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.     

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.     

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.     

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.     

Bioengineering nisin to overcome the nisin resistance protein

The emergence and dissemination of antibiotic resistant bacteria is a major medical challenge. Lantibiotics are highly modified bacterially produced antimicrobial peptides that have attracted considerable interest as alternatives or adjuncts to existing antibiotics. Nisin, the most widely studied and commercially exploited lantibiotic, exhibits high efficacy against many pathogens. However, some clinically relevant bacteria express highly specific membrane‐associated nisin resistance proteins. One notable example is the nisin resistance protein that acts by cleaving the peptide bond between ring E and the adjacent serine 29, resulting in a truncated peptide with significantly less activity. We utilised a complete bank of bioengineered nisin (nisin A) producers in which the serine 29 residue has been replaced with every alternative amino acid. The nisin A S29P derivative was found to be as active as nisin A against a variety of bacterial targets but, crucially, exhibited a 20‐fold increase in specific activity against a strain expressing the nisin resistance protein. Another derivative, nisin PV, exhibited similar properties but was much less prone to oxidation. This version of nisin with enhanced resistance to specific resistance mechanisms could prove useful in the fight against antibiotic resistant pathogens.     

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.     

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.     

Contributions of the σW, σM and σX regulons to the lantibiotic resistome of Bacillus subtilis

In Bacillus subtilis, the extracytoplasmic function (ECF) σ factors σ^M, σ^W and σ^X all contribute to resistance against lantibiotics. Nisin, a model lantibiotic, has a dual mode of action: it inhibits cell wall synthesis by binding lipid II, and this complex also forms pores in the cytoplasmic membrane. These activities can be separated in a nisin hinge‐region variant (N20P M21P) that binds lipid II, but no longer permeabilizes membranes. The major contribution of σ^M to nisin resistance is expression of ltaSa, encoding a stress‐activated lipoteichoic acid synthase, and σ^X functions primarily by activation of the dlt operon controlling d ‐alanylation of teichoic acids. Together, σ^M and σ^X regulate cell envelope structure to decrease access of nisin to its lipid II target. In contrast, σ^W is principally involved in protection against membrane permeabilization as it provides little protection against the nisin hinge region variant. σ^W contributes to nisin resistance by regulation of a signal peptide peptidase (SppA), phage shock proteins (PspA and YvlC, a PspC homologue) and tellurite resistance related proteins (YceGHI). These defensive mechanisms are also effective against other lantibiotics such as mersacidin, gallidermin and subtilin and comprise an important subset of the intrinsic antibiotic resistome of B. subtilis.     

Biosynthetic characterization and biochemical features of the third natural nisin variant,nisin Q,produced by Lactococcus lactis 61‐14

Aims: To characterize the genetic and biochemical features of nisin Q. Methods and Results: The nisin Q gene cluster was sequenced, and 11 putative orfs having 82% homology with the nisin A biosynthesis gene cluster were identified. Nisin Q production was confirmed from the nisQ‐introduced nisin Z producer. In the reporter assay, nisin Q exhibited an induction level that was threefold lower than that of nisin A. Nisin Q demonstrated an antimicrobial spectrum similar to those of the other nisins. Under oxidizing conditions, nisin Q retained a higher level of activity than nisin A. This higher oxidative tolerance could be attributed to the presence of only one methionine residue in nisin Q, in contrast to other nisins that contain two. Conclusions: The 11 orfs of the nisin producers were identical with regard to their functions. The antimicrobial spectra of the three natural nisins were similar. Nisin Q demonstrated higher oxidative tolerance than nisin A. Significance and Impact of the Study:  Genetic and biochemical features of nisin Q are similar to those of other variants. Moreover, owing to its higher oxidative tolerance, nisin Q is a potential alternative for nisin A.     

Site-directed mutagenesis of the hinge region of nisinZ and properties of nisinZ mutants

To study the role of the hinge region in nisin and to obtain mutants that exhibit altered or new biological activities and functional properties, we changed certain amino acids in the hinge region by performing site-directed mutagenesis with the nisinZ structural gene (nisZ). The results showed that the nisinZ mutants had decreased antimicrobial activities against Micrococcus flavus NCIB8166 and Streptococcus thermophilus. Interestingly, compared with wild nisinZ, mutant N20K nisinZ and M21K nisinZ displayed antimicrobial activity against gram-negative Shigella, Pseudomonas and Salmonella; and they had a higher solubility than wild-type nisinZ. At pH 8, the solubilities of N20K nisinZ and M21K nisinZ were, respectively, three-fold higher and five-fold higher than that of nisinZ. Mutant N20Q nisinZ and M21G nisinZ were considerably more stable than nisinZ at higher temperatures and neutral or alkaline pH. These mutants provided information that the central hinge region in nisinZ plays an important role in providing the conformational flexibility required for the antimicrobial activity on the membrane. Our finding documented that it may well be worth considering the construction of the new nisin mutants with changed inhibitory activity against a wide range of gram-negative bacteria and the improvement of functional properties by site-directed mutagenesis.     

A Bioengineered Nisin Derivative to Control Biofilms of Staphylococcus pseudintermedius

Antibiotic resistance and the shortage of novel antimicrobials are among the biggest challenges facing society. One of the major factors contributing to resistance is the use of frontline clinical antibiotics in veterinary practice. In order to properly manage dwindling antibiotic resources, we must identify antimicrobials that are specifically targeted to veterinary applications. Nisin is a member of the lantibiotic family of antimicrobial peptides that exhibit potent antibacterial activity against many gram-positive bacteria, including human and animal pathogens such as Staphylococcus, Bacillus, Listeria, and Clostridium. Although not currently used in human medicine, nisin is already employed commercially as an anti-mastitis product in the veterinary field. Recently we have used bioengineering strategies to enhance the activity of nisin against several high profile targets, including multi-drug resistant clinical pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) and also against staphylococci and streptococci associated with bovine mastitis. However, newly emerging pathogens such as methicillin resistant Staphylococcus pseudintermedius (MRSP) pose a significant threat in terms of veterinary health and as a reservoir for antibiotic resistance determinants. In this study we created a nisin derivative with enhanced antimicrobial activity against S. pseudintermedius. In addition, the novel nisin derivative exhibits an enhanced ability to impair biofilm formation and to reduce the density of established biofilms. The activities of this peptide represent a significant improvement over that of the wild-type nisin peptide and merit further investigation with a view to their use to treat S. pseudintermedius infections.     

Biomedical applications of nisin

Nisin is a bacteriocin produced by a group of Gram‐positive bacteria that belongs to Lactococcus and Streptococcus species. Nisin is classified as a Type A (I) lantibiotic that is synthesized from mRNA and the translated peptide contains several unusual amino acids due to post‐translational modifications. Over the past few decades, nisin has been used widely as a food biopreservative. Since then, many natural and genetically modified variants of nisin have been identified and studied for their unique antimicrobial properties. Nisin is FDA approved and generally regarded as a safe peptide with recognized potential for clinical use. Over the past two decades the application of nisin has been extended to biomedical fields. Studies have reported that nisin can prevent the growth of drug‐resistant bacterial strains, such as methicillin‐resistant Staphylococcus aureus, Streptococcus pneumoniae, Enterococci and Clostridium difficile. Nisin has now been shown to have antimicrobial activity against both Gram‐positive and Gram‐negative disease‐associated pathogens. Nisin has been reported to have anti‐biofilm properties and can work synergistically in combination with conventional therapeutic drugs. In addition, like host‐defence peptides, nisin may activate the adaptive immune response and have an immunomodulatory role. Increasing evidence indicates that nisin can influence the growth of tumours and exhibit selective cytotoxicity towards cancer cells. Collectively, the application of nisin has advanced beyond its role as a food biopreservative. Thus, this review will describe and compare studies on nisin and provide insight into its future biomedical applications.     

Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis

Streptococcus uberis is one of the principal causative agents of bovine mastitis. In this study, we report that S. uberis strain 42 produces a lantibiotic, nisin U, which is 78% identical (82% similar) to nisin A from Lactococcus lactis. The 15.6-kb nisin U locus comprises 11 open reading frames, similar in putative functionality but differing in arrangement from that of the nisin A biosynthetic cluster. The nisin U producer strain exhibits specific resistance (immunity) to nisin U and cross-resistance to nisin A, a finding consistent with the 55% sequence similarity of their respective immunity peptides. Homologues of the nisin U structural gene were identified in several additional S. uberis strains, and in each case cross-protective immunity was expressed to nisin A and to the other producers of nisin U and its variants. To our knowledge, this is the first report both of characterization of a bacteriocin by S. uberis, as well as of a member of the nisin family of peptides in a species other than L. lactis.     

Molecular and Genetic Characterization of a Novel Nisin Variant Produced by Streptococcus uberis

Streptococcus uberis is one of the principal causative agents of bovine mastitis. In this study, we report that S. uberis strain 42 produces a lantibiotic, nisin U, which is 78% identical (82% similar) to nisin A from Lactococcus lactis. The 15.6-kb nisin U locus comprises 11 open reading frames, similar in putative functionality but differing in arrangement from that of the nisin A biosynthetic cluster. The nisin U producer strain exhibits specific resistance (immunity) to nisin U and cross-resistance to nisin A, a finding consistent with the 55% sequence similarity of their respective immunity peptides. Homologues of the nisin U structural gene were identified in several additional S. uberis strains, and in each case cross-protective immunity was expressed to nisin A and to the other producers of nisin U and its variants. To our knowledge, this is the first report both of characterization of a bacteriocin by S. uberis, as well as of a member of the nisin family of peptides in a species other than L. lactis.     

Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity

Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation. Here we use genetically engineered nisin variants to identify the structural requirements for the interaction of the peptide with lipid II. Mutations affecting the conformation of the N-terminal part of nisin comprising rings A through C, e.g. [S3T]nisin, led to reduced binding and increased the peptide concentration necessary for pore formation. The binding constant for the S3T mutant was 0.043 x 10(7) m(-1) compared with 2 x 10(7) m(-1) for the wild-type peptide, and the minimum concentration for pore formation increased from the 1 nm to the 50 nm range. In contrast, peptides mutated in the flexible hinge region, e.g. [DeltaN20/DeltaM21]nisin, were completely inactive in the pore formation assay, but were reduced to some extent in their in vivo activity. We found the remaining in vivo activity to result from the unaltered capacity of the mutated peptide to bind to lipid II and thus to inhibit its incorporation into the peptidoglycan network. Therefore, through interaction with the membrane-bound cell wall precursor lipid II, nisin inhibits peptidoglycan synthesis and forms highly specific pores. The combination of two killing mechanisms in one molecule potentiates antibiotic activity and results in nanomolar MIC values, a strategy that may well be worth considering for the construction of novel antibiotics.     

Natural mutations in IFITM3 modulate post‐translational regulation and toggle antiviral specificity

The interferon‐induced transmembrane (IFITM) proteins protect host cells from diverse virus infections. IFITM proteins also incorporate into HIV‐1 virions and inhibit virus fusion and cell‐to‐cell spread, with IFITM3 showing the greatest potency. Here, we report that amino‐terminal mutants of IFITM3 preventing ubiquitination and endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV‐1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino‐terminal mutations that modify protein localization and function. This suggests that “runaway” IFITM3 variants could be selected for altered antiviral activity. Furthermore, we show that adaptations in IFITM3 result in a trade‐off in antiviral specificity, as variants exhibiting enhanced activity against HIV‐1 poorly restrict influenza A virus. Overall, we provide the first experimental evidence that diversification of IFITM3 genes may boost the antiviral coverage of host cells and provide selective functional advantages.     

Identification of Nisin-Producing Strains by Nisin-Controlled Gene Expression System

A specific method to identify nisin-producing strains was developed based on Nisin-Controlled gene Expression (NICE) vector pSec:Nuc. The plasmid pSec:Nuc was transformed into non-nisin-producing strain Lactococcus lactis NZ9000, a host commonly used for the NICE system. The generating strain L. lactis NZ9000/pSec:Nuc could sense extracellular inducer nisin and efficiently secrete a reporter protein Nuc, the staphylococcal nuclease (Nuc) into the medium. Instead of using purified nisin, the culture supernatants of nisin-producing strains were also used as inducers. Therefore, the NICE system could be used to identify nisin-producing strains. With this principle, 4 among 56 lactococci strains isolated from raw milk were identified as nisin producers. The results were further confirmed by polymerase chain reaction amplification with their genomic DNA as templates, and nucleotide sequencing revealed that three of them produced nisin A, and the others produced nisin Z. Those results made it possible to isolate and identify nisin-producing strains specifically and rapidly using NICE system.     

Expression of the Chlamydomonas reinhardtii Sedoheptulose‐1,7‐bisphosphatase in Dunaliella bardawil leads to enhanced photosynthesis and increased glycerol production

Bioengineering of photoautotrophic microalgae into CO₂ scrubbers and producers of value‐added metabolites is an appealing approach in low‐carbon economy. A strategy for microalgal bioengineering is to enhance the photosynthetic carbon assimilation through genetically modifying the photosynthetic pathways. The halotolerant microalgae Dunaliella posses an unique osmoregulatory mechanism, which accumulates intracellular glycerol in response to extracellular hyperosmotic stresses. In our study, the Calvin cycle enzyme sedoheptulose 1,7‐bisphosphatase from Chlamydomonas reinhardtii (CrSBPase) was transformed into Dunaliella bardawil, and the transformant CrSBP showed improved photosynthetic performance along with increased total organic carbon content and the osmoticum glycerol production. The results demonstrate that the potential of photosynthetic microalgae as CO₂ removers could be enhanced through modifying the photosynthetic carbon reduction cycle, with glycerol as the carbon sink.     

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.     

Chemical Modification of the Carboxyl Terminal of Nisin A with Biotin does not Abolish Antimicrobial Activity Against the Indicator Organism, Kocuria rhizophila

Nisin is an antimicrobial peptide that is widely used for food preservation. Although it has potent activity against a number of food pathogens, suggesting potential therapeutic applications, its potential for clinical use is limited by proteolytic susceptibility and poor oral bioavailability. Derivatization of nisin could overcome these issues; however, many nisin analogues, prepared by modification at the N-terminal and C-terminal have previously been shown to be inactive. A method for the C-terminal modification was developed using biotinylation as a model derivative. Purification of the modified nisin was carried out using reverse phase chromatography. Confirmation of nisin modification was confirmed by Mass Spectroscopy. The C-terminal modification of nisin resulted in only a twofold reduction in antimicrobial activity of the conjugate against the indicator organism, Kocuria rhizophila. The C-terminal modification could be used to increase the therapeutic potential of nisin by creating more favourable physicochemical characteristics. This is the first study that showed that nisin modification can be carried out successfully without destroying its antimicrobial activity.