Bypassing lantibiotic resistance by an effective nisin derivative

The need for new antibiotic compounds is rising and antimicrobial peptides are excellent candidates to fulfill this object. The bacteriocin subgroup lantibiotics, for example, are active in the nanomolar range and target the membranes of mainly Gram-positive bacteria. They bind to lipid II, inhibit cell growth and in some cases form pores within the bacterial membrane, inducing rapid cell death. Pharmaceutical usage of lantibiotics is however hampered by the presence of gene clusters in human pathogenic strains which, when expressed, confer resistance. The human pathogen Streptococcus agalactiae COH1, expresses several lantibiotic resistance proteins resulting in resistance against for example nisin.This study presents a highly potent, pore forming nisin variant as an alternative lantibiotic which bypasses the SaNSR protein. It is shown that this nisin derivate nisin_C28P keeps its nanomolar antibacterial activity against L. lactis NZ9000 cells but is not recognized by the nisin resistance protein SaNSR.Nisin_C28P is cleaved by SaNSR in vitro with a highly decreased efficiency, as shown by an cleavage assay. Furthermore, we show that nisin_C28P is still able to form pores in the membranes of L. lactis and is three times more efficient against SaNSR-expressing L. lactis cells than wildtype nisin.     

Influence of nisin hinge-region variants on lantibiotic immunity and resistance proteins

The rising existence of antimicrobial resistance, confirms the urgent need for new antimicrobial compounds. Lantibiotics are active in a low nanomolar range and represent good compound candidates. The lantibiotic nisin is well studied, thus it is a perfect origin for exploring novel lantibiotics via mutagenesis studies. However, some human pathogens like Streptococcus agalactiae COH1 already express resistance proteins against lantibiotics like nisin.This study presents three nisin variants with mutations in the hinge-region and determine their influence on both the growth inhibition as well as the pore-forming activity. Furthermore, we analyzed the effect of these mutants on the nisin immunity proteins NisI and NisFEG from Lactococcus lactis, as well as the nisin resistance proteins SaNSR and SaNsrFP from Streptococcus agalactiae COH1.We identified the nisin variant ₂₀NMKIV₂₄ with an extended hinge-region, to be an excellent candidate for further studies to eventually overcome the lantibiotic resistance in human pathogens, since these proteins do not recognize this variant well.     

The Solution Structure of the Lantibiotic Immunity Protein NisI and Its Interactions with Nisin

Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.     

大肠杆菌重组乳链菌肽抗性蛋白(NSR)的表达纯化及其功能分析

乳链菌肽(Nisin)是由某些乳酸菌产生的一种阳离子抗菌肽, 而Nisin抗性蛋白(Nisin resistance protein, NSR)的表达则使一些非 Nisin产生菌获得Nisin抗性。为深入探索NSR的作用机制, 本研究在大肠杆菌中表达了去除N末端38个氨基酸残基的NSR(NSRΔ38)与GST的融合蛋白GST-NSRΔ38。通过谷胱甘肽(GSH)亲和层析和GST标签的切除后, 得到纯化的NSRΔ38, 并测定了该蛋白可能的nisin降解活性。反应产物的抑菌活性测定结果表明被NSRΔ38作用     

Synergistic bacteriolysis by bacteriophage ϕEF24C endolysin ORF9 and lantibiotic nisin and its application to pulsed-field gel electrophoretic analysis of Enterococcus faecalis

Genotyping by pulsed-field gel electrophoresis (PFGE) is recognized as one of the most useful methods for epidemiological studies of drug-resistant Enterococcus faecalis nosocomial outbreaks. As the bacteriolysis procedure is a critical step in PFGE, an accurate and reliable alternative bacteriolytic method to the conventional protocol would be useful. In this study, we examined the applicability of endolysin ORF9, derived from the E. faecalis phage ?EF24C, and lantibiotic nisin in sample preparation for PFGE. The results show that the cooperative actions of nisin and ORF9 synergistically lysed E. faecalis, which was then amenable to PFGE analysis.     

Fragment screening and structural analyses highlight the ATP-assisted ligand binding for inhibitor discovery against type 1 methionyl-tRNA synthetase

Methionyl-tRNA synthetase (MetRS) charges tRNA^Met with l-methionine (L-Met) to decode the ATG codon for protein translation, making it indispensable for all cellular lives. Many gram-positive bacteria use a type 1 MetRS (MetRS1), which is considered a promising antimicrobial drug target due to its low sequence identity with human cytosolic MetRS (HcMetRS, which belongs to MetRS2). Here, we report crystal structures of a representative MetRS1 from Staphylococcus aureus (SaMetRS) in its apo and substrate-binding forms. The connecting peptide (CP) domain of SaMetRS differs from HcMetRS in structural organization and dynamic movement. We screened 1049 chemical fragments against SaMetRS preincubated with or without substrate ATP, and ten hits were identified. Four cocrystal structures revealed that the fragments bound to either the L-Met binding site or an auxiliary pocket near the tRNA CCA end binding site of SaMetRS. Interestingly, fragment binding was enhanced by ATP in most cases, suggesting a potential ATP-assisted ligand binding mechanism in MetRS1. Moreover, co-binding with ATP was also observed in our cocrystal structure of SaMetRS with a class of newly reported inhibitors that simultaneously occupied the auxiliary pocket, tRNA site and L-Met site. Our findings will inspire the development of new MetRS1 inhibitors for fighting microbial infections.     

Lantibiotic Immunity: Inhibition of Nisin Mediated Pore Formation by NisI

Nisin, a 3.4 kDa antimicrobial peptide produced by some Lactococcus lactis strains is the most prominent member of the lantibiotic family. Nisin can inhibit cell growth and penetrates the target Gram-positive bacterial membrane by binding to Lipid II, an essential cell wall synthesis precursor. The assembled nisin-Lipid II complex forms pores in the target membrane. To gain immunity against its own-produced nisin, Lactococcus lactis is expressing two immunity protein systems, NisI and NisFEG. Here, we show that the NisI expressing strain displays an IC₅₀ of 73±10 nM, an 8–10-fold increase when compared to the non-expressing sensitive strain. When the nisin concentration is raised above 70 nM, the cells expressing full-length NisI stop growing rather than being killed. NisI is inhibiting nisin mediated pore formation, even at nisin concentrations up to 1 µM. This effect is induced by the C-terminus of NisI that protects Lipid II. Its deletion showed pore formation again. The expression of NisI in combination with externally added nisin mediates an elongation of the chain length of the Lactococcus lactis cocci. While the sensitive strain cell-chains consist mainly of two cells, the NisI expressing cells display a length of up to 20 cells. Both results shed light on the immunity of lantibiotic producer strains, and their survival in high levels of their own lantibiotic in the habitat.     

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.     

Simplified lipid II-binding antimicrobial peptides: Design,synthesis and antimicrobial activity of bioconjugates of nisin rings A and B with pore-forming peptides

New designs of antimicrobial peptides are urgently needed in order to combat the threat posed by the recent increase of resistance to antibiotics. In this paper, we present a new series of antimicrobial peptides, based on the key structural features of the lantibiotic nisin. We have simplified the structure of nisin by conjugating the lipid II-binding motif at the N-terminus of nisin to a series of cationic peptides and peptoids with known antibacterial action and pore-forming properties. Hybrid peptides, where a hydrophilic PEG4 linker was used, showed good antibacterial activity against Micrococcus luteus.     

Mechanisms of nisin resistance in Gram-positive bacteria

Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain Gram-positive bacteria. However, the effectiveness of nisin is often affected by environmental factors such as pH, temperature, food composition, structure, as well as food microbiota. The development of nisin resistance has been seen among various Gram-positive bacteria. The mechanisms under the acquisition of nisin resistance are complicated and may differ among strains. This paper presents a brief review of possible mechanisms of the development of resistance to nisin among Gram-positive bacteria.     

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.     

Lipid II-Based Antimicrobial Activity of the Lantibiotic Plantaricin C

We analyzed the mode of action of the lantibiotic plantaricin C (PlnC), produced by Lactobacillus plantarum LL441. Compared to the well-characterized type A lantibiotic nisin and type B lantibiotic mersacidin, which are both able to interact with the cell wall precursor lipid II, PlnC displays structural features of both prototypes. In this regard, we found that lipid II plays a key role in the antimicrobial activity of PlnC besides that of pore formation. The pore forming activity of PlnC in whole cells was prevented by shielding lipid II on the cell surface. However, in contrast to nisin, PlnC was not able to permeabilize Lactococcus lactis cells or to form pores in 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes supplemented with 0.1 mol% purified lipid II. This emphasized the different requirements of these lantibiotics for pore formation. Using cell wall synthesis assays, we identified PlnC as a potent inhibitor of (i) lipid II synthesis and (ii) the FemX reaction, i.e., the addition of the first Gly to the pentapeptide side chain of lipid II. As revealed by thin-layer chromatography, both reactions were clearly blocked by the formation of a PlnC-lipid I and/or PlnC-lipid II complex. On the basis of the in vivo and in vitro activities of PlnC shown in this study and the structural lipid II binding motifs described for other lantibiotics, the specific interaction of PlnC with lipid II is discussed.     

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.     

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.     

Antimicrobial activity of nisin against the swine pathogen Streptococcus suis and its synergistic interaction with antibiotics

Streptococcus suis serotype 2 is known to cause severe infections in pigs, including meningitis, endocarditis and pneumonia. Furthermore, this bacterium is considered an emerging zoonotic agent. Recently, increased antibiotic resistance in S. suis has been reported worldwide. The objective of this study was to evaluate the potential of nisin, a bacteriocin of the lantibiotic class, as an antibacterial agent against the pathogen S. suis serotype 2. In addition, the synergistic activity of nisin in combination with conventional antibiotics was assessed. Using a plate assay, the nisin-producing strain Lactococcus lactis ATCC 11454 proved to be capable of inhibiting the growth of S. suis (n = 18) belonging to either sequence type (ST)1, ST25, or ST28. In a microdilution broth assay, the minimum inhibitory concentration (MIC) of purified nisin ranged between 1.25 and 5 μg/mL while the minimum bactericidal concentration (MBC) was between 5 and 10 μg/mL toward S. suis. The use of a capsule-deficient mutant of S. suis indicated that the presence of this polysaccharidic structure has no marked impact on susceptibility to nisin. Following treatment of S. suis with nisin, transmission electron microscopy observations revealed lysis of bacteria resulting from breakdown of the cell membrane. A time-killing curve showed a rapid bactericidal activity of nisin. Lastly, synergistic effects of nisin were observed in combination with several antibiotics, including penicillin, amoxicillin, tetracycline, streptomycin and ceftiofur. This study brought clear evidence supporting the potential of nisin for the prevention and treatment of S. suis infections in pigs.     

Characteristics and identification of bacteriocins produced by Lactococcus lactis subsp. lactis 194-K

The Lactococcus lactis subsp. lactis 194-K strain has been established to be able to produce two bacteriocins, one of which was identified as the known lantibiotic nisin A, and the other 194-D bacteriocin represents a polypeptide with a 2589-Da molecular mass and comprises 20 amino acid residues. Both bacteriocins were produced in varying proportions in all of the studied culture media, which support the growth of the producer. Depending on the cultivation medium, the nisin A content was 380- to 1123-fold lower in the 194-K stain culture broth than that of the 194-D peptide. In comparision to nisin A Bacteriocin 194-D possessed a wide range of antibacterial activity and suppressed the growth of both Gram-positive and Gram-negative bacteria. An optimal medium for 194-D bacteriocin synthesis was shown to be a fermentation medium which contained yeast extract, casein hydrolysate, and potassium phosphate. The biosynthesis of bacteriocin 194-D by the 194-K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture broth was observed at14–20 h of the strain’s growth.     

乳链菌肽(nisin)抗性机制的研究进展

乳链菌肽(nisin)是某些乳酸乳球菌产生的一种羊毛硫细菌素。其对包括食品腐败菌和致病菌在内的许多革兰氏阳性菌具有强烈的抑制作用,是目前世界上唯一被允许用作食品添加剂的细菌素。nisin的广泛使用虽未引发大范围的抗性,但在自然界或实验室的选择压力下,某些非nisin产生菌也获得了抵御nisin攻击的抗性机制。nisin抗性机制通常涉及两种方式,即非特异性的生理适应机制和特异性蛋白酶介导的主动防御机制。本文综述了近年来nisin抗性机制的研究进展。     

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.     

Comparison of the Potency of the Lipid II Targeting Antimicrobials Nisin, Lacticin 3147 and Vancomycin Against Gram-Positive Bacteria

While nisin (lantibiotic), lacticin 3147 (lantibiotic) and vancomycin (glycopeptides) are among the best studied lipid II-binding antimicrobials, their relative activities have never been compared. Nisin and lacticin 3147 have been employed/investigated primarily as food preservatives, although they do have potential in terms of veterinary and clinical applications. Vancomycin is used exclusively in clinical therapy. We reveal a higher potency for lacticin 3147 (MIC 0.95?C3.8???g/ml) and vancomycin (MIC 0.78?C1.56???g/ml) relative to that of nisin (MIC 6.28?C25.14???g/ml) against the food-borne pathogen Listeria monocytogenes. A comparison of the activity of the three antimicrobials against nisin resistance mutants of L. monocytogenes also reveals that their susceptibility to vancomycin and lacticin 3147 changed only slightly or not at all. A further assessment of relative activity against a selection of Bacillus cereus, Enterococcus and Staphylococcus aureus targets revealed that vancomycin MICs consistently ranged between 0.78 and 1.56???g/ml against all but one strain. Lacticin 3147 was found to be more effective than nisin against B. cereus (lacticin 3147 MIC 1.9?C3.8???g/ml; nisin MIC 4.1?C16.7???g/ml) and E. faecium and E. faecalis targets (lacticin 3147 MIC from 1.9 to 3.8???g/ml; nisin MIC ??8.3???g/ml). The greater effectiveness of lacticin 3147 is even more impressive when expressed as molar values. However, in agreement with the previous reports, nisin was the more effective of the two lantibiotics against S. aureus strains. This study highlights that in many instances the antimicrobial activity of these leading lantibiotics are comparable with that of vancomycin and emphasizes their particular value with respect to use in situations including foods and veterinary medicine, where the use of vancomycin is not permitted.