestingly, the lacticin 481 genetic locus, unlike that of most type AI lantibiotics, appears not to encode a two-component sensor-kinase response-regulator system, rather being regulated at the transcriptional level by pH control of P1 and P3 promoters located upstream of the structural gene lctA (Hindre et al. 2004). By contrast, the locus encoding SA-FF22 in S. pyogenes does have a two-component sensor-kinase response-regulator system, but this responds not to the inhibitory lantibiotic SA-FF22 but to another putative signal molecule (P.A. Wescombe, unpublished data). In fact, the molecular mechanisms of lantibiotic regulation are strikingly diverse, with examples of

1. negative regulation of members of the type AII (e.g., lactocin S; Rawlinson et al. 2002) and two-component lantibiotics (e.g., lacticin 3147, McAuliffe et al. 2001a; cytolysin, Haas et al. 2002),

2. no genes encoding regulatory elements within the locus (e.g., lacticin 481; Hindre et al. 2004),

3. homologous (auto) regulation (e.g., nisin, Kuipers et al. 1995; salivaricin A, Upton et al. 2001),

4. heterologous regulation using a signal peptide differing from the induced lantibiotic (e.g., SA-FF22; P.A. Wescombe, unpublished data), and

5. regulation by two peptides, each influencing expression of different genes within the locus (for example, the mersacidin locus encodes the response regulators MrsR1 and MrsR2, where MrsR1 regulates immunity gene expression and MrsR2 regulates lantibiotic biosynthesis; Guder et al. 2002).

In our laboratory, we have conducted extensive research on salivaricin A (SalA), a 22-aa type AII lantibiotic produced by S. salivarius (Ross et al.

1993). Five closely related variants of SalA have recently been described (Wescombe et al. 2006b), each shown to effect both auto- and heterologous induction of SalA production in the respective host strains. The novelty of SalA lies in the wide distribution of the SalA (and variant) locus in Streptococcus species, having now been detected in S. salivarius, S. pyogenes,

Streptococcus dygalactiae and Streptococcus agalactiae. Oddly, the structural gene salA1 was detected in S. pyogenes of 65 different M serotypes (Simpson et al. 1995). Only two strains (of serotypes 11 and 37) did not harbor salA1. At first glance, this appears to be anomalous, since the majority of S. pyogenes are inhibited by SalA when tested in vitro (Ross et al. 1993). However, it has now been demonstrated that, other than in serotype M4T4 S. pyogenes, all SalA1 loci are non-functional, due (at least in part) to either deletions in the genes encoding SalM and SalT, or frameshift mutations in the salT gene (Wescombe et al. 2006b). It is tempting to speculate that this very common retention, especially of the immunity-associated components of the SalA locus in S. pyogenes, may be ecologically driven, as both S. pyogenes and S. salivarius are inhabitants of the human oral cavity. The S. pyogenes serotype M11 and M37 prototype strains are unusual in that they do not possess the SalA immunity genes (P.A. Wescombe et al., unpublished data). The M11 strain is an A-variant S. pyogenes, thought to have lost the ability to assemble intact group A carbohydrate during the course of prolonged serial subculture in vitro (D. Johnson, personal communication). The lack of an obvious selective advantage associated with SalA immunity for S. pyogenes strains grown for prolonged periods as laboratory monocultures could favor the loss of immunity-related components of the locus. The M37 prototype strain is also very unusual, in that no other examples of strains of this serotype appear to have been isolated (D. Johnson, personal communication). Both of these observations are consistent with a survival advantage for S. pyogenes in situ being linked to their retention of at least the immunity-related components of the salA locus. Hence, in this case, the acquisition and retention of lantibi-otic genetic elements may have contributed to the adaptation and survival of a bacterial species.

It has recently been observed that the SalA locus is borne on large (>150 kb) plasmids in S. salivarius, whereas in S. pyogenes the locus appears typically to be chromosomally located. The ubiquitous presence of salA in S. pyogenes indicates that the acquisition of this locus was an early event in the establishment of the species, or at least that only strains of S. pyogenes that are capable of expressing SalA immunity have maintained associations with the human host. The large plasmids in bacteriocin-producing S. salivarius have been found to harbor loci for various combinations of streptococcal lantibiotics including salivaricin A, salivaricin B, streptin, and a variant of SA-FF22. The lantibiotic loci appear to be juxtaposed in contiguous segments, separated by no more than ca. 4 kb of non-lantibiotic-related DNA. Moreover, genes encoding Tra-like proteins (potentially involved in conjuga-tive transfer of the plasmids) have also been identified. These observations support the hypothesis that cassettes of lantibiotic loci could be disseminated together, thereby rapidly expanding the antimicrobial arsenal of the recipient strain. Indeed, in vivo transfer of the entire 180 kb of S. salivarius K12 bacteriocin-associated plasmid to indigenous S. salivarius has been demonstrated to occur in the oral cavity of subjects colonized with the probiotic

S. salivarius K12 (Wescombe et al. 2006a). The wide distribution of closely related lantibiotic loci throughout different oral streptococcal species indicates a high frequency of horizontal gene transfer. In the case of S. salivarius, the large plasmids appear to have been particularly effective at acquiring additional bacteriocin loci, and our preliminary findings indicate that most BLIS-producing S. salivarius strains have plasmids of size >40 kb.

4.2.3 Type B (Globular) Lantibiotics

The type B lantibiotics are more globular and compact in shape than those of type A, and generally are either uncharged or negatively charged at neutral pH. Mersacidin, the prototype for this group, is a 20-aa peptide (mass 1,825 Da) and its distinctive features include three MeLan rings, one Dha and a S-[(Z)-2-aminovinyl]-(3S)-3-methyl-D-cysteine (AviMeCys) residue (Chatterjee et al. 1992). Mersacidin, which derives its name from its potent activity against methicillin-resistant St. aureus (MRSA, the hospital-acquired "superbug"), is also the only lantibiotic of which the structure has been resolved by X-ray crystallography (Schneider et al. 2000). Mersacidin does not form pores in bacterial membranes, but rather inhibits peptidoglycan synthesis through a specific interaction with the peptidoglycan precursor lipid II (Brotz et al. 1997). The sequestering of lipid II prevents its utilization by the transpeptidase and transglycosylase enzymes that install the crosslinked network of the bacterial cell wall. Both nisin and mersacidin appear to bind to a different portion of lipid II than does vancomycin (the antimicrobial of last resort for the treatment of multiply antibiotic-resistant St. aureus), indicating that these molecules may prove to have important chemotherapeutic applications (Brotz et al. 1995; Breukink et al. 1999). Indeed, mersacidin has been shown to be very effective for the treatment of systemic staphylococcal infections, and in eliminating nasal carriage of St. aureus in a mouse model system (Chatterjee et al. 1992; Kruszewska et al. 2004). Lipid II, however, does not serve as a docking molecule for all lantibiotics, since Pep5 and epilancin K7 have been shown specifically not to bind lipid II. These molecules presumably have an alternative docking molecule or receptor, since they have greater activity than other pore-forming molecules against certain indicator bacteria (Brotz et al. 1998; Pag et al. 1999).

Cinnamycin is a 19-aa type B lantibiotic produced by Streptomyces cinna-moneus, and has also been purified as Ro 09-0918 (Kessler et al. 1987) and lanthiopeptin (Naruse et al. 1989; Palmer et al. 1989). Its structure is novel in that it has two MeLan residues in which the nucleophilic cysteine is positioned N-terminally to the Dhb. Although also found in some other type B lantibiotics, this direction of ring formation has so far not been observed in any of the type A lantibiotics other than the LtnA1 peptide of the lacticin 3147 two-component lantibiotic system. Another unusual feature of cinnamycin is a head-to-tail lysinoalanine bridge, the formation of which has so far not been ascribed to any particular gene product in the comprehensive array of putative ORFs identified in the cinnamycin locus. Intriguingly, the CinA prepeptide has a much longer leader peptide than that of other lantibiotics, and it has been proposed that cinnamycin may be secreted by a more general export mechanism such as the general secretory (Sec) pathway, once again illustrating the broad diversity of the lantibiotic class (Widdick et al. 2003). Cinnamycin has been shown to be a potent inhibitor of phospholipase A2 (an enzyme involved in the synthesis of prostaglandins and leukotrienes in the human immune system) through the sequestration of its substrate phos-phatidylethanolamine. Due to this activity, cinnamycin may prove to have a useful application as an anti-inflammatory and anti-allergy drug (Marki et al. 1991).

4.2.4 Type C (Multi-Component) Lantibiotics

Each of the multi-component lantibiotic consortia described to date comprise two post-translationally modified peptides that individually have little or no activity, but display strong synergistic antibacterial action. Lacticin 3147 produced by Lactococcus lactis DPC3147 is arguably the most intensively studied member of this group, and both component peptides (LtnA1 and LtnA2) have been structurally characterized (Ryan et al. 1999). Features of the peptides include Lan and MeLan residues, and also a D-Ala residue derived from L-Ser by post-translational modification. Interestingly, the structure of LtnA1 bears some resemblance to that of the type B lantibiotic mersacidin, and the LtnA2 peptide displays some similarity to lactocin S (a type AII lantibiotic). The obvious structural differences between LtnA1 and LtnA2 therefore require that the genetic locus for lacticin 3147 encode two LanM enzymes, each of which is presumably responsible for the post-translational modification of one of the peptides (McAuliffe et al. 2000). The mechanism of action of lacticin 3147 has recently been shown to result from the sequential action of the two peptides, on condition that LtnA1 be added prior to LtnA2 (Morgan et al. 2005). It was therefore inferred that LtnA1 binds lipid II (a reaction responsible for the independent inhibitory activity displayed by LtnA1), following which LtnA2 interacts with the LtnA1-lipid II complex to bring about more effective insertion into the target membrane and pore formation, with an associated 30-fold increase in inhibitory activity compared to that obtained by LtnA1 alone (Morgan et al. 2005).

Smb (Streptococcus mutans bacteriocin) was recently shown to be a two-component lantibiotic (Yonezawa and Kuramitsu 2005). Expression of SmbA and SmbB by the smb locus appears to be regulated in response to the external levels of a competence-stimulating peptide (the peptide that activates the development of competence for genetic transformation; see below). It is possible that the production of the lantibiotic has been coupled to the competence cascade to ensure that there is an abundance of exogenous DNA available for uptake by the newly competent bacteria. Alternatively, it may be that the apparent co-regulation is purely a consequence of the insertion of the lantibiotic locus into a region having the competence promoter upstream. A variant of the Smb lantibiotic, named BHT-A, was recently identified in Streptococcus rattus strain BHT, and shown to be composed of the two peptides BHT-Aa and BHT-AP (Hyink et al. 2005). Interestingly, the Smb/BHT-A locus appears to be closely linked to the locus for BHT-B, a class II bacteriocin, in all S. rattus strains examined to date (Hyink et al. 2005).

Cytolysin, produced by Enterococcus faecalis, consists of two lantibiotic subunits (CylLL and CylLS), and is the only lantibiotic confirmed to exhibit toxicity for both bacterial and eukaryotic cells. Although not all strains of E. faecalis are hemolytic, the occurrence of hemolysis is higher among clinical isolates, especially those from the bloodstream (Booth et al. 1996). As many as 60% of infection-associated E. faecalis elaborate cytolysin, and it has been shown to lower the LD50 of E. faecalis for mice and to contribute to tox-icity in experimental endocarditis and endophthalmitis models. In addition, cytolysin-positive strains are associated with a fivefold increased risk of acute terminal outcome in patients with nosocomial enterococcal bacteremia (Coburn et al. 1999). Interestingly, cytolysin is encoded on large pheromone-responsive conjugative plasmids, which may, at least in part, account for the high prevalence of the cytolysin locus in enterococci.

The CylLL and CylLS subunits are activated by a two-step process involving initial transport and GG site-specific cleavage (to CylLL' and CylLS') mediated by the ABC transporter CylB, followed by removal of a further six amino acids (forming CylLL'' and CylLS'') by the serine protease CylA. It was shown that in order for the cytolysin to efficiently lyse erythrocytes and bacterial cells, both subunits need to be fully processed by CylA (Booth et al. 1996).

Expression of the cytolysin locus is directly regulated by the synergistic action of two repressor proteins CylR1 and CylR2, both of which lack homologues of known function (Haas et al. 2002). De-repression occurs at a specific cell density when one of the cytolysin subunits (CylLS'') reaches an extracellular threshold concentration. These observations form the basis for a model of cytolysin auto-induction by a quorum-sensing mechanism involving a novel two-component regulatory system (Haas et al. 2002). CylLL and CylLS expression is further regulated in response to aerobiosis, with transcription being up-regulated under anaerobic conditions (Day et al. 2003).

Comparison of the cytolysin determinants with those of the type AII lan-tibiotic lactocin S (from Lactobacillus sake) indicates they may share a common ancestry (Gilmore et al. 1996). Although no cytotoxicity for eukaryotic cells has been reported for lactocin S, it seems prudent to perform toxicity tests on any lantibiotics (particularly two-component forms) that may have human or veterinary applications to assess their potential for disruption of eukaryotic membranes. Indeed, this revelation of the dual toxicity of cytolysin sounds a timely warning for those contemplating the engineering of novel lantibiotics, since it demonstrates the potential for these molecules to exhibit toxicity for eukaryotic cells, perhaps sometimes by forming multi-component membrane poration complexes in combination with het-erologous bacteriocins produced by indigenous bacteria (Wescombe et al. 2005).

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