Subclass IId bacteriocins targeting mannose phosphotransferase system-Structural diversity and implications for receptor interaction and antimicrobial activity.

The bacterial mannose phosphotransferase system (Man-PTS) mediates uptake of selected monosaccharides. Simultaneously, it is a receptor for diverse bacteriocins such as subclass IIa pediocin-like bacteriocins and some subclass IId ones (garvicins ABCQ, lactococcins ABZ, BacSJ, ubericin K, and angicin). So far, no attempt has been made to categorize this ever-expanding group of bacteriocins. Here, we identified Man-PTS as a receptor for a number of previously uncharacterized bacteriocins, and demonstrated that they all belong to a large family of Man-PTS-binding nonpediocin-like peptides, providing new insights into their structure and function. Based on amino acid sequence similarities between members of this family, we propose their classification into five groups. This classification conveniently distinguishes bacteriocins with specific structures and properties regarding their spectrum of antimicrobial activity and pattern of interaction with Man-PTS. With respect to the latter, we indicate individual amino acid residues or regions of Man-PTS and the bacteriocin responsible for their interaction. In Man-PTS, these residues localize to the exterior of the transport complex, specifically the extracellular loop of the so-called Vmotif domain-containing regions γ and/or γ+, and to the interior of the transport complex, specifically the interface between the Core and Vmotif domains. Finally, we propose that while the bacteriocins from separate groups display specific binding patterns to Man-PTS, the general mechanism of their interaction with the receptor is universal despite significant differences in their predicted structures, i.e. after initial docking on the bacterial cell through an interaction with the Man-PTS regions γ and/or γ+, they pull away its Core and Vmotif from one another to form a pore across the membrane.

Emerging lactic acid bacteria bacteriocins as anti-cancer and anti-tumor agents for human health.

Modern cancer diagnostics and treatment options have greatly improved survival rates; the illness remains a major cause of mortality worldwide. Current treatments for cancer, such as chemotherapy, are not cancer-specific and may cause harm to healthy cells; therefore, it is imperative that new drugs for cancer be developed that are both safe and effective. It has been found that lactic acid bacteria (LAB) have the potential to produce bacteriocins, which could potentially offer a promising alternative for cancer treatment. They have been shown in several studies to be effective against cancer cells while having no effect on healthy cells. More research is needed to fully understand the potential of LAB bacteriocins as anti-cancer medicines, to find the appropriate dose and delivery route, and to conduct clinical trials to evaluate the effectiveness and safety of the products in human patients, as is suggested by this work. Furthermore, LAB bacteriocins may evolve into a significant new class of anti-cancer drugs and food products. Patients with cancer may have a safe and effective alternative treatment option in the form of anti-cancer foods and drugs. Therefore, the aim of this study is to provide an in-depth analysis of the recent breakthroughs and potential future technical advancements of significant bacteriocins that are produced by LAB, how these bacteriocins function, and how these bacteriocins may be utilized as an anti-cancer agent. In addition, the current analysis emphasizes the significant constraints and boundaries that bacteriocins face when they are used as an anti-cancer factor.

Antibacterial Activity and Cytotoxicity of the Novel Bacteriocin Pkmh.

Bacteriocins are a class of proteins produced by bacteria that are toxic to other bacteria. These bacteriocins play a role in bacterial competition by helping to inhibit potential competitors. In this study, we isolated and purified a novel bacteriocin Pkmh, different from the previously reported bacteriocin PA166, from Pseudomonas sp. strain 166 by ammonium sulfate precipitation, dialysis membrane method, ion exchange chromatography, and gel filtration chromatography. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) revealed that the molecular weight of Pkmh is approximately 35 kDa. Pkmh exhibited potent antimicrobial activity against bovine Mannheimia haemolytica (M. haemolytica) with low cytotoxicity, and lower hemolytic activity was observed. In addition, Pkmh retained antimicrobial activity at different pH ranges (2-10) and temperature conditions (40, 60, 80, 100 °C). Our analysis of its antimicrobial mechanism showed that Pkmh acts on bacterial cell membranes, increasing their permeability and leading to cell membrane rupture and death. In conclusion, Pkmh exhibited low hemolytic activity, low toxicity, and potent antibacterial effects, suggesting its potential as a promising candidate for clinical therapeutic drugs.

Antibacterial and antibiofilm activities of bacteriocin produced by a new strain of Enterococcus faecalis BDR22.

A large number of recalcitrant bacterial pathogens cannot be easily treated by antibiotics due to the existence of biofilm. Hence, an alternative strategy needs to be adopted to remove the biofilm without the development of antibiotic resistance. Bacteriocins, ribosome-mediated proteinaceous toxins, having potential to inhibit the growth of closely or distantly related bacteria. In the present study, after screening a number of sources, a bacteriocin-producing strain, Enterococcus faecalis BDR22, was isolated that showed a significant reduction in the growth of planktonic cells of Gram-positive Staphylococcus aureus, Bacillus subtilis, and Gram-negative Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Enterobacter cloacae, and Klebsiella pneumoniae compared to the conventional antibiotic tetracycline. The considerable reduction of the biofilm-forming sessile cells of the test organisms S. aureus (ATCC 23235) and P. aeruginosa (ATCC 10145), with no significant cell revival even after withdrawal of the treatment, was also observed. The extracellular polymeric substance (EPS) content of the biofilm was also reduced, with around 84% total carbohydrate reduction found for both microorganisms. The antibiofilm activities of the strain against test organisms were clearly visible from scanning electron micrographs and confirmed by the changes in functional groups (C-H, -OH, C = C, C-N etc.) of biofilm matrices by Fourier transform infrared spectroscopy (FTIR) analysis. The molecular docking interactions with docking energies ∆G of - 54.40 kcal/mol and - 66.2373 kcal/mol validate the affinity of the bacteriocin towards the biofilm-forming protein, which confirms the competence of the bacteriocin-producing strain to act as an effective antimicrobial and antibiofilm agent, replacing antibiotics.

Synergistic antibacterial activity of Lactococcus lactis and Xylella phage MATE 2 for an effective biocontrol strategy against black rot disease in broccoli.

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is considered the most destructive disease affecting cruciferous vegetables, resulting in significant losses worldwide. The need for biocontrol agents against Xcc that can reduce reliance on chemical pesticides, enhance sustainability, and ensure crops and environmental health is crucial. Combining phages with other antibacterial agents (i.e., antibiotics and bacteriocins) to treat bacterial infections is gaining increased attention due to the frequently observed synergistic effects. This study introduces for the first time the combination of a lytic phage, i.e., Xylella phage MATE 2 (MATE 2) with nisin-producing Lactococcus lactis subsp. lactis (L. lactis) bacterium as an eco-friendly, cost-effective, and practical strategy for controlling Xcc in cruciferous vegetables. The antibacterial efficacy of MATE 2 and L. lactis, individually and in combination, against Xcc was investigated through a series of in vitro assays and in planta experiments conducted on broccoli plants. The time-killing curves results showed that under conditions of reduced Xcc population concentration (103 CFU/mL), MATE 2 at 108 PFU/mL exerted a persistent inhibitory effect on Xcc growth for 7 days. The Spot assays and v-qPCR analysis showed that both L. lactis and its bacteriocin nisin have significant antibacterial potential to contrast Xcc. Furthermore, combined application of MATE 2 and L. lactis in broccoli plants by foliar spraying generated significant synergistic efficacy in preventing Xcc infections, achieving a 71% reduction in symptoms, compared with 64 and 38% for single applications, respectively. In this study, the positive synergistic effect of the combined application of phage and beneficial bacteria in preventing black rot disease underscores this eco-friendly and cost-effective approach as a promising control measure against plant bacterial diseases.

Genomic Insights into Pediococcus pentosaceus ENM104: A Probiotic with Potential Antimicrobial and Cholesterol-Reducing Properties.

Pediococcus pentosaceus, which often occurs in fermented foods, is characterized by numerous positive effects on the human health, such as the presence of possible probiotic abilities, the reduction of cholesterol levels, satisfactory antimicrobial activity, and certain therapeutic functions. This study was conducted with the goal of describing the genomic content of Pediococcus pentosaceus ENM104, a strain known for its inhibitory effects against pathogenic bacteria and its remarkable probiotic potential, including the induction of significant reductions in cholesterol levels and the production of γ-aminobutyric acid (GABA). The P. pentosaceus ENM104 chromosome is circular. The chromosome is 1,734,928 bp with a GC content of 37.2%. P. pentosaceus also harbors a circular plasmid, pENM104, that is 71,811 bp with a GC content of 38.1%. Functional annotations identified numerous genes associated with probiotic traits, including those involved in stress adaptation (e.g., heat stress: htpX, dnaK, and dnaJ), bile tolerance (e.g., ppaC), vitamin biosynthesis (e.g., ribU, ribZ, ribF, and btuD), immunomodulation (e.g., dltA, dltC, and dltD), and bacteriocin production (e.g., pedA). Notably, genes responsible for lowering cholesterol levels (bile salt hydrolase, bsh) and GABA synthesis (glutamate/GABA antiporter, gadC) were also identified. The in vitro assay results using cell-free supernatants of P. pentosaceus ENM104 revealed antibacterial activity against carbapenem-resistant bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, and the inhibition zone diameter increased progressively over time. This comprehensive study provides valuable insights into the molecular characteristics of P. pentosaceus ENM104, emphasizing its potential as a probiotic. Its notable cholesterol-lowering, GABA-producing, and antimicrobial capabilities suggest promising applications in the pharmaceutical and food industries. Future research should focus on further exploring these functional properties and assessing the strain's efficacy in clinical settings.

Developing natural microcapsules by encapsulating peptides for preserving Zanthoxylum Bungeanum.

Natural edible microcapsules, were developed to improve the shelf life of Zanthoxylum bungeanum. Antimicrobial peptides, extracted from seeds of Sichuan pepper corn by ultrasound and microwave assisted extraction were encapsulated with nisin using water-in-oil-in-water (W/O/W) microencapsulation technique. Prepared microcapsules exhibited maximum encapsulation efficiency (ω %) of 30.20 at 3:1 ratio of extracted protein (EP) to gum Arabic (GA). After characterization, microcapsules were applied to Sichuan peppers by coating them during 10-days storage. Meanwhile, antimicrobial activity, total phenolic content (TPC), total flavonoid content (TFC) and radical scavenging activity (%) of treated pepper samples were evaluated; demonstrating that S3 and S4 microcapsules provided maximum antimicrobial activity (89.75 and 81.33 %), TPC (543.56 ± 3.87 and 481.40 ± 6.54 GAE/g), TFC (266.02 ± 2.64 QE/g and 306.96 ± 3.87 QE/g) and DPPH radical scavenging activity (78.06 ± 2.87 and 76.52 ± 1.67 %), respectively. Hence, S3 and S4 micro-capsules can be successfully employed as edible coating packaging to improve quality and shelf life of pepper.

Antibacterial action, proteolytic immunity, and in vivo activity of a Vibrio cholerae microcin.

Microcins are small antibacterial proteins that mediate interbacterial competition. Their narrow-spectrum activity provides opportunities to discover microbiome-sparing treatments. However, microcins have been found almost exclusively in Enterobacteriaceae. Their broader existence and potential implications in other pathogens remain unclear. Here, we identify and characterize a microcin active against pathogenic Vibrio cholerae: MvcC. We show that MvcC is reliant on the outer membrane porin OmpT to cross the outer membrane. MvcC then binds the periplasmic protein OppA to reach and disrupt the cytoplasmic membrane. We demonstrate that MvcC's cognate immunity protein is a protease, which precisely cleaves MvcC to neutralize its activity. Importantly, we show that MvcC is active against diverse cholera isolates and in a mouse model of V. cholerae colonization. Our results provide a detailed analysis of a microcin outside of Enterobacteriaceae and its potential to influence V. cholerae infection.

Fortification of Goat Milk Yogurts with Encapsulated Postbiotic Active Lactococci.

The species Lactococcus lactis is a bacterium extensively used in the dairy industry. This bacterium is Generally Recognized as Safe and was added to the European Food Safety Authority's Qualified Presumption of Safety list. The major functions of this species in dairy fermentation are the production of lactic acid from lactose, citric acid fermentation, and the hydrolysis of casein. But, the representatives of this species that produce bacteriocin substances can also exert an inhibitory effect against spoilage bacteria. The aims of this study were to test three lactococcal strains isolated from raw goat milk for their postbiotic activity and to test their stability in goat milk yogurts after their application in encapsulated form for their further application. To achieve these aims, validated methods were used. Three Lactococcus lactis strains (identified by Blastn 16S rRNA analysis) produced bacteriocin substances/postbiotics. These concentrated postbiotics inhibited the growth of enterococci and staphylococci (by up to 97.8%), reaching an inhibitory activity of up to 800 AU/mL. The encapsulated (freeze-dried) lactococci survived in the goat milk yogurts with sufficient stability. Strain MK2/8 fortified the yogurts in the highest amount (8.1 ± 0.0 cfu/g log 10). It did not influence the pH of the yogurt.

A Novel C-Terminal Truncated Bacteriocin Found by Comparison between Leuconostoc mesenteroides 406 and 213M0 Isolated from Mongolian Traditional Fermented Milk, Airag.

Bacteriocins produced by lactic acid bacteria are known to be useful tools for food biopreservation and fermentation control. Leuconostoc mesenteroides subsp. mesenteroides 406 and 213M0 isolated from different samples of Mongolian traditional fermented milk, airag, had been reported to produce listericidal bacteriocin-like inhibitory substances with similar but slightly different properties. In this study, the antibacterial properties and the related gene sequences of both strains were compared, and then their bacteriocins were purified and identified. Strain 406 was superior to strain 213M0 in cell growth and antibacterial activity against many strains. However, the activity of 213M0 was stronger than that of 406 against a few strains. DNA sequencing revealed two and three plasmids in 406 and 213M0, respectively, and each one of them harbored an almost identical mesentericin Y105-B105 gene cluster. Removal of these plasmids resulted in a complete loss of activity, indicating that the antibacterial activity of both strains was generated by bacteriocins encoded on the plasmids. Mesentericins Y105 and B105 were purified from both cultures, and another novel bacteriocin, named mesentericin M, was identified from the 213M0 culture only. Its structural gene was coded on a 213M0 plasmid and, surprisingly, its C-terminal three amino acid residues were post-translationally cleaved. To our knowledge, this is the first report of a C-terminal truncated bacteriocin. In conclusion, the novel bacteriocin should be mainly responsible for the difference in antibacterial properties between the two strains.

ParalichenysinDY4, a novel bacteriocin-like substance, is employed to control Clostridium perfringens.

Clostridium perfringens (C. perfringens) is an important pathogen that contributes to human and animal disease. At present, antibiotic therapy is one of the most effective strategies for C. perfringens. However, with the rise of antibacterial resistance, new agents with novel mechanisms of action are urgently needed. Bacteriocins are recognized as a viable alternative to antibiotics. In this study, the bacteriocin-like substance ParalichenysinDY4, derived from the Bacillus paralicheniformis (B. paralicheniformis) DY4 strain, is investigated as a potential alternative for combating Clostridium perfringens. The substance was isolated from B. paralicheniformis DY4 fermentation broth through a series of purification steps including methanol extraction, gel filtration, and high-performance liquid chromatography. Mass spectrometry analysis of ParalichenysinDY4 revealed that the detected peptide sequences did not match any previously known bacteriocins, indicating it is a novel bacteriocin-like substance. The novel bacteriocin-like substance exhibits effective antibacterial activity and broad antimicrobial spectrum against C. perfringens. Subsequent analyses utilizing methodologies including flow cytometry and scanning electron microscopy suggest that its mechanism of action is linked to its effects on the cell membrane. At the same time, due to its exceptional stability, safety, and efficient ability to remove pathogens both in vitro and in vivo, ParalichenysinDY4 holds promise as a valuable natural antimicrobial agent.

Fermentation starters and bacteriocins as biocontrol strategies for table olives preservation: a mini-review.

Biopreservation is a powerful strategy to prolong the shelf life of food products by applying naturally occurring microorganisms and/or their metabolites. Current food trends emphasise the need to develop alternatives for chemical or thermal preservation methods. In this line, different fermentation starters from table olives present the potential to control spoilage or pathogen-occurring microorganism in table olives storage. One of the most interesting family used as biopreservative culture is Lactobacillaceae and it has also been used in combination with yeasts as olive fermentation starter. Lactic acid bacteria, from Lactobacillaceae family, are characterised by the production of bacteriocins, proteins with the potential for preserving food by changing the organisation of the membrane of spoilage microorganisms. These bacteriocins-producing bacteria can be directly inoculated, although nanosystem technology is the most promising incorporation strategy. In table olives, the most commonly used starters are Lactiplantibacillus plantarum, Lactiplantibacillus pentosus, Saccharomyces cerevisiae, Wickerhamomyces anomalus, among others. These strains with biopreservation characteristics, inoculated alone or in mixed cultures, ensure food safety by conferring the product added value and prolonging product shelf life. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Bacteriocin distribution patterns in Enterococcus faecium and Enterococcus lactis: bioinformatic analysis using a tailored genomics framework.

Multidrug-resistant Enterococcus faecium strains represent a major concern due to their ability to thrive in diverse environments and cause life-threatening infections. While antimicrobial resistance and virulence mechanisms have been extensively studied, the contribution of bacteriocins to E. faecium's adaptability remains poorly explored. E. faecium, within the Bacillota phylum, is a prominent bacteriocin producer. Here, we developed a tailored database of 76 Bacillota bacteriocins (217 sequences, including 40 novel bacteriocins) and applied it to uncover bacteriocin distribution patterns in 997 quality-filtered E. faecium and Enterococcus lactis (former E. faecium clade B) genomes. Curated using computational pipelines and literature mining, our database demonstrates superior precision versus leading public tools in identifying diverse bacteriocins. Distinct bacteriocin profiles emerged between E. faecium and E. lactis, highlighting species-specific adaptations. E. faecium strains from hospitalized patients were significantly enriched in bacteriocins as enterocin A and bacteriocins 43 (or T8), AS5, and AS11. These bacteriocin genes were strongly associated with antibiotic resistance, particularly vancomycin and ampicillin, and Inc18 rep2_pRE25-derivative plasmids, classically associated with vancomycin resistance transposons. Such bacteriocin arsenal likely enhances the adaptability and competitive fitness of E. faecium in the nosocomial environment. By combining a novel tailored database, whole-genome sequencing, and epidemiological data, our work elucidates meaningful connections between bacteriocin determinants, antimicrobial resistance, mobile genetic elements, and ecological origins in E. faecium and provides a framework for elucidating bacteriocin landscapes in other organisms. Characterizing species- and strain-level differences in bacteriocin profiles may reveal determinants of ecological adaptation, and translating these discoveries could further inform strategies to exploit bacteriocins against high-risk clones. IMPORTANCE: This work significantly expands the knowledge on the understudied bacteriocin diversity in opportunistic enterococci, revealing their contribution in the adaptation to different environments. It underscores the importance of placing increased emphasis on genetic platforms carrying bacteriocins as well as on cryptic plasmids that often exclusively harbor bacteriocins since bacteriocin production can significantly contribute to plasmid maintenance, potentially facilitating their stable transmission across generations. Further characterization of strain-level bacteriocin landscapes could inform strategies to combat high-risk clones. Overall, these insights provide a framework for unraveling the therapeutic and biotechnological potential of bacteriocins.

Raffinocyclicin is a novel plasmid-encoded circular bacteriocin produced by Lactococcus raffinolactis with broad-spectrum activity against many gram-positive food pathogens.

This study describes the discovery and characterization of raffinocyclicin, a novel plasmid-encoded circular bacteriocin, produced by the raw milk isolate Lactococcus raffinolactis APC 3967. This bacteriocin has a molecular mass of 6,092 Da and contains 61 amino acids with a three-amino acid leader peptide. It shows the highest identity to the circular bacteriocins bacicyclicin XIN-1 (42.62%), aureocyclicin 4185 (42.62%), and garvicin ML (41.53%). A broad inhibitory spectrum includes strains from Staphylococcus, Enterococcus, Streptococcus, Micrococcus, Lactobacillus, Leuconostoc, and Listeria, in addition to a pronounced inhibitory effect against Lactococcus and Clostridium. It displays low sensitivity to trypsin, most likely as a result of its circular nature. The raffinocyclicin gene cluster is composed of 10 genes: 6 core genes, genes encoding an accessory three-component ABC transporter (rafCDE), and a putative transcriptional regulator related to the MutR family. A lack of inhibitory activity in the cell-free supernatant combined with the pronounced activity of cell extracts suggests that the majority of raffinocyclicin is associated with the cell rather than being released to the extracellular environment. This is the first report of a bacteriocin produced by the L. raffinolactis species.IMPORTANCEThe present study aimed to characterize raffinocyclicin, a novel circular bacteriocin produced by the lactic acid bacteria Lactococcus raffinolactis APC 3967. Bacteriocins are generally cationic and hydrophobic peptides with antimicrobial activity, which present diverse biotechnological properties of interest for the food industry. Raffinocyclicin inhibits a wide range of bacteria, including foodborne pathogens, and is stable against different treatments which suggest its potential as a natural biopreservative. Whole-genome sequencing and the genetic analysis of the raffinocyclicin gene cluster showed that it is encoded by plasmid that could be used in the future to transfer the ability to produce the bacteriocin to other lactic acid bacteria for industrial applications. These results together highlight the potential of this novel antimicrobial as a biopreservative to be used by the food industry.

Hyaluronic Acid/Gelatin-Based Multifunctional Bioadhesive Hydrogel Loaded with a Broad-Spectrum Bacteriocin for Enhancing Diabetic Wound Healing.

The development of multifunctional wound adhesives is critical in clinical settings due to the scarcity of dressings with effective adhesive properties while protecting against infection by drug-resistant bacteria. Polysaccharide and gelatin-based hydrogels, known for their biocompatibility and bioactivity, assist in wound healing. This study introduces a multifunctional bioadhesive hydrogel developed through dynamic covalent bonding and light-triggered covalent bonding, comprising oxidized hyaluronic acid, methacrylated gelatin, and the bacteriocin recently discovered by our lab, named jileicin (JC). The adhesion strength of the hydrogel, measured at 180 kPa, was 4.35 times higher than that of the fibrin glue. Furthermore, the hydrogel demonstrated robust platelet adhesion, procoagulant activity, and outstanding hemostatic properties in a mouse liver injury model. Incorporating JC significantly enhanced the phagocytosis and bactericidal capabilities of the macrophages. This immunomodulatory function on host cells, coupled with its potent bacterial membrane-disrupting ability, makes JC an effective killer against methicillin-resistant Staphylococcus aureus. In wound repair experiments on diabetic mice with infected full-thickness skin defects, the hydrogel treatment group showed a notable reduction in bacterial load, accelerated M2-type macrophage polarization, diminished inflammation, and hastened wound healing. Owing to its outstanding biocompatibility, antibacterial activity, and controlled adhesion, this hydrogel presents a promising therapeutic option for treating infected skin wounds.

Pseudocin 196, a novel lantibiotic produced by Bifidobacterium pseudocatenulatum elicits antimicrobial activity against clinically relevant pathogens.

Bacteriocins are broad or narrow-spectrum antimicrobial compounds that have received significant scientific attention due to their potential to treat infections caused by antibiotic-resistant pathogenic bacteria. The genome of Bifidobacterium pseudocatenulatum MM0196, an antimicrobial-producing, fecal isolate from a healthy pregnant woman, was shown to contain a gene cluster predicted to encode Pseudocin 196, a novel lantibiotic, in addition to proteins involved in its processing, transport and immunity. Following antimicrobial assessment against various indicator strains, protease-sensitive Pseudocin 196 was purified to homogeneity from cell-free supernatant. MALDI TOF mass spectrometry confirmed that the purified antimicrobial compound corresponds to a molecular mass of 2679 Da, which is consistent with that deduced from its genetic origin. Pseudocin 196 is classified as a lantibiotic based on its similarity to lacticin 481, a lanthionine ring-containing lantibiotic produced by Lactococcus lactis. Pseudocin 196, the first reported bacteriocin produced by a B. pseudocatenulatum species of human origin, was shown to inhibit clinically relevant pathogens, such as Clostridium spp. and Streptococcus spp. thereby highlighting the potential application of this strain as a probiotic to treat and prevent bacterial infections.

The microbiome-derived antibacterial lugdunin acts as a cation ionophore in synergy with host peptides.

Lugdunin is a microbiome-derived antibacterial agent with good activity against Gram-positive pathogens in vitro and in animal models of nose colonization and skin infection. We have previously shown that lugdunin depletes bacterial energy resources by dissipating the membrane potential of Staphylococcus aureus. Here, we explored the mechanism of action of lugdunin in more detail and show that lugdunin quickly depolarizes cytoplasmic membranes of different bacterial species and acidifies the cytoplasm of S. aureus within minutes due to protonophore activity. Varying the salt species and concentrations in buffers revealed that not only protons are transported, and we demonstrate the binding of the monovalent cations K+, Na+, and Li+ to lugdunin. By comparing known ionophores with various ion transport mechanisms, we conclude that the ion selectivity of lugdunin largely resembles that of 15-mer linear peptide gramicidin A. Direct interference with the main bacterial metabolic pathways including DNA, RNA, protein, and cell wall biosyntheses can be excluded. The previously observed synergism of lugdunin with dermcidin-derived peptides such as DCD-1 in killing S. aureus is mechanistically based on potentiated membrane depolarization. We also found that lugdunin was active against certain eukaryotic cells, however strongly depending on the cell line and growth conditions. While adherent lung epithelial cell lines were almost unaffected, more sensitive cells showed dissipation of the mitochondrial membrane potential. Lugdunin seems specifically adapted to its natural environment in the respiratory tract. The ionophore mechanism is refractory to resistance development and benefits from synergy with host-derived antimicrobial peptides. IMPORTANCE: The vast majority of antimicrobial peptides produced by members of the microbiome target the bacterial cell envelope by many different mechanisms. These compounds and their producers have evolved side-by-side with their host and were constantly challenged by the host's immune system. These molecules are optimized to be well tolerated at their physiological site of production, and their modes of action have proven efficient in vivo. Imbalancing the cellular ion homeostasis is a prominent mechanism among antibacterial natural products. For instance, over 120 naturally occurring polyether ionophores are known to date, and antimicrobial peptides with ionophore activity have also been detected in microbiomes. In this study, we elucidated the mechanism underlying the membrane potential-dissipating activity of the thiazolidine-containing cycloheptapeptide lugdunin, the first member of the fibupeptides discovered in a commensal bacterium from the human nose, which is a promising future probiotic candidate that is not prone to resistance development.

An insight into structure-activity relationships in subclass IIb bacteriocins: Plantaricin EvF.

This study reports the structure-activity relationships of a unique subclass IIb bacteriocin, plantaricin EvF, which consists of two peptide chains and possesses potent antimicrobial activity. Because the plantaricin Ev peptide chain lacks an α-helix structure, plantaricin EvF is unable to exert its antimicrobial activity through helix-helix interactions like typical subclass IIb bacteriocins. We have shown by various structural evaluation methods that plantaricin Ev can be stabilized by hydrogen bonding at amino acid residues R3, V12, and R13 to the N-terminal region of plantaricin F. This binding gives plantaricin EvF a special spade-shaped structure that exerts antimicrobial activity. In addition, the root-mean-square deviations (RMSDs) of the amino acid residues Y6, F8, and R13 of plantaricin Ev pre- and post-binding were 1.512, 1.723, and 1.369, respectively, indicating that they underwent large structural changes. The alanine scanning experiments demonstrated the important role of the above key amino acids in maintaining the structural integrity of plantaricin EvF. This study not only reveals the unique structural features of plantaricin EvF, but also provides an insight into the structure-activity relationships of subclass IIb bacteriocins.

Crystal structure of pectocin M1 reveals diverse conformations and interactions during its initial step via the ferredoxin uptake system.

Pectocin M1 (PM1), the bacteriocin from phytopathogenic Pectobacterium carotovorum which causes soft rot disease, has a unique ferredoxin domain that allows it to use FusA of the plant ferredoxin uptake system. To probe the structure-based mechanism of PM1 uptake, we determined the X-ray structure of full-length PM1, containing an N-terminal ferredoxin and C-terminal catalytic domain connected by helical linker, at 2.04 Å resolution. Based on published FusA structure and NMR data for PM1 ferredoxin domain titrated with FusA, we modeled docking of the ferredoxin domain with FusA. Combining the docking models with the X-ray structures of PM1 and FusA enables us to propose the mechanism by which PM1 undergoes dynamic domain rearrangement to translocate across the target cell outer membrane.

Effects of bacteriocin-producing Lactiplantibacillus plantarum on bacterial community and fermentation profile of whole-plant corn silage and its in vitro ruminal fermentation, microbiota, and CH4 emissions.

BACKGROUND: Silage is widely used to formulate dairy cattle rations, and the utilization of antibiotics and methane emissions are 2 major problems for a sustainable and environmentally beneficial ruminant production systems. Bacteriocin has received considerable attention because of its potential as an alternative to antibiotics in animal husbandry. However, the impact of bacteriocin-producing lactic acid bacteria on the microbiological conversion process of whole-plant corn silage and rumen fermentation remains limited. The purpose of this study was to assess the effect of 2 class IIa bacteriocin-producing strains Lactiplantibacillus plantarum ATCC14917 and CICC24194 on bacterial community composition and ensiling profiles of whole-plant corn silage and its in vitro rumen fermentation, microbiota, and CH4 emissions. RESULTS: Both bacteriocin-producing strains increased the lactic acid concentration in silage fermented for 7 d, whereas the lowest lactic acid was observed in the ATCC14917 inoculated silage fermented for 90 d (P < 0.05). The highest DM content was observed in the CICC24194 treatment (P < 0.05), and the silages treated with both strains had the lowest DM loss (P < 0.05). Bacteriocin-producing strains promoted the growth of Levilactobacillus brevis on d 60 of ensiling. In addition, treatment with bacteriocin-producing strains increased the in vitro DM digestibility (P < 0.05) and decreased the CH4 production (P < 0.05). The results of random forest and clustering analyses at the genus level showed that ATCC14917 increased the relative abundance of the influential variable Bacillus compared to that in the control group, whereas CICC24194 decreased the relative abundance of the influential variable Ruminococcaceae UCG-005. The CICC24194 treatment had the lowest total bacterial, fungal, protozoan, and methanogen populations (P < 0.05). CONCLUSIONS: Both class IIa bacteriocin-producing L. plantarum strains improved the fermentation quality of whole-plant corn silage by regulating the bacterial community composition during ensiling, with CICC24194 being the most effective. Both bacteriocin-producing strains mitigated CH4 production and improved digestibility by modulating the interactions among rumen bacteria, protozoa, methanogens, and the composition of fibrolytic bacteria.