An Engineered Nisin Analogue with a Hydrophobic Moiety Attached at Position 17 Selectively Inhibits Enterococcus faecium Strains

Antibiotic resistance is one of the most challenging global public health concerns. It results from the misuse and overuse of broad-spectrum antibiotics, which enhance the dissemination of resistance across diverse bacterial species. Antibiotics like nisin and teixobactin do not target an essential protein and employ a dual mode of action antibacterial mechanism, thereby being less prone to induce resistance. There is a need for the development of a potent narrow-spectrum dual-mode-acting antibiotic against human pathogens. Using nisin, a lantibiotic with potent antimicrobial activity against many pathogens, as a template, the unnatural amino acid azidohomoalanine was introduced at selected positions and subsequently modified using click chemistry with 14 alkyne-moiety containing tails. A novel nisin variant, compound 47, featuring a benzyl group-containing tail, exhibited potent activity against various (drug-resistant) E. faecium strains with an MIC value (3.8 mg/L) similar to nisin, whereas its activity toward other pathogens like Staphylococcus aureus and Bacillus cereus was significantly reduced. Like nisin, the mode of action of compound 47 results from the inhibition of cell wall synthesis by binding to lipid II and nisin–lipid II hybrid-pore formation in the outer membrane. The resistance of compound 47 against proteolytic degradation is markedly enhanced compared to nisin. Like nisin, compound 47 was hardly hemolytic even at a very high dose. Collectively, a modified nisin variant is presented with significantly enhanced target organism specificity and stability.


INTRODUCTION
Antimicrobial resistance poses a significant threat to the public health.A report from the United Kingdom government predicts that, without the development of new antimicrobial strategies, antibiotic-resistant infections could lead to 10 million deaths worldwide annually by 2050. 1 The acceleration of the rate of resistance development is attributed to the overuse and misuse of broad-spectrum antibiotics. 2These antibiotics not only naturally foster resistance but also promote the selection of resistance mechanisms in nontarget species, enabling their subsequent transfer to pathogenic bacteria.Additionally, the use of broad-spectrum antibiotics can disrupt the microbiota, which plays crucial roles in various aspects of human biology. 3To mitigate the risk of unintentional development of antibiotic resistance and microbiota disruption, employing a species-selective antimicrobial agent that specifically targets and eliminates the disease-causing strain is desired.
The issue of bacterial resistance is not uniformly distributed among all bacterial species. 4The Infectious Disease Society of America (IDSA) has identified six species with pronounced threats due to their potential mechanisms of multidrug resistance (MDR) and pathogenicity, collectively referred to as ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudo-monas aeruginosa, and Enterobacter species). 5E. faecium, in particular, is globally linked to hospital outbreaks involving bacteremia, urinary tract infections, and endocarditis, among other conditions. 6These outbreaks not only impose significant economic burdens on healthcare systems but also pose a severe risk to susceptible patients, potentially leading to fatal infections. 7The challenge is further exacerbated by the complexities of treatment associated with the development of high-level resistance to various antibiotics, whether intrinsic or acquired through the horizontal transfer of plasmids and transposons. 6,8Vancomycin-resistant E. faecium (VRE) has emerged as the predominant multidrug-resistant Enterococcus species in healthcare settings. 9Consequently, there is an urgent need to discover new therapeutic agents and develop alternative approaches to address this highly challenging drugresistant infection.
Protein-targeting antibiotics like penicillins are known to be most sensitive for inducing drug resistance. 10Antimicrobial peptides (AMPs) have emerged as a promising avenue for addressing antimicrobial resistance 11 as many, for example, vancomycin and mersacidin, do not target a protein but lipid II, an essential building block for cell wall synthesis.Other AMPs, like teixobactin, target both lipid II and lipid III, 10 whereas nisin targets lipid II, leading to the inhibition of the cell wall synthesis and forming nisin-hybrid pores in the cell membrane. 12Both teixobactin and nisin are very potent antimicrobials, and their dual mode of action on essential nonprotein targets renders the development of bacterial resistance challenging. 13,14Although many AMPs have proven efficacy, in many cases, they lack specificity and target a wide spectrum of Gram-positive and/or Gram-negative bacteria. 15,16−20 The strategy involves constructing a hybrid peptide that combines two functionally independent components, namely, a targeting peptide and a broad-spectrum AMP, linked by a short flexible linker.The targeting peptide imparts selectivity to the AMP domain by binding to specific determinants on the pathogen's surface, such as membrane charge, receptors, cell wall components, or distinctive virulent attributes.
Despite achieving some promising results, the method encountered certain limitations.Because of its gene-encoded character, nonpeptide molecular entities such as fatty acid tails or sugar groups cannot readily be included.Incorporating the desired moiety at positions other than those at the N-or Cterminus is not straightforward, while the termini of AMPs often play a crucial role in their function, making them unsuitable for modifications.Another limitation of the strategy is that these hybrid peptides are typically not well structured, making them susceptible for proteolytic degradation. 21n antibacterial nonribosomally produced lipopeptides, 22 nature is using a hydrophobic tail to enhance the activity.As cell membrane architecture and lipid composition differ between pathogen species, 23 engineering of the tail structure and its positioning within an AMP might be a strategy to enhance both activity and selectivity.Potent antibacterial nonribosomally produced lipopeptides like daptomycin contain ring structures and are rich in noncanonical amino acids, which make them more resilient to proteolytic degradation. 24any antibacterial ribosomally synthesized and post-translationally synthesized peptides (RiPPs) are also stabilized by ring structures and amino acid modifications and have the advantage of engineering their structure using molecular biology techniques. 25One of the best studied examples is nisin, a 34-residue long RiPP containing five lanthionine rings. 14 Koopmans et al. was the first to introduce a hydrophobic tail at the C-terminus of an antimicrobial RiPP. 26The low activity of a truncated nisin variant could be enhanced to wild-type nisin activity when a suitable tail was attached.In few other studies, similar results were observed using different RiPPs. 27,28In all of these studies, the tail was introduced at the N-or C-terminus.
Recently, we reported a Lactococcus lactis-based expression system for the efficient incorporation (>99.5%) of the Met analog azidoalanine (Aha) in nisin, together with a high expression yield of 9.5 mg/L pure peptide (Figure 1a). 29Aha possesses a unique azide functional group (Figure 1b) capable of reacting with alkyne substrates, commonly referred to as "click chemistry" (Figure 1c). 30This makes the system very suitable to introduce an alkyne moiety containing a tail molecule at a selected residue position in nisin, and this was demonstrated for a single alkyne (1-undecyne) introduced at four positions.In this study, we present 42 new nisin analogues by testing a diverse set of tail compounds, including linear and branched alkyl chains, phenyl groups, and polyprolines, introduced at four different positions of nisin.For these newly synthesized peptides, we report the antimicrobial activity profiles for a dozen pathogens, modes of action, stabilities, and toxicity toward human red blood cells.

RESULTS AND DISCUSSION
2.1.The Impact of the Tail Structure on the Antibacterial Activity of the Nisin Constructs.Nisin (Figure 2a) is one of the best studied antimicrobial RiPPs and has been documented since 1928. 14Derived from various strains, nisin exhibits potent activity against Gram-positive bacteria including Bacillus cereus, Listeria monocytogenes, enterococci, staphylococci, and streptococci. 14Nisin is characterized by the presence of five (methyl)lanthionine rings and various post-translational modifications (Figure 2a).The dual functionality of nisin emanates from two functional parts located at the N-and C-termini, respectively. 31The Nterminal segment, hosting three post-translationally incorporated (methyl)lanthionine rings (A, B, and C), connects to the C-terminal rings (D and E) through a flexible hinge region comprising three amino acids (Figure 2a).The collective (a) Schematic overview of the force-feeding method for noncanonical amino acid Aha incorporation into nisin.Translation of the genes for the nisin modification machinery nisBTC, controlled by the P czcD promoter, is initiated while cells are growing in rich medium.After the exchange of the growth medium to a chemically defined medium (CDM) without Met but supplemented with the Met analogue Aha, 10 ng/mL nisin (P nisA promotor inducer) is added to start expression of single Met nisin construct.In this way, the analog Aha instead of Met is incorporated into nisin with high incorporation efficiency and yield, at the same time not affecting the NisBTC enzymes needed for the peptide postmodifications, e.g., dehydration and cyclization.(b) Structures of Met and its analog.Met, methionine; Aha, azidohomoalanine.The side chain difference is indicated in blue/red.Aha possesses a unique azide functional group capable of reacting with alkyne substrates.(c) Scheme of Aha-labeled nisin attached with alkyne substrates using the copper (Cu + )catalyzed azide−alkyne click chemistry reaction.formation of the A, B, and C rings creates a "cage", facilitating the binding of lipid II's pyrophosphate moiety, thereby disrupting cell wall synthesis. 14This binding enhances the capability of the C-terminal segment, housing rings D and E, to generate pores in the cell membrane, resulting in the rapid efflux of ions and cytoplasmic solutes.Nisin's distinctive dual mode of action contributes to its remarkable efficacy and plays a crucial role in limiting the emergence of nisin resistance.
Recently, we presented a L. lactis-based expression system for the efficient labeling of four single-Met containing nisin mutants with Aha creating an attractive expression platform for subsequent labeling with an alkyne moiety containing tail molecule using click chemistry. 29Using this platform, in the first round of screening experiments, each single-Aha containing nisin construct was modified with eight different tail molecules, yielding compounds 6−37, and their structures are presented in Figures 2 and 3.The screening assay using E. faecium distinctly shows that different side chains influence nisin's antibacterial activity.The best modification site is Aha located at residue 17 (Figure 2b, no 4).When labeled with 1decyne, this yielded compound 16, for which the highest activity against E. faecium was observed.In compounds 18−21, 22−25, and 30−33, for which 1-dodecyne, 1-pentadecyne, and 3,7,11-trimethyl-1-dodecyn-3-ol were used as tail molecule, a decreased antibacterial activity was observed (Figure 3). Lee et al., attaching different acyl chain lengths (C8, C10, C12, C14, and C16) to a nonapeptide, also observed that the longest tails yielded the lowest antibacterial activity. 32Comparing compounds 34−37 with compounds 14−17, it is evident that introducing a carboxylic acid group at the end of the tail decreases the antimicrobial activity.Clicking with hydrophobic polyprolines yielded compounds 6−13, which also showed good activity, just somewhat less potent than its counterparts containing more hydrophobic alkyl tails.
Because for all eight tested tail structures explored in the first round, the nisin variant with Aha at position 17 gave the best result, two shorter alkynes (1-hexyne and 1-octyne) and a monosaturated undecyne were chosen to be introduced only at this Aha position.Nisin with Aha at the first residue position was also chosen for further study.The click chemistry products were tested against two strains, E. faecium and L. monocytogenes (Figure 4).A lower activity against E. faecium was observed in this round for all variants created compared to the most active variant, compound 16, discovered in the first round.Thus, shortening the chain length of compound 16 by two or four carbons lowers its activity (Figure 4).In this context, it is interesting to consider various clinically used lipopeptide antibiotics, including telavancin, dalbavancin, and daptomycin, that also contain chains of similar size as in compound 16. 22wo tail compounds containing a phenyl ring were also tested.Best results were obtained with 5-phenyl-1-pentyne to create compound 47, which shows a similar activity against L. monocytogenes as nisin and an activity comparable with compound 16 against E. faecium (Figure 4).Next, a more detailed investigation was conducted on compounds 14, 16, 46, and 47, which were formed by the click chemistry reaction between 1-decyne or 5-phenyl-1-pentyne and Aha at residue position 17 or at the N-terminus of nisin.

Selective Activity of Compounds 16 and 47 against E. faecium at Low Concentrations.
The four compounds mentioned above were purified (Figures S1 and  S2) (see the Experimental Section) and tested, together with nisin for comparison, in agar well diffusion assays using four pathogenic Gram-positive strains, namely, Staphylococcus aureus, Enterococcus faecium, Bacillus cereus, and Listeria monocytogenes.As expected, wild-type nisin demonstrated broad-spectrum antibacterial activity (Figure 5).However, introduction of the tails at the first residue position of nisin, yielding compounds 14 and 46, resulted in variants losing activity against Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes while retaining activity against Enterococcus faecium, albeit with weaker potency compared to wildtype nisin (Figure 5).Interestingly, a similar narrowing of activity spectrum was observed for compounds 16 and 47, but the high potency of nisin against Enterococcus faecium was retained.
Next, the specificity of compounds 16 and 47 was investigated in more detail using a panel of strains, including five Enterococcus faecium strains, one Enterococcus faecalis strain, two Listeria monocytogenes strains, two Staphylococcus aureus strains, one Bacillus cereus strain, and a Gram-negative Escherichia coli strain.Against different Enterococcus faecium strains, nisin and compounds 16 and 47 demonstrated high potency, whereas they showed lower activity against the remaining tested strains compared to wild-type nisin (Table 1, Figure S3).In the MIC assay (Table 2), both compounds 16   and 47 displayed potent activity against E. faecium, with a low concentration of 3.8 μg/mL, the same as that for nisin.However, compounds 16 and 47 exhibited 4 times higher MIC values against S. aureus and B. cereus compared to nisin, whereas all three compounds show the same MIC value against L. monocytogenes, which required a higher concentration of 15.3 μg/mL.Collectively, the newly developed compounds 16 and 47, modified at residue position 17 of nisin, exhibit a highly specific and potent lantibiotic activity against E. faecium strains.

Compound 47 Demonstrates Low Hemolytic Activity.
Considering the potential therapeutic application of a bacteriocin, ensuring its safety is of critical importance.Previous studies have identified hemolytic activities in certain bacteriocins, such as listeriolysin S from Listeria monocytogenes 33 and cytolysin from Enterococcus faecalis. 34Koopmans et al. reported that attaching fatty acid tails to the C-terminus of a nisin fragment, nisin (1−12), resulted in semisynthetic lipopeptides displaying antibacterial activities on par with that of the wild-type nisin, whereas an increase in hemolytic activities was observed. 26Therefore, a close evaluation of the safety of new bacteriocins is essential.In this study, a hemolytic activity assay was conducted to assess the safety of compounds 16 and 47.In this assay, human blood cells were incubated with varying concentrations of compound 16, compound 47, and nisin, ranging from 0.39 to 200 mg/L.Following a 1 h incubation at 37 °C, we measured the OD 414 of the supernatants, and hemolytic activities were calculated, with 10% Triton X-100 serving as a positive control.Notably, both compound 47 and nisin exhibited minimal hemolytic activities (below 5%) at the high concentration of 200 mg/L.In contrast, compound 16 demonstrated 65% hemolytic activity at 200 mg/L and 5% hemolytic activity at 100 mg/L.However, all three peptides showed no hemolytic activity against human red blood cells at concentrations below 100 mg/L (Figure 6).
It is interesting to note that the linear tail caused higher hemolytic activities (compound 16) compared to the phenylmoiety containing tail (compound 47); at the same time, it did not influence the overall activity.In summary, the selective and potent antimicrobial compound 47 against Enterococcus faecium shows minimal hemolytic activity at 200 mg/L, a concentration 50-fold higher than its MIC value against this pathogen.
2.4.Compound 47 Exhibits Superior Proteolytic Resistance Compared to Nisin.RiPPs have gained widespread applications, partly due to their resilience in harsh environments. 35To evaluate the stability of compound 47, we exposed the peptide to different temperature values (Figure S4) and proteolytic enzymes (Figure 7).
The stability of compound 47 and nisin was assessed across temperatures ranging from 20 to 90 °C (Figure S4).Less than 40% loss in activity was observed for the higher temperatures over an 8 h storage period (Figure S4).This suggests a good stability at physiological temperatures and being suitable for applications like high-temperature pasteurization. 36ext the resistance against the proteolytic enzymes trypsin, chymotrypsin, and protease K was investigated by adding these enzymes in separate experiments to assay wells followed by overnight incubation at 30 °C.Nisin exhibited a different resistance against trypsin and chymotrypsin degradation, with 32 and 49% activity loss, respectively (Figure 7a,b).Compound 47 showed a similar profile as nisin when exposed to trypsin degradation but demonstrated improved resistance to chymotrypsin compared to nisin, with only a 16% loss in activity.Particularly when exposed to proteinase K, compound   47 exhibited an 77% loss in activity, whereas proteinase K completely eradicated nisin activity (Figure 7b).
The peptidic character of nisin imposes certain constraints, notably its vulnerability to proteolytic degradation in vivo, 37 thereby restricting its broad applications.In previous studies, genome mining methods were utilized to discover new short nisin variants 29,38 while employing a dehydrated amino acid engineering approach and a hybrid peptide, both aimed at enhancing its stability against proteases. 39,40In this study, attaching a hydrophobic tail was introduced as another strategy to enhance resistance to proteolytic enzymes.Previous studies have shown that lipidation can increase the stability of AMPs by blocking vulnerable areas to proteases or forming supramolecular structures, thereby prolonging the drug effect time of lipidated AMPs. 41For example, Chionis et al. synthesized lipidated anoplin with fatty acid chains incorporated at the N-terminus, retaining antimicrobial activity even after a 4 h exposure to trypsin. 42Another study by Zhong et al. found that lipid-modified anoplin analogs exhibited high stability toward trypsin hydrolysis, and its antimicrobial activity was not significantly reduced after preincubation in mouse serum. 43These findings highlight lipidation as a viable strategy to enhance the resistance of AMPs to protease degradation.In  conclusion, compound 47 demonstrated high thermal stability and robust resistance to proteolytic enzymes.
2.5.Compound 47 Exhibits a Dual Mechanism against Bacteria Similar to Nisin.Nisin exerts its antimicrobial effects through pore formation and inhibition of cell wall synthesis by specifically binding to lipid II, a crucial precursor in peptidoglycan biosynthesis. 14To investigate the impact of the attached benzyl group in compound 47 on the mode of action compared to nisin (compound 1), we assessed its binding ability to lipid II (Figure 8a).Externally added purified lipid II reduced the antimicrobial activity of both nisin and the nisin variant against E. faecium and S. aureus, disrupting the typically circular antibiotic-induced halo.The non-lipid IIbinding antibiotic daptomycin maintained its antimicrobial activity against the tested strains after the addition of purified lipid II, resulting in a circular halo.Despite structural modifications, compound 47 retained its ability to bind to lipid II, similar to that of nisin.
To explore the potential impact of modifications on nisin's pore-forming activity, we conducted potassium ion efflux assays (Figure 8b).At the lowest concentration tested (4 μg/ mL), both peptides exhibited a slight potassium leakage.Higher lantibiotic concentrations resulted in stronger leakage for both wild-type nisin and the nisin variant.Interestingly, at the same concentration, nisin and its variant demonstrated comparable efficiency for potassium leakage against the two strains (Figure 8b) despite differences in their antibacterial efficacy (Table 2).In conclusion, the benzyl group modification to nisin did not influence its binding ability to lipid II.Furthermore, its pore-forming ability against E. faecium and S. aureus was retained, with efficiency unaffected, despite exhibiting different profiles against the two tested strains.Our understanding of the reasons for the different antimicrobial activities of RiPPs in various microorganisms is still developing, and this also applies to intensively studied nisin.Because the mechanism of action of the narrow-spectrum compound 47 parallels that of nisin, it is conceivable that variation in cell wall thickness and/or composition between bacterial strains has a significant impact on the effectiveness of these antimicrobial agents.

CONCLUSIONS
Nisin, a broad-spectrum lantibiotic active against many Grampositive bacteria, was synthetically modified to yield derivatives with a more focused antibacterial spectrum and enhanced proteolytic stability.The described approach offers a streamlined method for producing such compounds.Notably, among the analogues synthesized, compound 47 demonstrated the most potent antibacterial activity, particularly against drugresistant strains such as VRE.Their distinct mode of action and superior stability render these semisynthetic lipopeptides promising candidates for further refinement and development as innovative specific antibiotics.

Expression and Purification of Aha-Incorporated Nisin.
L. lactis NZ9000 cells containing the nisBTC plasmid with the plasmid harboring the nisin gene were plated on GM17 agar plates supplemented with 5 μg/mL chloramphenicol (Cm) and erythromycin (Em) and cultured overnight at 30 °C.A selected colony was inoculated into 25 mL of GM17CmEm medium for initial growth followed by transfer to 1 L of the same medium.At an OD 600 of approximately 0.4, 0.5 mM ZnSO 4 was introduced to induce the expression of the modification machinery NisBTC.After 3 h, cells were triple-washed with phosphate-buffered saline (PBS) buffer (pH 7.2) and resuspended in 1 L of CDM-P medium lacking tryptone 29 and Met.Aha (50 mg/L) and 10 ng/mL nisin were added for peptide expression.Following overnight growth, the supernatant was harvested through centrifugation at 8000g for 10 min.
The supernatant's pH was adjusted to 7.0, and it was incubated with purified NisP 29 at 37 °C for 3 h to cut off the leader; then the supernatant was applied to a C 18 open column (Spherical C 18 , 5 g, particle size: 40−75 μm, Sigma-Aldrich).The column was washed with 40 mL of different concentrations (25, 30, 35, 40, and 60%) of buffer B (buffer A, distilled water with 0.1% TFA; buffer B, acetonitrile with 0.1% TFA).The active fractions were lyophilized and further purified using an Agilent 1200 series high-performance liquid chromatograph (HPLC) equipped with a C 18 column (NUCLEODUR C 18 HTec, 5 μm, 250 × 4.6 mm, MACHEREY-NAGEL).The peak with correct molecular weight was collected, lyophilized, and stored at 4 °C until further use.
For the most potent analogs identified in this work, namely, compounds 14, 16, 46, and 47, the reaction volume was scaled up, and the compounds were purified using high-performance liquid chromatography (HPLC).Mass spectra of these purified compounds are presented in Figure S1b.To confirm that this procedure yields the modified nisin and at high purity, HPLC analysis of purified 47 was performed (Figure S2).Based on the area of the peaks, the purity of 47 is 96%.Subsequently, 30 μL of 0.1 mg mL −1 purified compound was added to the well to assess their activity.

Screening the Antibacterial Activity of Clicked
Peptides by a Agar Well Diffusion Assay.Cultures grown overnight were introduced into 0.8% LB agar for Listeria monocytogenes or GM17 agar for Enterococcus faecium at a temperature of 45 °C, reaching a final concentration of 0.1% (v/v).Subsequently, 30 mL of the mixture was poured onto the plate.Upon solidification of the agar, 8 mm wells were created, and 30 μL of the aforementioned clicked solutions was spotted into the wells.The agar plate underwent overnight incubation at 37 °C followed by measurement of inhibition zones.By comparing the antimicrobial impact of the click chemistry products in this screening assay, we assume that the yields of the different reactions are similar.Zone diameters were recorded in millimeters, with the zone area (πr 2 ) minus the well area (πr 2 ) measured in millimeters. 44.4.Minimal Inhibitory Concentration Assay.Minimal inhibitory concentration (MIC) values were determined through broth microdilution following standard guidelines.29 The inoculum was adjusted to approximately 5 × 10 5 CFU/mL, and the MIC was characterized as the lowest concentration of the HPLC purified antimicrobial compound that exhibited no visible growth after overnight incubation at 37 °C.
4.5.Hemolysis Assay.Erythrocytes were sourced from a healthy human volunteer donor, and whole human blood was subjected to centrifugation at 600g for 15 min.Subsequently, plasma was removed, and the erythrocytes were subjected to three washes with PBS (pH 7) by centrifugation at 600g for 15 min each.After the supernatant was discarded, the packed cells were stored on ice.HPLC purified peptides were then introduced at final concentrations of 200, 100, 50, 20, 25, 12.5, 6.25, 3.13, 1.56, 0.76, and 0.39 mg/L in PBS containing 2% (v/v) erythrocytes.The cells were incubated at 37 °C for 1 h and subsequently centrifuged for 5 min at 800g.The supernatant was transferred to a 96-well plate, and absorbance was measured at a wavelength of 414 nm using a Thermo Scientific Varioskan LUX multimode microplate reader.The absorbance, relative to the positive control treated with 10% Triton X-100, was defined as the percentage of hemolysis.
4.6.Effects of Proteolytic Enzymes and Temperature on the Antibacterial Activity of Nisin and Compound 47.The impact of proteolytic enzymes and temperature on antimicrobial activity was investigated using the representative strain L. lactis MG1363 through the agar well diffusion assay.Thirty microliters of the HPLC purified peptide (1 mg mL −1 ) was directly introduced into the agar well containing a final concentration of 1 mg mL −1 proteolytic enzymes (pH 7.2) or no proteolytic enzyme (control).The plates were incubated overnight at 30 °C followed by the measurement of inhibition zones.Temperature stability was assessed by incubating the peptide at 22, 55, 70, and 90 °C for the specified duration.
4.7.Spot-on-Lawn Assay to Measure Peptide−Lipid II Complex Formation.To assess the interaction between the HPLC purified peptide and lipid II, an overnight culture was introduced into 0.8% GM17 medium for E. faecium or LB medium for S. aureus (w/v, temperature 45 °C) at a final concentration of 1% (v/ v).Subsequently, this mixture was evenly distributed onto 10 mL plates.The binding affinity of the peptide and lipid II was further examined by applying purified lipid II (300 μM, 2 μL) to the periphery of the antibiotic inhibition halo.Briefly, HPLC purified antimicrobials were initially applied to the agar plate, and once the antimicrobial solution drops had dried, lipid II was spotted along the edge of the inhibition halo.The plates were then incubated overnight at 37 °C.
4.8.Potassium Ion Efflux Assays.For the K + release assay, the K + -specific fluorescent probe PBFI was employed.Strains of E. faecium were cultured in GM17 medium, whereas S. aureus strains were cultured in LB medium until reaching an OD 600 of 0.6.Subsequently, cells were harvested (5000g, 5 min) and washed twice with 10 mM HEPES buffer (pH 7.2) containing 0.5% glucose.The washed cells were then resuspended in the same buffer supplemented with 10 μM PBFI.Using a Varioskan LUX Multimode Microplate Reader, data were acquired by exciting cells at 346 nm, and the fluorescence emission was measured at 505 nm to establish a baseline signal.Following this, varied concentrations of HPLC purified antibiotics were added, and the data were collected.Nisin served as a positive control in these experiments.

Data Availability Statement
All data supporting the findings of this study are available within the paper and its Supporting Information files.

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.4c00337.Supplementary experimental section; mass spectra (MS) of purified nisin variants; HPLC profile of purified compound 47; spot-on-lawn assay for the compounds 1, 16, and 47 antibacterial spectrum; and thermal stability profile of compounds 1 and 47 (PDF)

Figure 1 .
Figure 1.(a) Schematic overview of the force-feeding method for noncanonical amino acid Aha incorporation into nisin.Translation of the genes for the nisin modification machinery nisBTC, controlled by the P czcD promoter, is initiated while cells are growing in rich medium.After the exchange of the growth medium to a chemically defined medium (CDM) without Met but supplemented with the Met analogue Aha, 10 ng/mL nisin (P nisA promotor inducer) is added to start expression of single Met nisin construct.In this way, the analog Aha instead of Met is incorporated into nisin with high incorporation efficiency and yield, at the same time not affecting the NisBTC enzymes needed for the peptide postmodifications, e.g., dehydration and cyclization.(b) Structures of Met and its analog.Met, methionine; Aha, azidohomoalanine.The side chain difference is indicated in blue/red.Aha possesses a unique azide functional group capable of reacting with alkyne substrates.(c) Scheme of Aha-labeled nisin attached with alkyne substrates using the copper (Cu + )catalyzed azide−alkyne click chemistry reaction.

Figure 2 .
Figure 2. (a) Structure of nisin A. Dha, dehydroalanine; Dhb, dehydrobutyrine; Ala-S-A, lanthionine; Abu-S-A, methyllanthionine.The functional domains, including the lipid II binding site, pore formation domain, and hinge region, are indicated.The positions that will be chemically modified are indicated in green.(b) Structure of various nisin constructs used in this study and each labeled produced using click chemistry.In gray, Met replaced by Ile or Val; in green, position of Aha incorporation and subsequent modification by click chemistry.

Figure 3 .
Figure 3. Antibacterial activities of nisin derivatives against Enterococcus faecium assessed through an agar well diffusion assay.Zone diameters were measured in millimeters, and the area of the inhibition zone (πr 2 ) minus the well area (πr 2 ) was calculated in mm 2 .The red color denotes the highest activity observed among the tested analogs.Experiments were performed in triplicate, and variations in zone diameters were 1 mm or less.

Figure 4 .
Figure 4. Antibacterial activities of nisin derivatives against Enterococcus faecium (labeled as Area 1 [mm 2 ]) and Listeria monocytogenes (Area 2 [mm 2 ]) evaluated using an agar well diffusion assay.The red color indicates the highest activity observed among the tested analogs against either Enterococcus faecium or Listeria monocytogenes.Experiments were performed in triplicate, and variations in zone diameters were 1 mm or less.

Figure 5 .
Figure 5. HPLC-purified nisin and nisin analogs tested against four pathogenic Gram-positive bacteria.Whereas wild-type nisin exhibits broad activity against all tested strains, the newly synthesized nisin variants demonstrate selective efficacy.The blue arrow indicates that the peptides exhibit antibacterial activity.

Figure 6 .
Figure 6.Hemolytic activity of nisin and two newly synthesized nisin variants.Human erythrocytes were incubated with nisin (1), compound 16, and compound 47 at concentrations ranging from 0.39 to 200 mg/L.Hemolytic activity was evaluated based on hemoglobin release.Cells treated without any tested compound served as the no lysis control, whereas cells treated with 10% Triton X-100 were used as completely lysed.The data represent three independent experiments and the standard deviation is indicated.

Figure 7 .
Figure 7. Investigation of the proteolytic stability profiles of nisin (compound 1) and compound 47.(a) Antimicrobial activity of the peptides evaluated against trypsin, chymotrypsin, and proteinase K.A representative image from three independent experiments is shown, with a control where no enzymes was added.(b) Relative antimicrobial activity of the peptides after exposure to different proteolytic enzymes.The relative activity was calculated as the area (enzyme added) divided by the area (control) multiplied by 100%.The standard deviation is indicated.

Figure 8 .
Figure 8. Mode of action of nisin (compound 1) and the benzyl group conjugated nisin variant (compound 47) against E. faecium and S. aureus.(a) A spot-on-lawn assay to assess the ability to bind to the cell wall synthesis precursor lipid II.Nisin was used as positive control, and daptomycin was used as negative control. 1, nisin; 2, compound 47; and 3, daptomycin.*The position lipid II was added (300 μM, 2 μL).(b) Potassium leakage, as detected by the increase in fluorescence of PBFI probe, after addition of different concentrations antimicrobials.At 5 min, antibiotics were added.

Table 1 .
Antimicrobial Activity of Wild-Type Nisin (Compound 1) and Compounds 16 and 47 against Pathogenic Microorganisms b

Table 2 .
Antimicrobial Profile of Nisin (1) and Compounds 16 and 47 against Selected Gram-Positive Strains a Presented MIC values are based on three experiments, yielding the same outcome for all tested conditions.