Des Field

The generation of nisin variants with enhanced activity against specific Gram‐positive pathogens

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

Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens

Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria.

The dawning of a ‘Golden era’ in lantibiotic bioengineering

There are many examples of highly modified antimicrobial peptides in nature, many of which are non‐ribosomally synthesized. However, the bacterial lantibiotics are produced as gene‐encoded pre‐peptides that are subsequently modified by dedicated enzyme systems to form extraordinarily potent inhibitors. Consequently, they are much more amenable to bioengineering which could lead to the generation of a new arsenal of potent antimicrobials. However, although bioengineering of these compounds has been underway for at least two decades, significant progress has only been reported in recent years. This review charts these recent developments which suggest that we are entering a ‘Golden era’ of lantibiotic bioengineering.

Technological characterization of bacteriocin producing Lactococcus lactis strains employed to control Listeria monocytogenes in Cottage cheese

In recent years, there has been a particular focus on the application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives to control the growth of spoilage and pathogenic bacteria in food. Bacteriocins are antimicrobial peptides which can be added to foods in concentrated forms as food preservatives, e.g. additives, or they can be produced in situ by starters or protective cultures. In this study, twenty Lactococcus lactis bacteriocin producers previously isolated from Italian fermented foods were subjected to a variety of physical and biochemical tests in order to identify those with the greatest potential as starter cultures in cheese production. Of these, four strains isolated from cheese (one nisin Z producer, one nisin A producer and two lacticin 481 producers) which fulfilled the desired technological criteria were assessed for their ability to control Listeria monocytogenes. The subsequent application of these bacteriocinogenic strains as starter cultures in Cottage cheese established that the nisin A producing Lact. lactis 40FEL3, and to a lesser extent the lacticin 481 producers 32FL1 and 32FL3, successfully controlled the growth of the pathogen. This is the first study to directly compare the ability of nisin A, nisin Z and lacticin 481 producing strains to control listerial growth during the manufacture and storage of Cottage cheese.

Studies with bioengineered Nisin peptides highlight the broad-spectrum potency of Nisin V.

Nisin A is the most thoroughly investigated member of the lantibiotic family of antimicrobial peptides. In addition to a long history of safe use as a food antimicrobial, its activity against multi‐drug resistant pathogens has resulted in a renewed interest in applying nisin as a chemotherapeutic to treat bacterial infections. The wealth of Nisin‐related information that has been generated has also led to the development of the biotechnological capacity to engineer novel Nisin variants with a view to improving the function and physicochemical properties of this already potent peptide. However, the identification of bioengineered Nisin derivatives with enhanced antimicrobial activity against Gram‐positive targets is a recent event. In this study, we created stable producers of the most promising derivatives of Nisin A generated to date [M21V (hereafter Nisin V) and K22T (hereafter Nisin T)] and assessed their potency against a range of drug‐resistant clinical, veterinary and food pathogens. Nisin T exhibited increased activity against all veterinary isolates, including streptococci and staphylococci, and against a number of multi‐drug resistant clinical isolates including MRSA, but not vancomycin‐resistant enterococci. In contrast, Nisin V displayed increased potency against all targets tested including hVISA strains and the hyper‐virulent Clostridium difficile ribotype 027 and against important food pathogens such as Listeria monocytogenes and Bacillus cereus. Significantly, this enhanced activity was validated in a model food system against L. monocytogenes. We conclude that Nisin V possesses significant potential as a novel preservative or chemotherapeutic compound.

Bioengineered nisin derivatives with enhanced activity in complex matrices

Nisin A is the best known and most extensively characterized lantibiotic. As it is ribosomally synthesized, bioengineering‐based strategies can be used to generate variants. We have previously demonstrated that bioengineering of the hinge region of nisin A can result in the generation of variants with enhanced anti‐microbial activity against Gram‐positive pathogens. Here we created a larger bank of hinge variant producers and screened for producers that exhibit enhanced bioactivity as assessed by agar‐based assays against a selection of target strains. Further analysis of 12 ‘lead’ variants reveals that in many cases enhanced bioactivity is not attributable to enhanced specific activity but is instead as a consequence of an enhanced ability to diffuse through complex polymers. In the case of two variants, which contain the residues SVA and NAK, respectively, within the hinge region, we demonstrate that this enhanced trait enables the peptides to dramatically outperform nisin A with respect to controlling Listeria monocytogenes in commercially produced chocolate milk that contains carrageenan as a stabilizer.

Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

It is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.

The gene encoded antimicrobial peptides, a template for the design of novel anti-mycobacterial drugs

Nisin A is the most widely characterized lantibiotic investigated to date. It represents one of the many antimicrobial peptides which have been the focus of much interest as potential therapeutic agents. This has resulted in the search for novel lantibiotics and more commonly, the engineering of novel variants from existing peptides with a view to increasing their activity, stability and solubility. The aim of this study was to compare the activities of nisin A and novel bioengineered hinge derivatives, nisin S, nisin T and nisin V. The microtitre alamar blue assay (MABA) was employed to identify the enhanced activity of these novel variants against M. tuberculosis (H37Ra), M. kansasii (CIT11/06), M. avium subsp. hominissuis(CIT05/03)and M. avium subsp. paratuberculosis (MAP) (ATCC 19698). All variants displayed greater anti-mycobacterial activity than nisin A. Nisin S was the most potent variant against M. tuberculosis, M. kansasii and M. avium subsp. hominissuis, retarding growth by a maximum of 29% when compared with nisin A. Sub-species variations of inhibition were also observed with nisin S reducing growth of Mycobacterium avium subsp hominissuis by 28% and Mycobacterium avium subsp paratuberculosis by 19% and nisin T contrastingly reducing growth of MAP by 27% and MAC by 16%. Nisin S, nisin T, and nisin V are potent novel anti-mycobacterial compounds, which have the capacity to be further modified, potentially generating compounds with additional beneficial characteristics. This is the first report to demonstrate an enhancement of efficacy by any bioengineered bacteriocin against mycobacteria.

Bioengineering Lantibiotics for Therapeutic Success.

Several examples of highly modified antimicrobial peptides have been described. While many such peptides are non-ribosomally synthesized, ribosomally synthesized equivalents are being discovered with increased frequency. Of the latter group, the lantibiotics continue to attract most attention. In the present review, we discuss the implementation of in vivo and in vitro engineering systems to alter, and even enhance, the antimicrobial activity, antibacterial spectrum and physico-chemical properties, including heat stability, solubility, diffusion and protease resistance, of these compounds. Additionally, we discuss the potential applications of these lantibiotics for use as therapeutics.

Intensive mutagenesis of the nisin hinge leads to the rational design of enhanced derivatives.

Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.