Antimicrobial peptides (AMPs) are favoured because of their broad-spectrum antimicrobial properties and because they do not easily develop microbial resistance. However, the low yield and difficult extraction processes of AMPs have become bottlenecks in large-scale industrial applications and scientific research. Microbial recombinant production may be the most economical and effective method of obtaining AMPs in large quantities. In this paper, we review the mechanism, summarize the current status of microbial recombinant production, and focus on strategies to improve the yield and activity of AMPs, in order to provide a reference for their large-scale production.

Infection-related cardiovascular diseases (CVDs) pose a significant health challenge, driving the need for novel therapeutic strategies to target key receptors involved in inflammation and infection. Antimicrobial peptides (AMPs) show the potential to disrupt pathogenic processes and offer a promising approach to CVD treatment. This study investigates the binding potential of selected AMPs with critical receptors implicated in CVDs, aiming to explore their therapeutic potential. A comprehensive computational approach was employed to assess AMP interactions with CVD-related receptors, including ACE2, CRP, MMP9, NLRP3, and TLR4. Molecular docking studies identified AMPs with high binding affinities to these targets, notably Tachystatin, Pleurocidin, and Subtilisin A, which showed strong interactions with ACE2, CRP, and MMP9. Following docking, 100 ns molecular dynamics (MD) simulations confirmed the stability of AMP-receptor complexes, and MM/PBSA calculations provided quantitative insights into binding energies, underscoring the potential of these AMPs to modulate receptor activity in infection and inflammation contexts. The study highlights the therapeutic potential of Tachystatin, Pleurocidin, and Subtilisin A in targeting infection-related pathways in CVDs. These AMPs demonstrate promising receptor binding properties and stability in computational models. Future research should focus on in vitro and in vivo studies to confirm their efficacy and safety, paving the way for potential clinical applications in managing infection-related cardiovascular conditions.

The aim of this study is to examine the functional role and resistance mechanisms of the Escherichia coli (E. coli) peptidoglycan transpeptidase gene, mrdA, in resistance to melittin.

The resistance of E. coli strains with either knockout or overexpression of the mrdA gene to melittin was initially assessed. The differences in melittin absorption between these two strains were evaluated following depletion and heterologous expression of the mrdA gene. Subsequently, peptidoglycan was extracted from the strains to determine its capacity to adsorb melittin. Finally, the morphological changes in different strains induced by melittin exposure were examined under scanning electron microscopy. These analyses served to validate the role of peptidoglycan transpeptidase mrdA in melittin resistance and to hypothesize its potential resistance mechanism.

The results clearly indicated a direct correlation between the degree of peptidoglycan cross-linking in E. coli and its enhanced resistance to melittin. Specifically, we found that increased cross-linking of peptidoglycan led to a thickening of the bacterial cell wall and a reduction in pore size. These structural changes potentially decrease the damage to the cell wall caused by melittin, as the thicker cell wall and smaller pores reduce the ability of melittin to penetrate and access the interior of bacterial cells. This mechanism effectively limits the contact between melittin and bacterial components, minimizing its destructive effects, and thereby conferring resistance to melittin in the bacteria.

This study is the first to elucidate the role of peptidoglycan in the cell wall of E. coli in the context of antimicrobial peptide resistance. Novel ideas have been proposed for the development of antibacterial drugs targeting the peptidoglycan of Gram-negative bacteria.

Cyclic antimicrobial peptides (CAMPs) are gaining attention as promising candidates in advanced drug delivery systems due to their structural stability, resistance to proteolytic degradation, and versatile therapeutic potential. Their unique properties enable applications that extend beyond combating multidrug-resistant (MDR) pathogens. Their amphipathic and cell-penetrating properties allow them to efficiently transport drugs across cellular membranes.

This review explores the structural advantages and mechanisms of action of CAMPs, emphasizing their role in drug delivery. The literature analysis (2010-2024) from PubMed, Scopus, and Web of Science highlights developments in CAMP-conjugated therapies, liposomal formulations, and encapsulation systems. The review also examines their antimicrobial potency, amphipathic and cell-penetrating properties, and integration into nanocarrier technologies to enhance drug stability, bioavailability, and precision targeting. Challenges such as toxicity, scalability, and cost are also discussed. CAMPs have the potential to revolutionize drug delivery through their robustness and multifunctionality, particularly in precision medicine.

Future advancements in peptide engineering, nanotechnology, and AI-driven design are expected to enhance CAMPs' therapeutic specificity, reduce toxicity, and broaden their applications, including oncology and gene therapy, paving the way for their integration into next-generation therapeutics.

This review highlights several basic problems associated with bacterial drug resistance, including the decreasing efficacy of commercially available antimicrobials as well as the related problem of microbiome irregularity and dysbiosis. The article explains that this present situation is addressable through LAB species, such as Streptococcus salivarius and Ligilactobacillus salivarius, which are well established synthesizers of both broad- and narrow-spectrum antimicrobials. The sheer number of antimicrobials produced by LAB species and the breadth of their biological effects, both in terms of their bacteriostatic/bactericidal abilities and their immunomodulation, make them prime candidates for new probiotics and antibiotics. Given the ease with which several of the molecules can be biochemically engineered and the fact that many of these compounds target evolutionarily constrained target sites, it seems apparent that these compounds and their producing organisms ought to be looked at as the next generation of robust dual action symbiotic drugs.

Background/objectives: Bacteriocins can be considered a novel source of natural alternatives to antibiotics or chemical food additives with the potential to fight against clinical and food pathogens. A number have already been commercialised as food preservatives, but they also have the potential to treat drug-resistant clinical pathogens and can play a role in immune modulation. To achieve their full potential, an understanding of their mode of action is required. Methods: Bactofencin A and nisin A were purified to homogeneity by reversed-phase HPLC and their effect on the mastitis pathogen Staphylococcus aureus DPC5246 was assessed by cell viability assays and flow cytometry. Results: We report that bactofencin A displays a delayed inhibitory effect against the mastitis pathogen, Staphylococcus aureus DPC5246, suggesting an unusual mode of action. This characteristic was clearly visible on BHI plate media, where formation of inhibition zones against the staphylococcal strain took 23 h compared to 6 h for the well-characterised nisin. This delayed killing and injury was also demonstrated using flow cytometry, where damage was evident 4 h after bacteriocin addition. Treatment with 2 μM bactofencin A resulted in approximately 20-fold higher numbers of injured and 50-fold higher numbers of dead cells when compared to untreated cells. Combining bactofencin A with the lantibiotic nisin A resulted in faster killing at lower bacteriocin concentrations. When combined in an equal ratio, the combination exhibited a 4-fold increase in inhibition compared to nisin A alone. These results demonstrate that the combination may be very effective in therapeutic applications against pathogenic staphylococci.

Antimicrobial peptides (AMPs) are a diverse group of naturally occurring molecules produced by eukaryotes and prokaryotes. They have an important role in innate immunity via their direct microbicidal properties or immunomodulatory activities against pathogens. With the widespread occurrence of antimicrobial resistance (AMR), AMPs are considered as viable alternatives for the treatment of multidrug-resistant microbes, inflammation, and, wound healing. The broad-spectrum antibacterial activity of AMPs is predominantly attributed to membrane disruption, leading to the formation of transmembrane pores and, eventually, cell lysis. However, mechanisms related to inhibition of cell wall synthesis, nucleic acid synthesis, protein synthesis, or enzymatic activity are also associated with these peptides. In this review, we discuss our current understanding, therapeutic uses and challenges associated with the clinical applications of AMPs.

The emergence of antibiotic resistance among the clinically important bacterial pathogens has increased healthcare costs and reduced patient safety and quality of life. Lantibiotics is a large class of ribosomally synthesized, and posttranslationally modified peptides have been the primary focus of numerous research aimed at discovering compounds for treating bacterial infections.

The article explains the most up to date hierarchy of methods followed in the field for high throughput screening of lantibiotics/analogs with improved therapeutic properties. Herein, we explain how the structure and the biosynthesis of lantibiotics can be manipulated for the expansion of the horizon of lantibiotic potency. Furthermore, we discuss the lantibiotic analogs that have demonstrated the efficacy against bacterial pathogens of interest in animal models.

In this current age of rapidly advancing antimicrobial resistance, there is a dire need for the development of therapeutic agents that possess distinct mechanisms of action to existing modes of treatment. Recent advances in the understanding of many of the lantibiotic biosynthesis systems and the discovery of new analogs with superior properties to the native compound may have paved the way for the development of a much-needed novel potent class of antibiotic.

Cyclic peptides have emerged as an essential tool in the advancement of biomedical nanotechnologies, offering unique structural and functional advantages over linear peptides. This review article aims to highlight the roles of cyclic peptides in the development of biomedical fields, with a particular focus on their application in drug discovery and delivery. Cyclic peptides exhibit exceptional stability, bioavailability, and binding specificity, making them ideal candidates for therapeutic and diagnostic applications. We explore the synthesis and design strategies that enable the precise control of cyclic peptide structures, leading to enhanced performance in targeting specific cellular pathways. The article also highlights recent breakthroughs in the use of cyclic peptides for creating innovative drug delivery systems, including nanoparticle conjugates and peptide-drug conjugates, which have shown promise in improving the efficacy and safety profiles of existing traditional treatments. The integration of cyclic peptides into nanotechnological frameworks holds significant promise for addressing unmet medical needs, providing a foundation for future advancements in personalized medicine and targeted drug delivery.

Fresh meat is highly perishable, presenting challenges in spoilage mitigation and waste reduction globally. Despite the efforts, foodborne outbreaks from meat consumption persist. Biopreservation offers a natural solution to extend shelf life by managing microbial communities. However, challenges include the effective diffusion of bacteriocins through the meat matrix and the potential inhibition of starter cultures by bacteriocins targeting closely related lactic acid bacteria (LAB). LAB, predominant in meat, produce bacteriocins - small, stable peptides with broad antimicrobial properties effective across varying pH and temperature conditions. This review highlights the recent advances in the optimization of bacteriocin use, considering its structure and mode of action. Moreover, the strengths and weaknesses of different techniques for bacteriocin screening, including novel bioengineering methods, are described. Finally, we discuss the advantages and limitations of the modes of application of bacteriocins toward the preservation of fresh, cured, and novel meat products.

The rapid emergence of antibiotic-resistant strains of Staphylococcus aureus presents a substantial challenge to global public health, underscoring the urgent need for novel antibiotics with diverse mechanisms of action. In this study, we conducted mutagenesis on the C-terminal region of the lantibiotic ripcin C to enhance its antimicrobial efficacy against S. aureus. The resulting optimized variant, ripcin CP23A, demonstrated potent and selective antimicrobial activity, with a minimal inhibitory concentration of 2-4 mg/L against S. aureus. Beyond its strong antimicrobial properties, ripcin CP23A exhibited significant antibiofilm activity against methicillin-resistant S. aureus (MRSA). Mechanistic studies revealed that, in addition to targeting lipid II, ripcin CP23A disrupts bacterial membranes, a capability absent in ripcin C, which may contribute to its superior antimicrobial and antibiofilm effects. Moreover, ripcin CP23A displayed favorable biosafety and plasma stability profiles. Notably, in a mouse model of MRSA-induced mastitis, ripcin CP23A effectively reduced bacterial load, alleviated inflammation, and preserved the normal histomorphology of mammary glands. This study introduces ripcin CP23A as a promising antibiotic candidate for the treatment of MRSA-related infections.

The lantibiotic pore-forming peptide nisin is a promising candidate in the fight against multidrug-resistant bacteria due to its unique structure, which allows it to disrupt bacteria in two distinct ways─Lipid II trafficking and transmembrane pore formation. However, exactly how nisin and Lipid II assemble into oligomeric pore structures in the bacterial membrane is not known. Spontaneous peptide assembly into pores is difficult to observe in even the very long-time scale molecular dynamics (MD) simulations. In this study, we adopted an MD-guided modeling approach to investigate the nisin-nisin and nisin-Lipid II associations in the membrane environment. Through extensive microsecond-time scale all-atom MD simulations, we established that nisin monomers dimerize by forming β-sheets in a POPE:POPG lipid bilayer and oligomerize further to form stable transmembrane channels. We determined that these nisin dimers use Lipid II as a dimer interface to incur enhanced stability. Our results provide a clearer understanding of the self-assembly of nisin monomers within the membrane and insights into the role of Lipid II in the structural integrity of oligomeric structures.

Lanthipeptides, a group of ribosomally synthesized and post-translationally modified peptides (RiPPs), exhibit diverse structures and bioactivities. Their biosynthetic enzymes serve as valuable tools for peptide bioengineering. Here, we report a class II lanthipeptide biosynthetic gene cluster in a Bacillus strain, driving the biosynthesis of a two-component lanthipeptide, termed rodencin, featured by the presence of two different d-amino acids, i.e., d-Ala and d-Abu. Rodencin displays synergistic antimicrobial activity against food-borne pathogens such as Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes. The α-peptide of rodencin contains one d-Ala and the β-peptide features both d-Ala and d-Abu. These are installed by dehydratases RodM1 and RodM2 and dehydrogenase RodJA, the activities of which were successfully reconstituted using a dedicated E. coli expression system. To illustrate the unusual d-Abu incorporation potential of the enzymes, analogous to the d-amino acid-containing β peptide of lacticin 3147, was successfully produced with the rodencin heterologous expression system, by employing RodM2 and the dehydrogenase RodJA.

Antimicrobial peptides (AMPs) are a critical component of the innate immune system, playing a key role in defending against a variety of pathogenic microorganisms. While many AMPs act primarily on the cell membrane of target pathogens, leading to lysis and subsequent cell death, less is known about their nonlytic membrane activity. This nonlytic activity allows AMPs to target and disrupt bacterial cells without causing lysis, leading to bacterial death through alternative mechanisms.Understanding these nonlytic properties of AMPs is crucial, as they present a promising alternative to traditional antibiotics, which can induce bacterial resistance and have adverse effects on human health and the environment. The mechanisms by which AMPs exhibit nonlytic membrane activity are still being explored. However, it is believed that AMPs penetrate the bacterial membrane and interact directly with internal cellular components such as DNA, RNA, and various enzymes essential for microbial survival and replication. This interaction disrupts metabolic homeostasis, ultimately resulting in bacterial death.The nonlytic activity of AMPs also results in minimal damage to host cells and tissues, making them attractive candidates for the development of new, more effective antibiotics. This review emphasizes the mechanisms by which AMPs nonlytically target cellular components, including DNA, proteins, RNA, and other biomolecules, and discusses their clinical significance. Understanding these mechanisms may pave the way for developing alternatives to conventional antibiotics, offering a solution to the growing issue of antibiotic resistance.

Small pentacyclic peptides, represented by nisin, have been successfully utilized as preservatives in the food industry and have evolved into a paradigm for understanding the genetic structure, expression, and control of genes created by lantibiotics. Due to the ever-increasing antibiotic resistance, nisin-relevant antimicrobial peptides have received much attention, which calls for a summarization of their synthesis, modification and applications. In this review, we first provided a timeline of select highlights in nisin biosynthesis and engineering. Then, we outlined the current developments in nisin synthesis. We also provided an overview of the engineering, screening, and production of nisin-relevant antimicrobial peptides based on enzyme alteration, substrate modification, and sequence mining. Furthermore, an updated summary of applications of nisin-relevant antimicrobial peptides has been developed for food applications. Finally, this study offers insights into emerging technologies, limitations and the future development of nisin-relevant antimicrobial peptides for pathogen inhibition, food preservatives, and improved health.

Solid lipid nanoparticles (SLNs) can protect and deliver naturally derived or synthetic biologically active products to target sites in vivo. Here, an SLN formulation produces a measured four-fold reduction in inhibitory concentration of an antimicrobial peptide nisin Z against S. aureus as compared to the free peptide, indicating the successful delivery and enhanced effectiveness of the SLN-encapsulated bacteriocin. Spherical SLNs of size 79.47 ± 2.01 nm and zeta potential of -9.8 ± 0.3 mV were synthesised. The lipid formulation maximizes the membrane-damaging mode of action of the free peptide with more and larger-sized pores formed on bacterial membranes treated with nisin Z SLNs as measured from scanning electron microscopy and transmission electron microscopy. Flow cytometry measurements precisely quantified an enhanced dye leakage from pre-labeled bacterial cells when treated with nisin Z-loaded SLNs compared to free peptide. The lipid formulation accelerated cell death by killing all the cells within half an hour compared to the equivalent concentration of free peptide which was not bactericidal. Molecular dynamics simulations revealed a mechanism of SLN facilitated binding to the lipid II bacterial cell wall precursor via enhanced adsorption of nisin Z at the inner bacterial cell membrane bilayer. These findings confirmed the potential of SLN formulations for the effective delivery of therapeutic peptides for next-generation antibiotics that are active at low concentrations with the potential to mitigate antimicrobial resistance.

Bacteria have developed diverse strategies for defending their cell envelopes from external threats. In Firmicutes, one widespread strategy is to use Bce modules-membrane protein complexes that unite a peptide-detoxifying ABC transporter with a stress response coordinating two-component system. These modules provide specific, front-line defense for a wide variety of antimicrobial peptides and small molecule antibiotics as well as coordinate responses for heat, acid, and oxidative stress. Because of these abilities, Bce modules play important roles in virulence and the development of antibiotic resistance in a variety of pathogens, including Staphylococcus, Streptococcus, and Enterococcus species. Despite their importance, Bce modules are still poorly understood, with scattered functional data in only a small number of species. In this review, we will discuss Bce module structure in light of recent cryo-electron microscopy structures of the B. subtilis BceABRS module and explore the common threads and variations-on-a-theme in Bce module mechanisms across species. We also highlight the many remaining questions about Bce module function. Understanding these multifunctional membrane complexes will enhance our understanding of bacterial stress sensing and may point toward new therapeutic targets for highly resistant pathogens.

Viridisin A1 and A2 were previously heterologously expressed, purified, and characterized as ribosomally produced and post-translationally modified lanthipeptides. Such lanthipeptide operons are surprisingly common in Gram-negative bacteria, although their expression seems to be predominantly cryptic under laboratory conditions. However, the bioactivity and biological role of most lanthipeptide operons originating from marine-associated Pseudomonadota, such asThalassomonas viridans XOM25T, have not been described. Therefore, marine-associated Gram-negative lanthipeptide operons represent an untapped resource for novel structures, biochemistries, and bioactivities. Here, the upscaled production of viridisin A1 and A2 was performed for (methyl)lanthionine stereochemistry characterization, antibacterial, antifungal, and larval zebrafish behavioral screening. While antimicrobial activity was not observed, the VirBC modification machinery was found to install both dl- and ll-lanthionine stereoisomers. The VdsA1 and VdsA2 peptides induced sedative and stimulatory effects in zebrafish larvae, respectively, which is a bioactivity not previously reported from lanthipeptides. When combined, VdsA1 and VdsA2 counteracted the sedative and stimulatory effects observed when used individually.

Resistance to available antibiotics poses a growing challenge to modern medicine, as this often disallows infections to be controlled. This problem can only be alleviated by the development of new drugs. Nisin, a natural lantibiotic with broad antimicrobial activity, has shown promise as a potential candidate for combating antibiotic-resistant bacteria. However, nisin is poorly soluble and barely stable at physiological pH, which despite attempts to address these issues through mutant design has restricted its use as an antibacterial drug. Therefore, gaining a deeper understanding of the antimicrobial effectiveness, which relies in part on its ability to form pores, is crucial for finding innovative ways to manage infections caused by resistant bacteria. Using large-scale molecular dynamics simulations, we find that the bacterial membrane-specific lipid II increases the stability of pores formed by nisin and that the interplay of nisin and lipid II reduces the overall integrity of bacterial membranes by changing the local thickness and viscosity.

Recent advances in sequencing techniques unveiled the vast potential of ribosomally synthesized and post-translationally modified peptides (RiPPs) encoded in microbiomes. Class I lantibiotics such as nisin A, widely used as a food preservative, have been investigated for their efficacy in killing pathogens. However, the impact of nisin and nisin-like class I lantibiotics on commensal bacteria residing in the human gut remains unclear. Here, we report six gut-derived class I lantibiotics that are close homologues of nisin, four of which are novel. We applied an improved lantibiotic expression platform to produce and purify these lantibiotics for antimicrobial assays. We determined their minimal inhibitory concentration (MIC) against both Gram-positive human pathogens and gut commensals and profiled the lantibiotic resistance genes in these pathogens and commensals. Structure-activity relationship (SAR) studies with analogs revealed key regions and residues that impact their antimicrobial properties. Our characterization and SAR studies of nisin-like lantibiotics against both pathogens and human gut commensals could shed light on the future development of lantibiotic-based therapeutics and food preservatives.

The emergence of multidrug-resistant (MDR) pathogens is a major problem in the therapeutic management of infectious diseases. Among the bacterial resistance mechanisms is the development of an enveloped protein and polysaccharide-hydrated matrix called a biofilm. Polyphenolics have demonstrated beneficial antibacterial effects. Phenolic compounds mediate their antibiofilm effects via disruption of the bacterial membrane, deprivation of substrate, protein binding, binding to adhesion complex, viral fusion blockage and interactions with eukaryotic DNA. However, these compounds have limitations of chemical instability, low bioavailability, poor water solubility and short half-lives. Nanoformulations offer a promising solution to overcome these challenges by enhancing their antibacterial potential. This review summarizes the antibiofilm role of polyphenolics, their underlying mechanisms and their potential role as resistance-modifying agents.

Nisin, with its unique mode of action and potent antimicrobial activity, serves as a remarkable inspiration for the design of novel antibiotics. However, peptides possess inherent weaknesses, particularly their susceptibility to proteolytic degradation, such as by trypsin, which limits their broader applications. This led us to speculate that natural variants of nisin produced by underexplored bacterial species can potentially overcome these limitations. We carried out genome mining of two Romboutsia sedimentorum strains, RC001 and RC002, leading to the discovery of rombocin A, which is a 25 amino acid residue short nisin variant that is predicted to have only four macrocycles compared to the known 31-35 amino acids long nisin variants with five macrocycles. Using the nisin-controlled expression system, we heterologously expressed fully modified and functional rombocin A in Lactococcus lactis and demonstrated its selective antimicrobial activity against Listeria monocytogenes. Rombocin A uses a dual mode of action involving lipid II binding activity and dissipation of the membrane potential to kill target bacteria. Stability tests confirmed its high stability at different pH values, temperatures, and in particular, against enzymatic degradation. With its gene-encoded characteristic, rombocin A is amenable to bioengineering to generate novel derivatives. Further mutation studies led to the identification of rombocin K, a mutant with enhanced bioactivity against L. monocytogenes. Our findings suggest that rombocin A and its bioengineered variant, rombocin K, are promising candidates for development as food preservatives or antibiotics against L. monocytogenes.

Many bacteria produce antimicrobial compounds such as lantibiotics to gain advantage in the competitive natural environments of microbiomes. Epilancins constitute an until now underexplored family of lantibiotics with an unknown ecological role and unresolved mode of action. We discovered production of an epilancin in the nasal isolate Staphylococcus epidermidis A37. Using bioinformatic tools, we found that epilancins are frequently encoded within staphylococcal genomes, highlighting their ecological relevance. We demonstrate that production of epilancin A37 contributes to Staphylococcus epidermidis competition specifically against natural corynebacterial competitors. Combining microbiological approaches with quantitative in vivo and in vitro fluorescence microscopy and cryo-electron tomography, we show that A37 enters the corynebacterial cytoplasm through a partially transmembrane-potential-driven uptake without impairing the cell membrane function. Upon intracellular aggregation, A37 induces the formation of intracellular membrane vesicles, which are heavily loaded with the compound and are essential for the antibacterial activity of the epilancin. Our work sheds light on the ecological role of epilancins for staphylococci mediated by a mode of action previously unknown for lantibiotics.

Gram-positive Enterococcus faecium exhibited higher susceptibility (>4-fold) to polymyxin B (PMB), the canonical antimicrobial peptide against Gram-negative bacteria, under anaerobic condition than aerobic condition. Anaerobically grown E. faecium exhibited high vulnerability to PMB, leading to alteration of cell surface and morphology, as observed based on their high dansyl-PMB affinity (>2.9-fold), a proportion (>8.5-fold) of propidium iodide-stained cells, and observation of scanning electron microscopy results. Interestingly, our transcriptomic and chemical analyses revealed that enterocin B, produced anaerobically, imposes a burden on the cellular envelope when cells are exposed to PMB. This scenario was also supported by PMB susceptibility tests and killing curves, which showed that ΔentB knockout mutant cells were more resistant to PMB (32 µg/mL) compared to wild-type cells (4 µg/mL) under anaerobic condition. Fluorescent D-amino acid and BOCILLIN™-fluorescent profiling of transpeptidase activities in ΔentB mutant cells under anaerobic condition revealed similar levels of activity to those observed in WT cells under aerobic condition. The high level of secreted bacteriocins in WT under anaerobic condition likely led to significant membrane depolarization and loosening of the peptidoglycan layer, making the cells more permeable to PMB. Overall, our findings suggest that anaerobically produced bacteriocins, in conjunction with PMB, contribute to the killing of E. faecium by destabilizing its cell envelope.

Addressing the current antibiotic-resistance challenge would be aided by the identification of compounds with novel mechanisms of action. Epilancin 15X, a lantibiotic produced by Staphylococcus epidermidis 15 × 154, displays antimicrobial activity in the submicromolar range against a subset of pathogenic Gram-positive bacteria. S. epidermidis is a common member of the human skin or mucosal microbiota. We here investigated the mechanism of action of epilancin 15X. The compound is bactericidal against Staphylococcus carnosus as well as Bacillus subtilis and appears to kill these bacteria by membrane disruption. Structure-activity relationship studies using engineered analogs show that its conserved positively charged residues and dehydroamino acids are important for bioactivity, but the N-terminal lactyl group is tolerant of changes. Epilancin 15X treatment negatively affects fatty acid synthesis, RNA translation, and DNA replication and transcription without affecting cell wall biosynthesis. The compound appears localized to the surface of bacteria and is most potent in disrupting the membranes of liposomes composed of negatively charged membrane lipids in a lipid II independent manner. Epilancin 15X does not elicit a LiaRS response in B. subtilis but did upregulate VraRS in S. carnosus. Treatment of S. carnosus or B. subtilis with epilancin 15X resulted in an aggregation phenotype in microscopy experiments. Collectively these studies provide new information on epilancin 15X activity.

Lipopeptides are surface active, natural products of bacteria, fungi and green-blue algae origin, having diverse structures and functionalities. In analogy, a number of chemical synthesis techniques generated new designer lipopeptides with desirable features and functions. Lipopetides are self-assembly guided, supramolecular compounds which have the capacity of high-density presentation of the functional epitopes at the surface of the nanostructures. This feature contributes to their successful application in several industry sectors, including food, feed, personal care, and pharmaceutics. In this comprehensive review, the novel class of ribosomally synthesized lipopeptides is introduced alongside the more commonly occuring non-ribosomal lipopeptides. We highlight key representatives of the most researched as well as recently described lipopeptide families, with emphasis on structural features, self-assembly and associated functions. The common biological, chemical and hybrid production routes of lipopeptides, including prominent analogues and derivatives are also discussed. Furthermore, genetic engineering strategies aimed at increasing lipopeptide yields, diversity and biological activity are summarized and exemplified. With respect to application, this work mainly details the potential of lipopeptides in personal care and cosmetics industry as cleansing agents, moisturizer, anti-aging/anti-wrinkling, skin whitening and preservative agents as well as the pharmaceutical industry as anitimicrobial agents, vaccines, immunotherapy, and cancer drugs. Given that this review addresses human applications, we conclude on the topic of safety of lipopeptide formulations and their sustainable production.

Nisin is a widely used lantibiotic owing to its potent antimicrobial activity and its food-grade status. Its mode of action includes cell wall synthesis inhibition and pore formation, which are attributed to the lipid II binding and pore-forming domains, respectively. We discovered cesin, a short natural variant of nisin, produced by the psychrophilic anaerobe Clostridium estertheticum. Unlike other natural nisin variants, cesin lacks the two terminal macrocycles constituting the pore-forming domain. The current study aimed at heterologous expression and characterization of the antimicrobial activity and physicochemical properties of cesin. Following the successful heterologous expression of cesin in Lactococcus lactis, the lantibiotic demonstrated a broad and potent antimicrobial profile comparable to that of nisin. Determination of its mode of action using lipid II and lipoteichoic acid binding assays linked the potent antimicrobial activity to lipid II binding and electrostatic interactions with teichoic acids. Fluorescence microscopy showed that cesin lacks pore-forming ability in its natural form. Stability tests have shown the lantibiotic is highly stable at different pH values and temperature conditions, but that it can be degraded by trypsin. However, a bioengineered analog, cesin R15G, overcame the trypsin degradation, while keeping full antimicrobial activity. This study shows that cesin is a novel (small) nisin variant that efficiently kills target bacteria by inhibiting cell wall synthesis without pore formation. IMPORTANCE The current increase in antibiotic-resistant pathogens necessitates the discovery and application of novel antimicrobials. In this regard, we recently discovered cesin, which is a short natural variant of nisin produced by the psychrophilic Clostridium estertheticum. However, its suitability as an antimicrobial compound was in doubt due to its structural resemblance to nisin(1-22), a bioengineered short variant of nisin with low antimicrobial activity. Here, we show by heterologous expression, purification, and characterization that the potency of cesin is not only much higher than that of nisin(1-22), but that it is even comparable to the full-length nisin, despite lacking two C-terminal rings that are essential for nisin's activity. We show that cesin is a suitable scaffold for bioengineering to improve its applicability, such as resistance to trypsin. This study demonstrates the suitability of cesin for future application in food and/or for health as a potent and stable antimicrobial compound.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

In the present study, the antimicrobial peptide nisin was successfully conjugated onto the surface of sulfonated polyetheretherketone (SPEEK), which was decorated with graphene oxide (GO) to investigate its biofilm resistance and antibacterial properties. The PEEK was activated with sulfuric acid, resulting in a porous structure. The GO deposition fully covered the porous SPEEK specimen. The nisin conjugation was accomplished using the crosslinker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) through a dip-coating method. The surface micrographs of the SPEEK-GO-nisin sample indicated that nisin formed discrete islets on the flat GO surface, allowing both the GO and nisin to perform a bactericidal effect. The developed materials were tested for bactericidal efficacy against Staphylococcus aureus (S. aureus). The SPEEK-GO-nisin sample had the highest antibacterial activity with an inhibition zone diameter of 27 mm, which was larger than those of the SPEEK-nisin (19 mm) and SPEEK-GO (10 mm) samples. Conversely, no inhibitory zone was observed for the PEEK and SPEEK samples. The surface micrographs of the bacteria-loaded SPEEK-GO-nisin sample demonstrated no bacterial adhesion and no biofilm formation. The SPEEK-nisin and SPEEK-GO samples showed some bacterial attachment, whereas the pure PEEK and SPEEK samples had abundant bacterial colonies and thick biofilm formation. These results confirmed the good biofilm resistance and antibacterial efficacy of the SPEEK-GO-nisin sample, which is promising for implantable orthopedic applications.

Antibiotic resistance remains a global threat to human and animal health. Staphylococcus aureus is an opportunistic pathogen that causes minor to life-threatening infections. The widespread use of antibiotics in the clinical, veterinary, and agricultural setting combined with the increasing prevalence of antibiotic-resistant S. aureus strains makes it abundantly clear that alternatives to antibiotics are urgently needed. Bacteriocins represent one potential alternative therapeutic. They are antimicrobial peptides that are produced by bacteria that are generally nontoxic and have a relatively narrow target spectrum, and they leave many commensals and most mammalian cells unperturbed. Multiple studies involving bacteriocins (e.g., nisin, epidermicin, mersacidin, and lysostaphin) have demonstrated their efficacy at eliminating or treating a wide variety of S. aureus infections in animal models. This review provides a comprehensive and updated evaluation of animal studies involving bacteriocins and highlights their translational potential. The strengths and limitations associated with bacteriocin treatments compared with traditional antibiotic therapies are evaluated, and the challenges that are involved with implementing novel therapeutics are discussed.

Lantibiotics are believed to have a conceivable potential to be used as therapeutics, especially against clinically resistant bacterial strains. However, their low solubility and poor stability under physiological conditions limit their availability for clinical studies and further pharmaceutical commercialization. Nisin is a readily available and cheap lanthipeptide and thus serves as a good model in the search for the tools to engineer lantibiotics with improved pharmacological properties. This review aims to address technologies that can be applied to alter and enhance the antimicrobial activity, antibacterial spectrum and physicochemical properties (solubility, solution stability and protease resistance) of nisin. There are basically two general means to obtain nisin analogs-protein engineering and chemical functionalization of this antibiotic. Although bioengineering techniques have been well developed and enable the creation of nisin mutants of variable structures and properties, they are lacking spectacular effects so far. Chemical modifications of nisin based on utilization of the reactivity of its free amino and carboxylic moieties, as well as reactivity of the double bonds of its dehydroamino acids, are in their infancy.

Bacillus licheniformis produces several classes of antimicrobial substances, including bacteriocins, which are peptides or proteins with different structural composition and molecular mass: ribosomally synthesized by bacteria (1.4-20 kDa), non-ribosomally synthesized peptides and cyclic lipopeptides (0.8-42 kDa) and exopolysaccharides (>1000 kDa). Different bacteriocins act against Gram-positive or Gram-negative bacteria, fungal pathogens and amoeba cells. The main mechanisms of bacteriocin lytic activity include interaction of peptides with membranes of target cells resulting in structural alterations, pore-forming, and inhibition of cell wall biosynthesis. DNase and RNase activity for some bacteriocines are also postulated. Non-ribosomal peptides are synthesized by special non-ribosomal multimodular peptide synthetases and contain unnatural amino acids or fatty acids. Their harmful effect is due to their ability to form pores in biological membranes, destabilize lipid packaging, and disrupt the peptidoglycan layer. Lipopeptides, as biosurfactants, are able to destroy bacterial biofilms. Secreted polysaccharides are high molecular weight compounds, composed of repeated units of sugar moieties attached to a carrier lipid. Their antagonistic action was revealed in relation to bacteria, viruses, and fungi. Exopolysaccharides also inhibit the formation of biofilms by pathogenic bacteria and prevent their colonization on various surfaces. However, mechanism of the harmful effect for many secreted antibacterial substances remains unknown. The antimicrobial activity for most substances has been studied in vitro only, but some substances have been characterized in vivo and they have found practical applications in medicine and veterinary. The cyclic lipopeptides that have surfactant properties are used in some industries. In this review, special attention is paid to the antimycobacterials produced by B. licheniformis as a possible approach to combat multidrug-resistant and latent tuberculosis. In particular, licheniformins and bacitracins have shown strong antimycobacterial activity. However, the medical application of some antibacterials with promising in vitro antimycobacterial activity has been limited by their toxicity to animals and humans. As such, similar to the enhancement in the antimycobacterial activity of natural bacteriocins achieved using genetic engineering, the reduction in toxicity using the same approach appears feasible. The unique capability of B. licheniformis to synthesize and produce a range of different antibacterial compounds means that this organism can act as a natural universal vehicle for antibiotic substances in the form of probiotic cultures and strains to combat various types of pathogens, including mycobacteria.

The increase in antibiotic resistance calls for accelerated molecular engineering strategies to diversify natural products for drug discovery. The incorporation of non-canonical amino acids (ncAAs) is an elegant strategy for this purpose, offering a diverse pool of building blocks to introduce desired properties into antimicrobial lanthipeptides. We here report an expression system using Lactococcus lactis as a host for non-canonical amino acid incorporation with high efficiency and yield. We show that incorporating the more hydrophobic analog ethionine (instead of methionine) into nisin improves its bioactivity against several Gram-positive strains we tested. New-to-nature variants were further created by click chemistry. By azidohomoalanine (Aha) incorporation and subsequent click chemistry, we obtained lipidated variants at different positions in nisin or in truncated nisin variants. Some of them show improved bioactivity and specificity against several pathogenic bacterial strains. These results highlight the ability of this methodology for lanthipeptide multi-site lipidation, to create new-to-nature antimicrobial products with diverse features, and extend the toolbox for (lanthi)peptide drug improvement and discovery.

It is almost a century since nisin was discovered in fermented milk cultures, coincidentally in the same year that penicillin was first described. Over the last 100 years this small, highly modified pentacyclic peptide has not only found success in the food industry as a preservative but has also served as the paradigm for our understanding of the genetic organization, expression, and regulation of genes involved in lantibiotic biosynthesis-one of the few cases of extensive post-translation modification in prokaryotes. Recent developments in understanding the complex biosynthesis of nisin have shed light on the cellular location of the modification and transport machinery and the co-ordinated series of spatio-temporal events required to produce active nisin and provide resistance and immunity. The continued unearthing of new natural variants from within human and animal gastrointestinal tracts has sparked interest in the potential application of nisin to influence the microbiome, given the growing recognition of the role the gastrointestinal microbiota plays in health and disease. Moreover, interdisciplinary approaches have taken advantage of biotechnological advancements to bioengineer nisin to produce novel variants and expand nisin functionality for applications in the biomedical field. This review will discuss the latest progress in these aspects of nisin research.

Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55 PP, Lipid II, Lipid WTA ). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.

The growth of bacteria and fungi may cause disease inf human or spoilage of food. New antimicrobial substances need to be discovered. Lactoferricin (LFcin) is a group of antimicrobial peptides derived from the N-terminal region of the milk protein lactoferrin (LF). LFcin has antimicrobial ability against a variety of microorganisms, which is significantly better than that of its parent version. Here, we review the sequences, structures, and antimicrobial activities of this family and elucidated the motifs of structural and functional significance, as well as its application in food. Using sequence and structural similarity searches, we identified 43 new LFcins from the mammalian LFs deposited in the protein databases, which are grouped into six families according to their origins (Primates, Rodentia, Artiodactyla, Perissodactyla, Pholidota, and Carnivora). This work expands the LFcin family and will facilitate further characterization of novel peptides with antimicrobial potential. Considering the antimicrobial effect of LFcin on foodborne pathogens, we describe the application of these peptides from the prospective of food preservation.

Nisin is a posttranslationally modified antimicrobial peptide that is widely used as a food preservative. It contains five cyclic thioethers of varying sizes. Nisin activity and stability are closely related to its primary and three dimensional structures. It has nine reported natural variants. Nisin A is the most studied nisin as it was the first one purified. Here, we review the sequence feature of nisin A and its natural variants, and their biosynthesis pathway, mode of action and application as a meat preservative. We systematically illustrate the functional domains of the main enzymes (NisB, NisC, and NisP) involved in nisin synthesis. NisB was shown to dehydrate its substrate NisA via a tRNA associated glutamylation mechanism. NisC catalysed the cyclization of the didehydro amino acids with the neighboring cysteine residues. After cyclization, the leader peptide is removed by the protease NisP. According to multiple sequence alignments, we detected five conserved sites Dha⁵, Pro⁹, Gly¹⁴, Leu¹⁶, and Lys²². These residues are probably the structural and functional important ones that can be modified to produce peptides versions with enhanced antimicrobial activity. Through comparing various application methods of nisin in different meats, the antimicrobial effects of nisin used individually or in combination with other natural substances were clarified.

Helicobacter pylori infects approximately 50% of the world's population and is considered the major etiological agent of severe gastric diseases, such as peptic ulcers and gastric carcinoma. Increasing resistance to standard antibiotics has now led to an ever-decreasing efficacy of eradication therapies and the development of novel and improved regimens for treatment is urgently required. Substantial progress has been made over the past few years in the identification of molecular mechanisms which are conducive to resistant phenotypes as well as for efficient strategies to counteract strain resistance and to avoid the use of ineffective antibiotics. These involve molecular testing methods, improved salvage therapies, and the discovery of novel and potent antimicrobial compounds. High rates of prevalence and gastric cancer are currently observed in Asian countries, including Japan, China, Korea, and Taiwan, where concomitantly intensive research efforts were initiated to explore advanced eradication regimens aimed at reducing the risk of gastric cancer. In this review, we present an overview of the known molecular mechanisms of antibiotic resistance and discuss recent intervention strategies for H. pylori diseases, with a view of the research progress in Asian countries.

A growing body of evidence from multiple areas proposes that periodontal disease, accompanied by oral inflammation and pathological changes in the microbiome, induces gut dysbiosis and is involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). A subgroup of NAFLD patients have a severely progressive form, namely nonalcoholic steatohepatitis (NASH), which is characterized by histological findings that include inflammatory cell infiltration and fibrosis. NASH has a high risk of further progression to cirrhosis and hepatocellular carcinoma. The oral microbiota may serve as an endogenous reservoir for gut microbiota, and transport of oral bacteria through the gastro-intestinal tract can set up a gut microbiome dysbiosis. Gut dysbiosis increases the production of potential hepatotoxins, including lipopolysaccharide, ethanol, and other volatile organic compounds such as acetone, phenol and cyclopentane. Moreover, gut dysbiosis increases intestinal permeability by disrupting tight junctions in the intestinal wall, leading to enhanced translocation of these hepatotoxins and enteric bacteria into the liver through the portal circulation. In particular, many animal studies support that oral administration of Porphyromonas gingivalis, a typical periodontopathic bacterium, induces disturbances in glycolipid metabolism and inflammation in the liver with gut dysbiosis. NAFLD, also known as the hepatic phenotype of metabolic syndrome, is strongly associated with metabolic complications, such as obesity and diabetes. Periodontal disease also has a bidirectional relationship with metabolic syndrome, and both diseases may induce oral and gut microbiome dysbiosis with insulin resistance and systemic chronic inflammation cooperatively. In this review, we will describe the link between periodontal disease and NAFLD with a focus on basic, epidemiological, and clinical studies, and discuss potential mechanisms linking the two diseases and possible therapeutic approaches focused on the microbiome. In conclusion, it is presumed that the pathogenesis of NAFLD involves a complex crosstalk between periodontal disease, gut microbiota, and metabolic syndrome. Thus, the conventional periodontal treatment and novel microbiome-targeted therapies that include probiotics, prebiotics and bacteriocins would hold great promise for preventing the onset and progression of NAFLD and subsequent complications in patients with periodontal disease.

Throughout the ages the world has witnessed the outbreak of many infectious diseases. Emerging microbial diseases pose a serious threat to public health. Increasing resistance of microorganisms towards the existing drugs makes them ineffective. In fact, anti-microbial resistance is declared as one of the top public health threats by WHO. Hence, there is an urge for the discovery of novel antimicrobial drugs to combat with this challenge. Structural diversity and unique pharmacological effects make natural products a prime source of novel drugs. Staggeringly, in spite of its extensive biodiversity, a prominent portion of microorganism species remains unexplored for the identification of bioactives. Microorganisms are a predominant source of new chemical entities and there are remarkable number of antimicrobial drugs developed from it. In this review, we discuss the contributions of microorganism based natural products as effective antibacterial agents, studied during the period of 2010-2020. The review encompasses over 140 structures which are either natural products or semi-synthetic derivatives of microbial natural products. 65 of them are identified as newly discovered natural products. All the compounds discussed herein, have exhibited promising efficacy against various bacterial strains.

The rapid emergence of microbial multi-resistance against antibiotics has led to intense search for alternatives. One of these alternatives are ribosomally synthesized and post-translationally modified peptides (RiPPs), especially lantibiotics. They are active in a low nanomolar range and their high stability is due to the presence of characteristic (methyl-) lanthionine rings, which makes them promising candidates as bacteriocides. However, innate resistance against lantibiotics exists in nature, emphasizing the need for artificial or tailor-made lantibiotics. Obviously, such an approach requires an in-depth mechanistic understanding of the modification enzymes, which catalyze the formation of (methyl-)lanthionine rings. Here, we determined the structure of a class I cyclase (MadC), involved in the modification of maddinglicin (MadA) via X-ray crystallography at a resolution of 1.7 Å, revealing new insights about the structural composition of the catalytical site. These structural features and substrate binding were analyzed by mutational analyses of the leader peptide as well as of the cyclase, shedding light into the mode of action of MadC.

To date, a number of lantibiotics have been shown to use lipid II-a highly conserved peptidoglycan precursor in the cytoplasmic membrane of bacteria-as their molecular target. The α-component (Lchα) of the two-component lantibiotic lichenicidin, previously isolated from the Bacillus licheniformis VK21 strain, seems to contain two putative lipid II binding sites in its N-terminal and C-terminal domains. Using NMR spectroscopy in DPC micelles, we obtained convincing evidence that the C-terminal mersacidin-like site is involved in the interaction with lipid II. These data were confirmed by the MD simulations. The contact area of lipid II includes pyrophosphate and disaccharide residues along with the first isoprene units of bactoprenol. MD also showed the potential for the formation of a stable N-terminal nisin-like complex; however, the conditions necessary for its implementation in vitro remain unknown. Overall, our results clarify the picture of two component lantibiotics mechanism of antimicrobial action.