Cross-immunity and immune mimicry as mechanisms of resistance to the lantibiotic lacticin 3147

Authors


*E-mail p.cotter@ucc.ie; Tel. (+353) 21 4903146; Fax (+353) 21 4903101;

**E-mail c.hill@ucc.ie; Tel. (+353) 21 4901373; Fax (+353) 21 4903101.

Summary

Lantibiotics are antimicrobial peptides that possess great potential as clinical therapeutic agents. These peptides exhibit many beneficial traits and in many cases the emergence of resistance is extremely rare. In contrast, producers of lantibiotics synthesize dedicated immunity proteins to provide self-protection. These proteins have very specific activities and cross-immunity is rare. However, producers of two peptide lantibiotics, such as lacticin 3147, face the unusual challenge of exposure to two active peptides (α and β). Here, in addition to establishing the contribution of LtnI and LtnFE to lacticin 3147 immunity, investigations were carried out to determine if production of a closely related lantibiotic (i.e. staphylococcin C55) or possession of LtnI/LtnFE homologues could provide protection. Here we establish that not only are staphylococcin C55 producers cross-immune to lacticin 3147, and therefore represent a natural repository of Staphylococcus aureus strains that are protected against lacticin 3147, but that functional immunity homologues are also produced by strains of Bacillus licheniformis and Enterococcus faecium. This result raises the spectre of resistance through immune mimicry, i.e. the emergence of lantibiotic-resistant strains from the environment resulting from the possession/acquisition of immunity gene homologues. These phenomena will have to be considered carefully when developing lantibiotics for clinical application.

Introduction

Lantibiotics are potent post-translationally modified antimicrobial peptides produced by many Gram-positive bacteria (Guder et al., 2000; McAuliffe et al., 2001a; Pag and Sahl, 2002; Chatterjee et al., 2005; Cotter et al., 2005). A number of these antimicrobials target the essential peptidoglycan precursor lipid II, are active at nanomolar concentrations, have a wide spectrum of activity (including activity against multidrug resistant pathogens), and have been the subject of much research focusing on their application in clinical and food settings (Galvin et al., 1999; Wiedemann et al., 2001; Brumfitt et al., 2002; Kruszewska et al., 2004; Cotter et al., 2005; Breukink and de Kruijff, 2006). Encouragingly, the development of spontaneous resistance is, in many cases, rare (Guinane et al., 2006). In contrast, lantibiotic-producing strains need to withstand high concentrations of the antimicrobial that they produce, i.e. it must be immune. This is achieved through the production of immunity proteins, typically consisting of either an individual small immunity protein (generically designated as LanI) or an ABC transporter [usually composed of either two or three different subunits, generically designated as LanFE(G)], which, to date, have only been identified in lantibiotic-producing strains. Indeed, some lantibiotic-producers, such as those that produce nisin, subtilin or lacticin 3147, possess both LanI and LanFE(G) systems while, in the case of the gallidermin producer, an additional nonessential factor, LanH, which acts as an ancillary protein for the assembly of the LanFE(G) transporter, has also been reported (Peschel et al., 1997) (for review see Draper et al., 2008). In all cases the genes responsible for these immunity determinants have been found to be colocated with the corresponding biosynthetic genes. Although the LanI peptides are thought to function by lantibiotic interception or target shielding (Hoffmann et al., 2004; Stein et al., 2005), they share little homology and cross-immunity is rare, even between closely related lantibiotics. While homology between ABC transporter components, which reduce the concentration of cell-associated lantibiotic through peptide transport, is greater, this is usually limited to the ATP-binding (LanF) component and cross-immunity is again rare (Heidrich et al., 1998; Aso et al., 2005).

The majority of research on lantibiotic immunity has focused on immunity to single peptide lantibiotics (e.g. Hoffmann et al., 2004; Stein et al., 2005; Takala and Saris, 2006; Okuda et al., 2008). However, there also exists a subset of strains which produce and are immune to lantibiotics which act through the synergistic activities of two antimicrobial peptides, i.e. two peptide lantibiotics such as lacticin 3147 (Lactococcus lactis) and staphylococcin C55 (Staphylococcus aureus) (for review see Lawton et al., 2007). Lacticin 3147, as a consequence of the combined activity of Ltnα and β (formerly termed LtnA1 and A2), is active at a single nanomolar level (Morgan et al., 2005; Cotter et al., 2006a; Wiedemann et al., 2006). Lacticin 3147 targets lipid II (Wiedemann et al., 2006), with binding facilitating a dual mechanism of action, i.e. the inhibition of cell wall synthesis followed by the dissipation of the membrane potential of target organisms. A 60.2 kb lactococcal plasmid pMRC01 contains the genes encoding Ltnαβ (i.e. ltnA1 and A2 respectively), and these are colocated in a 12.6 kb region with other genes associated with lacticin 3147 biosynthesis and immunity in two divergent operons, ltnA1A2M1TM2J and ltnRIFE respectively. It has been previously established that both the LtnI (LanI) and LtnR proteins play a role in lacticin 3147 immunity (McAuliffe et al., 2000; 2001b). LtnI is a predominantly hydrophobic, 116-amino-acid protein which, like the majority of LanI peptides discussed above, shows no homology to any other characterized immunity peptides (McAuliffe et al., 2000). LtnR functions as a regulator of the promoter driving the immunity operon, Pimm (McAuliffe et al., 2001b). Surprisingly, despite apparently encoding LanFE-like proteins, previous results have suggested that ltnFE are not involved in the provision of protection (McAuliffe et al., 2000), and thus that immunity to lacticin 3147 is provided by LtnI alone.

In this study we carry out the most detailed and systematic investigation of immunity to a two-peptide lantibiotic to date. We prove that LtnFE contributes to lacticin 3147 immunity and quantify the contribution of LtnF, E and I to the protection provided. Furthermore, in addition to establishing that producers of staphylococcin C55 are cross-immune to lacticin 3147, we reveal for the first time the potential for the emergence of lantibiotic resistance through immune mimicry, i.e. the spread of non-lantibiotic-producing strains which are lantibiotic resistant by virtue of possessing immunity gene homologues.

Results

Sequencing of the plasmid pOM01

In previous studies, the contribution of individual genes to lacticin 3147 immunity was assessed with the help of a number of subclones. As pOM01 (containing ltnRIFE) and pOM14 (ltnRI) provide an equal level of protection against lacticin 3147, it was assumed that ltnFE did not contribute to immunity and that LtnI was the sole immunity determinant (McAuliffe et al., 2000). We can now report that recent sequencing of pOM01 has revealed the presence of errors within all three subcloned immunity genes. A single point mutation within LtnI results in a Y66C change, while an F59S change occurs in LtnF and the introduction of a stop codon within ltnE resulted in a truncated protein at amino acid position 109. While these misincorporations (a PCR-based strategy was used in the initial cloning experiments) do not impact on the protection provided by LtnI, they provided an explanation for the apparent non-involvement of LtnFE in immunity.

Determination of the sensitivity of deletion mutants to lacticin 3147

In light of the errors in pOM01, we sought to reassess the contribution of the individual ltnI, F and E genes, through the non-polar deletion of different combinations of these genes from within the natural pMRC01 plasmid. The deletions were generated in a non-producing strain (MG1363) containing a pMRC01 derivative lacking the structural genes, ltnA1A2[pMRC01(ΔA1A2); Cotter et al., 2003], to ensure that any impact on the immunity of the strain would not lead to self-killing by lacticin 3147. The net result was the generations of ΔltnI, ΔltnF, ΔltnFE and ΔltnIFE mutations were made against the pMRC01(ΔA1A2) background. The relative contribution of LtnI and FE to lacticin 3147 immunity was then investigated through minimum inhibitory concentration (MIC) determination studies using equimolar concentrations of Ltnα and β. It was established that the MICs of lacticin 3147 against the sensitive indicator strains L. lactis HP and L. lactis MG1363 are 7.8 nM and 31.25 nM respectively, i.e. substantially more sensitive than the pMRC01(ΔA1A2) control, the MIC of which was 625 nM (Table 1, Fig. S1), thereby highlighting the highly effective nature of natural immunity. Curiously, the MIC for a MG1363 derivative containing pMRC01 (MG1363pMRC01) was even greater (2.5 μM; Table 1, Fig. S1) indicating that the production of Ltnα and Ltnβ had an impact on the level of protection provided raising the possibility that intracellular levels of Ltnα and/or Ltnβ (or precursors thereof) influence immunity gene expression, a possibility which will be the focus of targeted research in the future. When the relative sensitivities of the strains lacking ltnI, F, FE or IFE were quantified it was notable that although deletion of the ltnI gene has a detrimental impact on immunity (250 nM; a 10-fold reduction in immunity relative to MG1363pMRC01), this strain remained more immune than MG1363 (Table 1, Fig. S1), thus establishing for the first time that LtnI is not the sole provider of immunity. It became apparent that this additional protection is provided by the LtnFE proteins as their absence (MG1363pMRC01(ΔA1A2) ΔltnFE) results in a 20-fold reduction in immunity relative to MG1363pMRC01 (MIC of 125 nM). Thus, LtnFE provide immunity to lacticin 3147 and this protection is greater than that provided by LtnI. As the level of sensitivity of the ltnF mutant corresponds to that of its ΔltnFE counterpart (Table 1, Fig. S1), it is apparent that the presence of both components of the ABC transporter is necessary for functionality. It would appear that no other pMRC01-associated immunity genes exist as the combined absence of ltnIFE resulted in the complete loss of immunity, i.e. a MIC of 30 nM which equates to that of L. lactis MG1363 (Table 1, Fig. S1). While both LtnI and LtnFE are obviously capable of functioning in isolation, it is apparent from MIC values that they can function synergistically to provide even greater protection.

Table 1.  MIC determination of L. lactis strains and gene deletion mutants.
L. lactis strainaLtnαβ MICLtnα MICLtnβ MIC
  • a. 

    MG1363 derivative.

HP7.8 nM1.25 μM2.5 μM
MG136331.25 nM7.5 μM4.37 μM
pMRC01a2.5 μM15 μM35 μM
pMRC01(ΔA1A2)a625 nM7.5 μM15 μM
pMRC01(ΔA1A2) ΔltnIa250 nM15 μM17.5 μM
pMRC01(ΔA1A2) ΔltnFa125 nM15 μM35 μM
pMRC01(ΔA1A2) ΔltnFEa125 nM15 μM35 μM
pMRC01(ΔA1A2) ΔltnIFEa31.25 nM3.75 μM2.5 μM

The two-peptide nature of lacticin 3147 provided a unique opportunity to assess whether immunity is mediated by specific targeting of Ltnα and/or Ltnβ. It has previously been established that Ltnα exhibits activity, albeit greatly reduced, in the absence of its companion peptide against the indicator strain L. lactis HP (Morgan et al., 2005; Cotter et al., 2006a; Wiedemann et al., 2006). Here we confirm this result (MIC of Ltnα is 1.25 μM) and establish that the corresponding MG1363 value is 7.5 μM. Surprisingly, the ΔIFE mutant is more sensitive to Ltnα than MG1363 (3.75 μM). With respect to the individual immunity proteins, each system is equally capable of providing native levels of protection against Ltnα and there is no benefit from the combined presence of both systems [i.e. MICs for MG1363pMRC01, MG1363pMRC01(ΔA1A2) ΔltnI, -ΔFE and -ΔF are all 15 μM]. Ltnβ is half as potent as Ltnα with respect to MG1363pMRC01 and HP (in line with previous observations in the latter case; Cotter et al., 2006a) but is approximately twice as potent as its companion peptide against MG1363 and MG1363pMRC01(ΔA1A2) ΔltnIFE. Curiously, the strain containing pMRC01(ΔA1A2) ΔltnIFE (2.5 μM) was again more sensitive than MG1363 (4.37 μM). Although, this enhanced sensitivity may be as a consequence of the metabolic load associated with the maintenance of the pMRC01 plasmid, it is unclear why this impact is not evident when the strain is exposed to both peptides in combination.

Investigating the immunity of staphylococcin C55 producers to lacticin 3147

The two-peptide lantibiotic staphylococcin C55 closely resembles lacticin 3147, with the α peptide differing with respect to only four residues while the β peptides share 55% identity (O'Connor et al., 2007). The staphylococcin C55 genes are associated with a variety of large natural S. aureus plasmids which also possess genes encoding exfoliative toxin B (ETB) (Navaratna et al., 1999; Yamaguchi et al., 2001). The similarity of the two lantibiotics and observations by O'Connor et al. (2007) suggested that cross-immunity could be an issue. As lantibiotic immunity genes are always colocated with those encoding the structural peptides and biosynthetic machinery, it was deemed highly likely that the C55 immunity genes are also plasmid-located (the S. aureus C55 32 kb plasmid is hereafter referred to as pC55i), although genes which closely resemble ltnI, E or F could not be located on the plasmid. To test if immunity genes were indeed plasmid located, the immunity of S. aureus C55 (C55 pC55i+) and a pC55i cured derivative (C55 pC55i-) to staphylococcin C55 was assessed. It was apparent that possession of pC55i did provide protection against the lantibiotic as in its absence the strain became sensitized (Fig. 1). This contrasts with previous observations involving another staphylococcin C55 producer (TY4) and a plasmid-cured derivative thereof strains suggesting the presence of additional resistance determinants on the genome of TY4. The parental and cured strains of S. aureus C55 were also tested to determine if pC55i also provided cross-immunity to lacticin 3147. The enhanced sensitivity of the cured strain confirmed that possession of the plasmid bestowed a cross-immune phenotype (Fig. 1). Lacticin 3147 MIC determination studies confirmed this phenomenon, with the MIC decreasing from 3.75 μM to 1.87 μM as a consequence of plasmid curing.

Figure 1.

Agar well diffusion assays comparing the resistance of Staphylococcus aureus C55 strains possessing (pC55i+) and lacking (pC55i-) pC55i to staphylococcin C55 (acell-free supernatant) and lacticin 3147 (bpartially purified preparation).

The in silico identification of homologues of LtnI, F and E

It has been highlighted that there is little homology between the LanI and LanE equivalents in various different lantibiotic clusters (McAuliffe et al., 2001a; Twomey et al., 2002; Chatterjee et al., 2005). However, due to the enormous volume of bacterial DNA sequence information that has been generated in recent years and the re-evaluation of the role of LtnFE, an in silico approach was taken to re-investigate the possibility that lacticin 3147 immunity protein homologues exist. Homology searches were performed using BLASTP (National Centre for Biotechnology Information; Altschul et al., 1997) and, surprisingly, hypothetical proteins with significant homology to LtnI were identified in Bacillus sp. NRRL B-14911, Bacillus licheniformis DSM 13, Bacillus cereus G9241 and Bacillus thuringiensis serovar konkukian str. 97-27 (Table 3, Fig. S2). Due to the abundance of ABC transporters in nature and the highly conserved nature of the associated ATPase domains, it was unsurprising that a vast number of proteins with homology to LtnF were identified. Of these the proteins with the highest percentage identity and E values were encoded within the S. aureus ssp. aureus NCTC 8325, Enterococcus faecium DO, Lactobacillus casei ATCC 334 and Lactobacillus plantarum WCFS1 genomes (Table 3, Fig. S2). As expected, because the integral membrane components of ABC transporters are much less conserved, fewer LtnE homologues were located. The most closely related of these were encoded within the genomes of E. faecium DO-, Lb. plantarum WCFS1-, Staphylococcus epidermidis ATCC 12225- and S. aureus ssp. aureus NCTC 8325-located genes (Table 3, Fig. S2). Thus, three of the four most homologous proteins in each case are associated with the same strain, and indeed it was established that in these three cases, the two ABC transporter-associated genes were contiguous suggesting that they combine to form LtnFE-like ABC transporters. All of the LtnF-like proteins contain a conserved sequence representative of COG1131, which is associated with the ATPase component of ABC-type multidrug transport systems. In contrast no specific functional domain was identified in the LtnE-like proteins.

Table 3.  Proteins homologous to LtnI, F or E.
Strain producing homologue% identityE value
LtnI homologues  
 Bacillus sp. NRRL B-14911434e-22
 Bacillus licheniformis DSM 13371e-16
 Bacillus cereus G9241314e-07
 Bacillus thuringiensis serovar konkukian str. 97-27304e-06
LtnF homologues  
 S. aureus ssp. aureus NCTC 8325465e-49
 Enterococcus faecium DO456e-53
 Lactobacillus casei ATCC 334421e-54
 Lactobacillus plantarum WCFS1403e-56
LtnE homologues  
 Enterococcus faecium DO382e-44
 Lactobacillus plantarum WCFS1361e-44
 Staphylococcus epidermidis ATCC 12225359e-41
 S. aureus ssp. aureus NCTC 8325323e-37

Assessment of the protection provided by LtnI- and LtnFE-like proteins

Heterologous production was selected as a means to determine if any of the newly identified LtnI- and LtnFE-like proteins protected against lacticin 3147. However, in order to establish the significance of any protection that might be provided, it was necessary to generate a number of control constructs for comparative purposes. To this end ltnI, ltnFE and ltnIFE were cloned into pNZ44 under the control of the P44 promoter and introduced into MG1363 and the individual MG1363pMRC01(ΔA1A2) immunity mutants. As the addition of these genes on a multicopy vector such as pNZ44 has the potential to enhance immunity, the constructs were also introduced into MG1363pMRC01 to determine if a hyper-immune phenotype could be achieved. Having first confirmed that the presence of pNZ44 does not impact on the sensitivity of MG1363 to lacticin 3147 (Table 2, Fig. S1), it was then established that the introduction of pNZ44ltnI increases the immunity of MG1363 by 40-fold, to a level half of that conferred by pMRC01 (Table 2, Fig. S1) but that the presence of pNZ44ltnFE had an even more dramatic impact, resulting in a 160-fold increase in immunity to twice that conferred by pMRC01 (Table 2, Fig. S1). This was also the case for pNZ44ltnIFE, thus demonstrating that the additional presence of ltnI on this construct provides no further protection. The reintroduction of these constructs into the pMRC01 deletion mutants resulted in complementation but failed to enhance immunity beyond that of the corresponding MG1363 transformants. However, immunity was enhanced further when the constructs were introduced into a strain containing pMRC01 (Table 2, Fig. S1). In these circumstances pNZ44ltnI increased immunity by at least twofold relative to that associated with either pNZ44ltnI or pMRC01 alone and in the situations where additional copies of ltnFE were present (either as pNZ44ltnFE or pNZ44ltnIFE), immunity is increased fourfold relative to MG1363pMRC01 and by 320-fold relative to MG1363 (Table 2, Fig. S1). Having established the levels of protection provided by ltnI, F and E when heterologously expressed in this manner, strains with genomes containing an ltnI-like gene, i.e. Blicheniformis DSM 13, and all three that contain both ltnF and E-like genes, i.e. Lbplantarum WCFS1, S. aureus ssp. aureus NCTC 8325 and E. faecium DO, were acquired and the corresponding genes BLi00574 (hereafter bliI), lp_ 1791–92 (hereafter lpwFE), SAOUHSC_02820–21 (hereafter sanFE) and EfaeDRAFT_ 1830–31 (hereafter efdFE), respectively, were amplified by PCR and introduced into pNZ44 in the same manner. Once their integrity was confirmed, the resultant recombinant plasmids were introduced into MG1363 and the protection provided against lacticin 3147 was assessed by well diffusion assays (Fig. 2). These assays revealed that although the MG1363pNZ44sanFE and pNZ44lpwFE strains remained sensitive to lacticin 3147, the strains containing either pNZ44bliI or pNZ44efdFE exhibited enhanced resistance and thus provide protection through a previously unreported phenomenon. Unfortunately, multiple attempts to assess if the corollary was also true, i.e. does mutation of bliI or efdFE in their natural background result in enhanced lacticin 3147 sensitivity, failed due to an inability to create the mutations targeted. Further investigations established, again through agar well diffusion assays, that all strains expressing LtnI and LtnFE homologues remained sensitive to the distantly related one peptide lantibiotic nisin (data not shown) and thus that the protection provided is not as a consequence of general non-specific enhanced lantibiotic resistance. As a consequence of the interesting phenomenon observed, the resistance of each strain to lacticin 3147 was ascertained more precisely by MIC determination (Table 2, Fig. S1). Expression of bliI or efdFE from pNZ44 both results in levels of resistance just fourfold less than the immunity provided by the plasmid pMRC01 and 20-fold greater than that of L. lactis MG1363. This level of self-protection is also just half of that of MG1363pNZ44ltnI, indicating that a relatively high level of protection is provided by these genes. It is noteworthy that in contrast to the immunity provided by LtnFE/LtnI, in this instance the relative protection provided by the ABC transporter (EfdFE) does not exceed that of the LtnI homologue, BliI. As expected the MIC values for the sanFE and lpwFE-expressing strains were equal to that of MG1363, therefore confirming that no protection was provided. To establish if coproduced BliI and EfdFE could function synergistically, bliI was cloned into pNZ44E, a derivative of pNZ44, and introduced into MG1363pNZ44efdFE cells. MIC determination revealed a 12 μM concentration of lacticin 3147 was required in order to inhibit growth of this strain (Table 2, Fig. S1). This MIC value is even higher than that for strains containing pNZ44ltnIFE or pMRC01·pNZ44ltnIFE. The protection provided by the heterologous expression of bliI and efdFE is not restricted to lactococci alone and can extend to other Gram positive microorganisms including food pathogens. Using Listeria monocytogenes strain F2365 as a model, it was established that the sensitivity of this strain to lacticin 3147 (MIC 625 nM) decreased fourfold and eightfold in the presence of pNZ44bliI (2.5 μM) and pNZ44efdFE (5 μM) respectively.

Table 2.  Lacticin 3147 MIC determination of L. lactis strains expressing immunity genes.
L. lactis MG1363 derivativeMIC
pNZ4431.25 nM
pNZ44ltnI1.25 μM
pNZ44ltnFE5 μM
pNZ44ltnIFE5 μM
pMRC01(ΔA1A2) ΔltnI. pNZ44ltnI1.25 μM
pMRC01(ΔA1A2) ΔltnFE. pNZ44ltnFE5 μM
pMRC01(ΔA1A2) ΔltnIFE. pNZ44ltnIFE5 μM
pMRC01.pNZ44ltnI5 μM
pMRC01.pNZ44ltnFE10 μM
pMRC01.pNZ44ltnIFE10 μM
pNZ44bliI0.63 μM
pNZ44efdFE0.63 μM
pNZ44sanFE31.25 nM
pNZ44lpwFE31.25 nM
pNZ44efdFE.pNZ44EbliI12 μM
pNZ44sanFltnE0.63 μM
pNZ44lpwFltnE2.5 μM
pNZ44ltnFlpwE78 nM
Figure 2.

Agar well diffusion assays revealing the protection provided by pNZ44 constructs containing ltnI, ltnFE and/or homologues thereof (% identity of resulting proteins is indicated) when expressed in MG1363 against cell-free supernatant from a lacticin 3147-producing strain.

Hybrid ABC transporters

The observation that EfdFE is the only transporter homologue capable of conferring protection correlates with the fact that the EfdE protein shares a higher level of identity with LtnE than any of the other LtnE-like proteins and supports the role of the membrane-bound portion of the ABC transporter in substrate identification and binding. Having established that constructs generated through sequential cloning of the individual ABC transporter genes (pNZ44ltnFltnE) provide an equal amount of protection as those in which the genes were introduced in one step (pNZ44ltnFE; data not shown), a number of ‘hybrid’ constructs were prepared to test the substrate recognition role of the LtnE-like proteins, i.e. ltnE was expressed in combination with lpwF and sanF, the ATP-binding portion of the ABC transporters which did not provide protection. In both cases a protective effect was observed which, although lower than that when LtnE and F were produced, was between 8-fold (SanFLtnE) and 32-fold (LpwFLtnE) greater than a LtnFLpwE-producing control, i.e. a strain producing the LanE protein from a non-effective ABC transporter. This latter combination is partially effective, enhancing the resistance of MG1363 twofold (Table 3, Fig. S1).

Discussion

This study represents the most thorough investigation of immunity to two peptide lantibiotics to date, resulting in the relative contribution of LtnI, F and E to lacticin 3147 immunity being definitively established. While the involvement of LtnI had previously been recognized, the role of the LtnFE ABC transporter had heretofore been unclear (McAuliffe et al., 2000). Here, not only has this been rectified, but the individual contribution of each of the immunity systems has been established. The erroneous conclusion that LtnFE played no role in immunity is explained by the existence of errors generated during the construction of pOM01. It is now apparent that this ABC transporter contributes greatly to the immunity phenotype of lacticin 3147-producing strains, to an even greater degree than LtnI. Unsurprisingly, in line with data from LanFE(G) systems (Stein et al., 2005), such protection only occurs when all components of the ABC transporter are present. From precise measurements of the immunity provided it is apparent that when all three proteins are present the combined immunity is greater than the additive contribution of both systems individually. This suggests that both systems act cooperatively, resulting in a heightened self-protective capability. With respect to the individual contributions of the immunity proteins, it was apparent that although LtnFE has a greater ability to protect the cell against lacticin 3147 (i.e. Ltnα and β combined) than LtnI, LtnI and LtnFE provide an equal level of protection when exposed to Ltnα only and LtnI exceeds LtnFE in its ability to protect the cell from the action of Ltnβ. In fact the absence of LtnFE in these circumstances is only apparent in cells already devoid of LtnI. This infers that the ABC transporter functions most efficiently in transporting an Ltnα:Ltnβ complex. In addition, as the coproduction of LtnI and FE has neither a synergistic nor an additive impact on immunity when exposed to the individual α and β peptides, the existence of an Ltnα:Ltnβ complex would appear to be a prerequisite for complementary functionalities. While further investigations are required to disclose the precise mechanisms via which these proteins provide protection, the definitive identification of the lacticin 3147 immunity determinants and the quantification of the levels of protection provided were crucial in assessing the cross-immunity/resistance provided by other immunity proteins and by homologues of LtnI, F and E.

Cross-immunity between lantibiotic producers is rare and has only been observed between producers of closely related lantibiotics, and even then only in exceptional cases, e.g. between producers of nukacin ISK-1 and lacticin 481 or Pep5 and epicidin (Heidrich et al., 1998; Aso et al., 2005). However, it is apparent that producers of staphylococcin C55 are cross-immune to lacticin 3147 and this cross-immunity is mediated by possession of pC55i. In silico examination of another staphylococcin C55-encoding plasmid, pETB, for which the entire sequence is available, suggests that orfs 46–48 are most likely to encode the immunity determinants. These are located immediately downstream of the lantibiotic biosynthesis and export genes and are predicted to encode the individual components of an ABC transporter (orf 46–47) and a small potentially LanI-encoding gene (orf 48). However, while, as is the case with all ABC binding components of ABC transporters, orf 46 and LtnF share homology (33% identity, E value 1e-29), this is low relative to the homology between LtnF and EfdF, SanD and LpwF. Furthermore, orfs 47 and 48 show no significant homology to LtnE or LtnI respectively, and thus further investigation is required to establish if these are indeed the staphylococcin C55 immunity proteins. The fact that S. aureus strains possessing ETB determinants are naturally cross-immune to lacticin 3147 is a cause for concern. Of exfoliative toxin producers, ETB-producing strains are most frequently isolated from children with the generalized form of staphylococcal scalded skin syndrome (Mulligan et al., 1993) and is directly associated with an increase in the number and size of lesions on children suffering from non-bullous impetigo (Koning et al., 2003). It has been speculated that the colocation of genes encoding the mono-ADP-ribosyltransferase EDIN-C on ETB- (and staphylococcin C55-) encoding plasmids may be responsible for the exacerbation of symptoms, relative to those caused by exfoliative toxin A (Yamaguchi et al., 2001). While these plasmids do not possess genes that could facilitate their spread through conjugation (Yamaguchi et al., 2001), transduction may be possible and has been observed in a laboratory setting (Rogolsky et al., 1986). The emergence of populations of staphylococci that possess or acquire such plasmids needs to be considered carefully with respect to the clinical application of lacticin 3147 against the pathogen.

Given the absence of closely related homologues of lanI, F and E on pC55i, it was particularly surprising to identify lacticin 3147 immunity gene homologues in non-lacticin 3147-producing strains. This development has implications that extend beyond simply a fundamental investigation of lantibiotic immunity, especially as two such systems (BliI and EfdFE) actually provided protection against lacticin 3147. Given that in a bacteriocin context the term immunity refers to the ability of lantibiotic-producing strains to protect themselves from which they produce, we suggest that the protection provided here represents a new method of protection, i.e. resistance as a consequence of the mimicking of lantibiotic immunity or immune mimicry. The description of bliI as an orphan immunity gene may be the subject of debate by virtue of the presence of genes encoding the structural and modification genes associated with the lantibiotic lichenicidin on the DSM 13 chromosome (Veith et al., 2004). However, to date, lantibiotic immunity genes have always been colocated with the associated biosynthetic determinants whereas in this case the putative lantibiotic-encoding genes are found within a different region of the chromosome. Furthermore, no lantibiotic biosynthetic genes have been identified in Bacillus sp. NRRL B-14911, which possesses a protein with even greater identity with LtnI. Thus the presence of lantibiotic-associated genes in strain DSM 13 may be coincidental. No lantibiotic biosynthetic genes were identified in the E. faecium DO genome. While we were unfortunately unable to mutate these genes in their natural hosts, their ability to provide protection is beyond doubt with heterologous expression of bliI or efdFE being equivalent to 50% of that provided by ltnI. It is also notable that the various immunity homologues are, like LtnE and F, capable of functioning synergistically, therefore establishing that such synergism is not the preserve of LtnFE/I combination alone. In fact MG1363 containing both pNZ44efdFE and pNZ44EbliI was the least sensitive of all the strains, though this extreme protection has almost certainly been amplified as a consequence of the involvement of two multicopy vectors. From the study of LtnFE, EfdFE and other LtnFE homologues, it is apparent that the integral membrane domain component of these ABC transporters represents the substrate recognition domain and it is thus logical that of the transporters identified, it is EfdFE, which includes the most closely related membrane domain, that provides protection. However, the margin between success and failure in this regard is quite slim in that although EfdE and LpwE differ with respect to identity to LtnE by just 2%, this small divergence results in the loss of the immunity phenotype. Although there exist a number of residues that are present in LtnE-EfdE but absent from LpwE/SanE, in silico analysis could not explain the success of the former pair of proteins and the lack of success of the latter two (Fig. S2). This is not surprising as attempts to determine the substrate binding location within integral membrane domains of ABC transporters have, in general, remained unsuccessful despite extensive research. As epitope binding efficiency can be diminished as a result of a single amino acid change (Ito et al., 2001; Mealey et al., 2006), it is possible that these differences in activity could be attributed to a single amino acid difference. Should the corollary also be true, exposure to lacticin 3147 could also select for spontaneous mutations of SanE or LpwE genes resulting in a resistant phenotype. The adaptable nature of lantibiotic immunity proteins has been demonstrated in Streptococcus pyogenes where proteins, initially dedicated to lantibiotic immunity, have evolved such that they now provide protection against macrophage-associated peptides, thereby contributing to virulence (Phelps and Neely, 2007). In this context the evolution of SanE/LpwE such that they could provide protection against lacticin 3147 does not seem an outrageous suggestion.

The production of functional LtnFE homologues by an Efaecium strain is notable. The tendency for enterococci to acquire antibiotic resistance is already well documented and poses huge therapeutic challenges (Murray, 1998). The hardiness of this pathogen also facilitates their survival and thus the spread of drug-resistant clones. Lacticin 3147 has demonstrated potential as an antimicrobial against vancomycin-resistant enterococci (VRE) (Galvin et al., 1999), but its further development as an anti-VRE agent is obviously dependent on the continuing sensitivity of the pathogen. The distribution of efdFE among enterococci will need to be assessed in order to determine whether these genes are rare, in which case restricting their emergence would be a priority, or common, in which case an approach could be developed to further sensitize a population which are at present relatively susceptible to the antimicrobial action of lacticin 3147. In either case it is to be anticipated that the utilization of lacticin 3147 in conjunction with ABC transporter inhibitors would be beneficial.

To date, as a consequence of the rarity with which spontaneously lacticin 3147-resistant mutants emerge and the low level resistance of such mutants (Guinane et al., 2006), resistance to lacticin 3147 has not been an issue. Furthermore, although there have been reports of the development of high level resistance to nisin in the laboratory (Kramer et al., 2006), it has been used in the food industry for over 50 years without such a problem emerging. Care needs to be taken, however, to ensure that these results do not lead to false confidence. The absence of widespread lantibiotic immunity needs to be viewed in the context that lantibiotics have not had widespread use in a clinical setting, i.e. situations where the rapid development of antimicrobial resistance is common. The recently renewed interest in applying lantibiotics in such settings means that lantibiotic researchers also need to anticipate where and how problems may arise and how to negate them. In this context the existence of subpopulations of strains which are resistant to a lantibiotic as a consequence of cross-immunity or immune mimicry is worrying and needs to be carefully considered.

Experimental procedures

Growth conditions

Strains utilized during this study are listed in Table 4. Lactococci were routinely grown at 30°C without aeration in M17 broth (Oxoid, Basingstoke, Hampshire, England) supplemented with 0.5% (w/v) glucose (GM17) or GM17 agar unless otherwise stated. Escherichia coli and B. licheniformis DSM13 were grown in Luria–Bertani broth (Sambrook et al., 1989) at 37°C with vigorous agitation. E. faecium DO and Li. monocytogenes F2365 were grown in brain heart infusion broth (Oxoid) at 37°C without aeration, while S. aureus ssp. aureus NCTC 8325 and S. aureus C55 were grown in Mueller–Hinton broth (Oxoid) at 37°C with vigorous agitation. Lb. plantarum WCFS1 was cultured in MRS broth (Oxoid) at 37°C without aeration. Antibiotics were used, where indicated, at the following concentrations: chloramphenicol, 10 μg ml−1 for Es. coli and 5 μg ml−1 for L. lactis; erythromycin, 300 μg ml−1 for Es. coli and 5 μg ml−1 for L. lactis.

Table 4.  Bacterial strains used in this study.
StrainDescriptionSource/reference
L. lactis  
 HPBacteriocin-sensitive strainDPC collection
 MG1363Plasmid free, bacteriocin-sensitive strainGasson (1983)
 NZ9700Nisin producerKuipers et al. (1993; 1995)
Es. coli  
 Top10Intermediate cloning hostInvitrogen
 EC101Intermediate cloning host (RepA+)Law et al. (1995)
Bacillus licheniformis DSM 13Strain with LtnI-like peptideAmerican Type Culture Collection
Enterococcus. faecium DOGenome sequenced strain with ltnFE homologuesArduino et al. (1994)
Staphylococcus aureus ssp. aureus NCTC 8325Strain with ltnFE homologuesNovick et al. (1967)
Lactobacillus plantarum WCFS1Strain with ltnFE-like peptidesKleerebezem et al. (2003)
Staphylococcus aureus C55Producer of C55α and C55β peptidesNavaratna et al. (1999)
Listeria monocytogenes F2365Listeriosis outbreak isolateLinnan et al. (1988)

General molecular biology techniques

Plasmid DNA was isolated from Es. coli strains using the RBC HiYield plasmid mini kit as recommended by the manufacturer. Plasmids isolated from L. lactis were isolated in the same way following treatment with protoplast buffer (5 mM EDTA, 50 U ml−1 mutanolysin, 10 mg ml−1 lysozyme, 0.75 M sucrose, 20 mM Tris-HCl pH 7.5). Total cell DNA was isolated using Roche high pure PCR template preparation kit (Roche Diagnostics, Mannheim, Germany). Chemically competent Es. coli Top10 (Invitrogen) was used as an immediate host for the plasmids pNZ44 and pNZ44E (Table 5) following manufacturer's guidelines for transformation, while Es. coli EC101 were made electrocompetent as described by standard methods. L. lactis strains were made electrocompetent following the procedure described by Holo and Nes (1995). Li. monocytogenes F2365 was made electrocompetent following the procedure described by Park and Stewart (1990). In all cases electrotransformation was performed with a Gene-Pulser (Bio-Rad) and transformation was carried out by electroporation using similar apparatus described for Es. coli EC101. PCR was performed according to standard procedures using BioTaq DNA (Bioline), Vent polymerase (New England Biolabs), Extensor Hi-Fidelity PCR Master Mix (Abgene) or PWO polymerase (Roche) in a PTC-200 DNA Engine (MJ Research, USA). For colony PCR genomic DNA was accessed through lysis of cells in 10% Igepal CA-630 (Sigma-Alrich) at 94°C for 10 min. Extraction of DNA from agarose gels was performed using the RBC HiYield gel/PCR DNA extraction kit as recommended by the manufacturer. Restriction digests and DNA ligations were executed according to established procedures using restriction enzymes, HindIII, KpnI, SphI and XbaI, PstI, EcoRI and T4 ligase supplied by Roche Diagnostics. DNA sequencing was performed by MWG Biotech AG or GATC Biotech.

Table 5.  Plasmids used in this study.
PlasmidDescriptionSource/reference
pMRC01(60.2 kb) Ltn+, Imm+, originally isolated from L. lactis DPC3147Ryan et al. (1996)
pMRC01.pOM02Facilitates overproducer of lacticin 3147Cotter et al. (2006b)
pMRC01(ΔA1A2)pMRC01 with ltnA1/ltnA2 deletionsCotter et al. (2003)
pMRC01(ΔA1A2) ΔltnIpMRC01 with ltnA1,ltnA2 with an introduced ltnI deletionThis study
pMRC01(ΔA1A2) ΔltnFpMRC01 with ltnA1,ltnA2 with an introduced ltnF deletionThis study
pMRC01(ΔA1A2) ΔltnFEpMRC01 with ltnA1,ltnA2 with an introduced ltnFE deletionThis study
pMRC01(ΔA1A2) ΔltnIFEpMRC01 with ltnA1,ltnA2 and ltnIFE deletionsThis study
pORI280(5.2 kb) RepA, ori+, β-galactosidase+, Eryr integration vectorLeenhouts et al. (1996)
pVE6007Temperature sensitive, Cmr, Rep+Maguin et al. (1992)
pORI280ltnIsoepORI280 containing spliced ltnISOEAD fragment created using primers P13, 14, 15 and 16.This study
pORI280ltnFsoepORI280 containing spliced ltnFSOEAD fragment created using primers P9, 10, 11 and 12This study
pORI280ltnFEsoepORI280 containing spliced ltnFESOEAD fragment created using primers P3, 4, 5 and 6This study
pNZ44Chloramphenicol-resistant, Es. coliL. lactis shuttle vectorMcGrath et al. (2001)
pNZ44EErythromycin-resistant, Es. coliL. lactis shuttle vectorThis study
pNZ44ltnIpNZ44 constitutively expressing ltnIL.H. Deegan et al. (unpublished)
pNZ44ltnFEltnFE PCR product amplified using P17 and P18 and cloned into pNZ44This study
pNZ44ltnIFEltnIFE PCR product amplified using P18 and P19 and cloned into pNZ44This study
pNZ44bliIbliI PCR product amplified using P20 and P21 and cloned into pNZ44This study
pNZ44efdFEefdFE PCR product amplified using P24 and P25 and cloned into pNZ44This study
pNZ44sanFEsanFE PCR product amplified using P22 and P23 and cloned into pNZ44This study
pNZ44lpwFElpwFE PCR product amplified using P26 and P27 and cloned into pNZ44This study
pNZ44EbliIbliI PCR product amplified using P20 and P21 and cloned into pNZ44EThis study
pNZ44ltnEiIntermediate vector for creation of pNZ44lpwFltnE and pNZ44sanFltnE,
ltnE PCR product amplified using P29 and P18 and cloned into pNZ44
This study
pNZ44ltnEiiIntermediate vector for creation of pNZ44ltnFltnE, ltnE PCR product amplified using P33 and P18 and cloned into pNZ44This study
pNZ44lpwEIntermediate vector for creation of pNZ44ltnFlpwE
lpwE PCR product amplified using P30 and P31
This study
pNZ44lpwFltnElpwF PCR product amplified using P26 and P30, and cloned into pNZ44ltnEiThis study
pNZ44ltnFlpwEltnF PCR product amplified using P17 and P32, and cloned into pNZ44lpwEThis study
pNZ44sanFltnEsanF PCR product amplified using P22 and P28, and cloned into pNZ44ltnEiThis study
pNZ44ltnFltnEltnF PCR product amplified using P17 and P32, and cloned into pNZ44ltnEiiThis study

Non-polar deletion of selected ltn genes

To investigate the functionality of ltn (lacticin) genes, non-polar deletions were made in pMRC01(ΔA1A2). This was carried out using a SOEing (splicing by overlap extension) procedure (Cotter et al., 2003). In each case, four primers, termed SOE A, -B, -C and -D, were designed to amplify fragments of equal size on either side of the portion of the gene to be deleted. Vent polymerase (New England Biolabs) was used to amplify fragments for cloning to minimize mutation due to nucleotide misincorporation. The relevant DNA fragments were amplified from MG1363pMRC01 to create SOE-AB and SOE-CD. These were gel extracted and mixed in a 1:1 ratio before being re-amplified by PCR with SOEA and SOED primers resulting in the production of a spliced A–D fragment. The final A–D PCR products were digested with PstI and EcoRI, sites incorporated into the A and D primers respectively, and ligated with similarly digested pORI280 before being transformed into Es. coli EC101 (Rep+) (Law et al., 1995). Blue colonies on Luria–Bertani agar plates containing Ery (100 μg ml−1) and Xgal (50 μg ml−1) were selected and the primer pair pORI280 F/R (P1 and P2), situated on either side of the multiple cloning site of pORI280, was used to confirm the insert was of correct size and plasmid inserts were sequenced. The relevant pORI280(AD) was subsequently electroporated into L. lactis MG1363 (Gasson, 1983) containing pMRC01(ΔA1A2) and the temperature-sensitive Cmr, RepA+, helper plasmid, pVE6007 (Maguin et al., 1992). Chromosomal integration of pORI280 into pMRC01 took advantage of the temperature-sensitive nature of pVE6007, in that transformants were selected and serially passaged at 39°C in pre-warmed GM17 broth containing erythromycin and replica-plated on pre-warmed GM17 agar containing chloramphenicol and erythromycin respectively, to check for loss of pVE6007 (Cmr), i.e. temperature induced curing. Due to the RepA- nature of pORI280, an Eryr phenotype can only arise in cells where pORI280 has integrated into pMRC01. Excision and loss of pORI280 were then achieved by continuous subculturing and plating (37°C) and potential mutants appearing as white eryS colonies on GM17. To distinguish between mutants and wild-type revertants, a PCR utilizing primers located upstream of SOEA primer and downstream of SOED primer, respectively, was utilized.

Plasmid construction

Specific genes of interest were amplified from E. faecium, S. aureus, Lb. plantarum, B. licheniformis or MG1363pMRC01 using the primers listed in Table S1. These PCR products and pNZ44, an Es. coli–L. lactis shuttle/expression vector with a constitutive P44 promoter, were digested using the appropriate restriction enzymes and subsequently ligated. These transcriptional fusions were ultimately introduced into lactococcal strains (following initial isolation from Es. coli Top10 and sequencing to ensure integrity). Using the same approach ltnF, sanF and lpwF were introduced into pNZ44lpwE or pNZ44ltnEi to form pNZ44ltnFltnE, pNZ44ltnFlpwE, pNZ44sanFltnE and pNZ44lpwFltnE. pNZ44E was generated by first amplifying the pNZ44 backbone, minus the cat gene, with primers P34-35, digesting with BglII and EcoRI, and ligating with similarly digested eryC amplified from the vector pMG36e with the primers P36-37. bliI was cloned into this vector using the same approach as described above but with the use of erythromycin rather than chloramphenicol for selection.

Curing of pC55i from S. aureus C55

To induce the loss of pC55i S. aureus C55 was grown overnight at 37°C with vigorous shaking before being subcultured into preheated broth and incubated at 42°C (shaking). Following a second subculture at 42°C, the culture was serially diluted and plated on Mueller–Hinton agar. The resultant colonies were tested to identify those lacking pC55i on the basis of negative PCR results and the absence of a lantibiotic-producing phenotype.

Lacticin 3147 purification

TYG media (tryptone, 2.5 g l−1; yeast extract, 5.0 g l−1; glucose, 10 g l−1; β-glycerophosphate, 19.0 g l−1; MgSO4·7H2O, 0.25 g l−1; MnSO4·4H2O, 0.05 g l−1) was passed through 500 g XAD-16 beads (Sigma-Aldrich Company, Dorset, UK) in order to remove all hydrophobic components. An overnight culture of MG1363pMRC01.pOM02 was then used to inoculate 1 l of the modified TYG broth (1% inoculum) and incubated at 30°C overnight. The cells were subsequently harvested by centrifugation (7000 r.p.m. for 20 min) and resuspended in 250 ml 70% propan-2-ol, pH 2 (adjusted to pH 2 with addition of conc. HCl). Following stirring at 4°C for 4 h, the cell debris was removed by centrifugation and the supernatant was subjected to rotary evaporation (50 mbar at 40°C) to reduce the volume to ∼60 ml via removal of propan-2-ol. The resultant preparation was applied to a 10 g/60 ml Strata C-18E Giga-Tube (Phenomenex, Cheshire, UK) after pre-equilibration with 60 ml methanol followed by 60 ml water. The column was subsequently washed with 120 ml of 30% ethanol and the lantibiotic was then eluted from the column via addition of 100 ml of 70% propan-2-ol, pH 2. From the 100 ml preparation, 20 ml volumes were subjected to rotary evaporation in order to reduce them to ∼1.7 ml through removal of propan-2-ol. Aliquots of 1650 μl were then applied to a Phenomenex (Phenomenex, Cheshire, UK) C12 reverse-phase high-performance liquid chromatography (HPLC) column (Jupiter 4 μ 90 Å 250 × 10.0 mm, 4 μm) previously equilibrated with 25% propan-2-ol containing 0.1% trifluoroacetic acid (TFA). The column was then developed in a gradient of 30% propan-2-ol containing 0.1% TFA to 60% propan-2-ol containing 0.1% TFA in 4–40 min at a flow rate of 1.2 ml min−1. Fractions containing Ltnα and Ltnβ were collected after each HPLC run and stored under nitrogen gas. The Ltnα and Ltnβ-containing fractions were pooled separately and subsequently subjected to rotary evaporation to remove all propan-2-ol before freeze-drying of the peptides. The Ltnα and Ltnβ peptides were weighed in μg quantities using a Mettler UMT2 microbalance.

Well diffusion assays

Agar well diffusion assays were performed to preliminarily assess the relative immunity of individual strains. Briefly, 20 ml molten agar at 48°C was seeded (50 μl) with fresh overnight grown cells (approximately 1 × 106 ml−1) of the strain of interest, dispensed into sterile Petri dishes and allowed to solidify. Wells of approximately 4.6 mm in diameter were made, into which 50 μl of lantibiotic-containing cell-free supernatant from an overnight culture of L. lactis MG1363pMRC01 (lacticin 3147), S. aureus C55 (staphylococcin C55) or L. lactis NZ9700 (nisin) was placed. After overnight incubation at the appropriate temperature, immunity was assessed.

Minimum inhibitory concentration determination

Minimum inhibitory concentration determinations were carried out as described by Wiedemann et al. (2006). L. lactis, S. aureus and Li. monocytogenes strains assayed were grown in M17 broth supplemented with 0.5% (w/v) glucose (GM17), Mueller–Hinton broth and brain heart infusion broth (Oxoid, Basingstoke, Hampshire, UK) respectively. Serial twofold dilutions of the peptides were made in the growth medium of the respective strain. Bacteria were added to give a final inoculum of 105 cfu ml−1 in a volume of 0.2 ml. After incubation for 16 h at 30°C (L. lactis) or 37°C (S. aureus, Li. monocytogenes) the MIC was read as the lowest peptide concentration causing inhibition of visible growth.

Acknowledgements

The authors would like to thank Barbara E. Murray and Michiel Kleerebezem for supplying strains E. faecium DO and Lb. plantarum WCFS1 respectively. This material is based upon works supported by the Science Foundation Ireland under Grant No. 06/IN.1/B98.

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