Lorraine A. Draper

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

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

Listeriolysin S, a Novel Peptide Haemolysin Associated with a Subset of Lineage I Listeria monocytogenes

Streptolysin S (SLS) is a bacteriocin-like haemolytic and cytotoxic virulence factor that plays a key role in the virulence of Group A Streptococcus (GAS), the causative agent of pharyngitis, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. Although it has long been thought that SLS and related peptides are produced by GAS and related streptococci only, there is evidence to suggest that a number of the most notorious Gram-positive pathogenic bacteria, including Listeria monocytogenes, Clostridium botulinum and Staphylococcus aureus, produce related peptides. The distribution of the L. monocytogenes cluster is particularly noteworthy in that it is found exclusively among a subset of lineage I strains; i.e., those responsible for the majority of outbreaks of listeriosis. Expression of these genes results in the production of a haemolytic and cytotoxic factor, designated Listeriolysin S, which contributes to virulence of the pathogen as assessed by murine- and human polymorphonuclear neutrophil–based studies. Thus, in the process of establishing the existence of an extended family of SLS-like modified virulence peptides (MVPs), the genetic basis for the enhanced virulence of a proportion of lineage I L. monocytogenes may have been revealed.

A comparison of the activities of lacticin 3147 and nisin against drug-resistant Staphylococcus aureus and Enterococcus species

OBJECTIVES Our goal was to compare the activities of lacticin 3147 and nisin, two of the most well characterized lantibiotics, against antibiotic-resistant staphylococci and enterococci. METHODS We determined the MICs of lacticin 3147 and nisin for 20 strains of methicillin-resistant Staphylococcus aureus (MRSA), 20 strains of vancomycin-resistant enterococci (VRE), 6 strains of S. aureus with intermediate resistance to vancomycin (VISA), 5 strains of heterogeneous vancomycin-intermediate S. aureus (hVISA) and 4 strains of S. aureus that are susceptible to methicillin. RESULTS Lacticin 3147 displayed potent activity against VRE with MIC values between 1.9 and 7.7 mg/L, and varying levels of activity against S. aureus strains (MRSA, 1.9-15.4 mg/L; laboratory strains, >or=15.4 mg/L; hVISA, 15.4-30.9 mg/L; VISA, >or=61.8 mg/L). Nisin was more active against the S. aureus strains in general (MRSA and laboratory strains, 0.5-4.1 mg/L; VISA and hVISA, 2 to >or=8.3 mg/L), but was less effective than lacticin 3147 against VRE (2 to >or=8.3 mg/L). CONCLUSIONS Nisin is more effective against S. aureus whereas lacticin 3147 possesses greater potency against VRE. The modifications responsible for the vancomycin-resistant phenotypes of hVISA and VISA strains also provide protection against the two lantibiotics.

Production of the Bsa Lantibiotic by Community-Acquired Staphylococcus aureus Strains

Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.

Comparison of the activities of the lantibiotics nisin and lacticin 3147 against clinically significant mycobacteria

The aim of this study was to use the microtitre alamarBlue assay to investigate and compare the antimycobacterial potential of the lantibiotics nisin and lacticin 3147 against a representative cohort of clinically significant mycobacteria, i.e. Mycobacterium tuberculosis H37Ra, Mycobacterium avium subsp. paratuberculosis (MAP) ATCC 19698 and Mycobacterium kansasii CIT11/06. Lacticin 3147 displayed potent activity against all strains of mycobacteria, with MIC(90) values (lowest concentration of lantibiotic that prevented growth of >90% of the bacterial population) of 60 mg/L and 15 mg/L for M. kansasii and MAP, respectively. Lacticin 3147 was particularly effective against M. tuberculosis H37Ra, with a MIC(90) value of 7.5mg/L. Nisin, although inhibitory, was generally less potent against all strains of mycobacteria, with MIC(90) values of 60 mg/L for M. kansasii and >60 mg/L for MAP and M. tuberculosis H37Ra. Thus, lacticin 3147 is a potent antimycobacterial peptide that shows superior activity compared with nisin at physiological pH.

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

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

The two peptide lantibiotic lacticin 3147 acts synergistically with polymyxin to inhibit Gram negative bacteria

BackgroundThe emergence of bacterial drug resistance encourages the re-evaluation of the potential of existing antimicrobials. Lantibiotics are post-translationally modified, ribosomally synthesised antimicrobial peptides with a broad spectrum antimicrobial activity. Here, we focussed on expanding the potential of lacticin 3147, one of the most studied lantibiotics and one which possesses potent activity against a wide range of Gram positive species including many nosocomial pathogens. More specifically, our aim was to investigate if lacticin 3147 activity could be enhanced when combined with a range of different clinical antibiotics.ResultsInitial screening revealed that polymyxin B and polymyxin E (colistin) exhibited synergistic activity with lacticin 3147. Checkerboard assays were performed against a number of strains, including both Gram positive and Gram negative species. The resultant fractional inhibitory concentration (FIC) index values established that, while partial synergy was detected against Gram positive targets, synergy was obvious against Gram negative species, including Cronobacter and E. coli.ConclusionsCombining lacticin 3147 with low levels of a polymyxin could provide a means of broadening target specificity of the lantibiotic, while also reducing polymyxin use due to the lower concentrations required as a result of synergy.

Overproduction of Wild-Type and Bioengineered Derivatives of the Lantibiotic Lacticin 3147

ABSTRACT Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e., ltnA1A2) is not.

Manipulation of charged residues within the two-peptide lantibiotic lacticin 3147

Lantibiotics are antimicrobial peptides which contain a high percentage of post‐translationally modified residues. While most attention has been paid to the role of these critical structural features, evidence continues to emerge that charged amino acids also play a key role in these peptides. Here 16 ‘charge’ mutants of the two‐peptide lantibiotic lacticin 3147 [composed of Ltnα (2+, 2−) and Ltnβ (2+)] were constructed which, when supplemented with previously generated peptides, results in a total bank of 23 derivatives altered in one or more charged residues. When examined individually, in combination with a wild‐type partner or, in some instances, in combination with one another, these mutants reveal the importance of charge at specific locations within Ltnα and Ltnβ, confirm the critical role of the negatively charged glutamate residue in Ltnα and facilitate an investigation of the contribution of positively charged residues to the cationic Ltnβ. From these investigations it is also apparent that the relative importance of the overall charge of lacticin 3147 varies depending on the target bacteria and is most evident when strains with more negatively charged cell envelopes are targeted. These studies also result in, for the first time, the creation of a derivative of a lacticin 3147 peptide (LtnβR27A) which displays enhanced specific activity.

Saturation mutagenesis of selected residues of the α-peptide of the lantibiotic lacticin 3147 yields a derivative with enhanced antimicrobial activity.

The lantibiotic lacticin 3147 consists of two ribosomally synthesized and post‐translationally modified antimicrobial peptides, Ltnα and Ltnβ, which act synergistically against a wide range of Gram‐positive microorganisms. We performed saturation mutagenesis of specific residues of Ltnα to determine their functional importance. The results establish that Ltnα is more tolerant to change than previously suggested by alanine scanning mutagenesis. One substitution, LtnαH23S, was identified which improved the specific activity of lacticin 3147 against one pathogenic strain, Staphylococcus aureus NCDO1499. This represents the first occasion upon which the activity of a two peptide lantibiotic has been enhanced through bioengineering.