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Candida albicans is a common opportunistic fungus in humans, whose morphological switch between yeast and hyphae forms represents a key virulence trait. Developing strategies to inhibit C. albicans hyphal growth may provide insights into designs of novel antivirulent therapeutics. Importantly, the gut commensal bacterium, Enterococcus faecalis , secretes a bacteriocin EntV which has potent antivirulent and antifungal effects against C. albicans in infection models; however, hampered by the challenges to access large quantities of bioactive EntV, the detailed understanding of its mechanisms on C. albicans has remained elusive. In this work, we biochemically reconstituted the proteolytic cleavage reaction to obtain recombinant EntV 88 -His 6 on a large preparative scale, providing facile access to the C-terminal EntV construct. Under in vitro C. albicans hyphal assay with specific inducers, we demonstrated that EntV 88 -His 6 exhibits potent bioactivity against GlcNAc-triggered hyphal growth. Moreover, with fluorescent FITC-EntV 88 -His 6 , we revealed that EntV 88 -His 6 enters C. albicans via endocytosis and perturbs the proper localization of the polarisome scaffolding Spa2 protein. Our findings provide important clues on EntV's mechanism of action. Surprisingly, we showed that EntV 88 -His 6 does not affect C. albicans yeast cell growth but potently exerts cytotoxicity against C. albicans under hyphal-inducing conditions in vitro . The combination of EntV 88 -His 6 and GlcNAc displays rapid killing of C. albicans , rendering it a promising antivirulent and antifungal agent.

A novel bacteriocin, plantaricin ZFM9, was purified from Lactiplantibacillus plantarum ZFM9 using a combination of ammonium sulfate precipitation, XAD-2 macroporous resin, Sephadex G-50, Sephadex LH-20, and reversed-phase high performance liquid chromatography. The molecular mass of plantaricin ZFM9 was 1151.606 Da, and the purity was 98.3%. Plantaricin ZFM9 has thermal stability (95.6% retention at 120 °C for 30 min), pH stability (pH ≤ 5), and sensitivity to the pepsin, trypsin, papain, and proteinase K. Plantaricin ZFM9 exhibited broad-spectrum antimicrobial activity and notably inhibit methicillin-resistant Staphylococcus aureus D48 (MRSA). According to the results of electron microscopy and fluorescence leakage assay, it was found that plantaricin ZFM9 caused damage to the cells membrane and leakage of the contents of S. aureus D48. In addition, Lipid II was not the anti-MRSA target of plantaricin ZFM9. This study underscores the potential of plantaricin ZFM9 for applications in the food field and biopharmaceuticals against MRSA infection., Competing Interests: Declaration of competing interest All author declares that there are no potential commercial or financial conflicts of interest in this study., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bacteriocins are potent antimicrobial peptides that are produced by bacteria. Since their discovery almost a century ago, diverse peptides have been discovered and described, and some are currently used as commercial food preservatives. Many bacteriocins exhibit extensively post-translationally modified structures encoded on complex gene clusters, whereas others have simple linear structures. The molecular structures, mechanisms of action and resistance have been determined for a number of bacteriocins, but most remain incompletely characterized. These gene-encoded peptides are amenable to bioengineering strategies and heterologous expression, enabling metagenomic mining and modification of novel antimicrobials. The ongoing global antimicrobial resistance crisis demands that novel therapeutics be developed to combat infectious pathogens. New compounds that are target-specific and compatible with the resident microbiota would be valuable alternatives to current antimicrobials. As bacteriocins can be broad or narrow spectrum in nature, they are promising tools for this purpose. However, few bacteriocins have gone beyond preclinical trials and none is currently used therapeutically in humans. In this Review, we explore the broad diversity in bacteriocin structure and function, describe identification and optimization methods and discuss the reasons behind the lack of translation beyond the laboratory of these potentially valuable antimicrobials., (© 2024. Springer Nature Limited.)

The cell wall is an indispensable element of bacterial cells and a long-known target of many antibiotics. Penicillin, the first discovered beta-lactam antibiotic inhibiting the synthesis of cell walls, was successfully used to cure many bacterial infections. Unfortunately, pathogens eventually developed resistance to it. This started an arms race, and while novel beta-lactams, either natural or (semi)synthetic, were discovered, soon upon their application, bacteria were developing resistance. Currently, we are facing the threat of losing the race since more and more multidrug-resistant (MDR) pathogens are emerging. Therefore, there is an urgent need for developing novel approaches to combat MDR bacteria. The cell wall is a reasonable candidate for a target as it differentiates not only bacterial and human cells but also has a specific composition unique to various groups of bacteria. This ensures the safety and specificity of novel antibacterial agents that target this structure. Due to the shortage of low-molecular-weight candidates for novel antibiotics, attention was focused on peptides and proteins that possess antibacterial activity. Here, we describe proteinaceous agents of various origins that target bacterial cell wall, including bacteriocins and phage and bacterial lysins, as alternatives to classic antibiotic candidates for antimicrobial drugs. Moreover, advancements in protein chemistry and engineering currently allow for the production of stable, specific, and effective drugs. Finally, we introduce the concept of selective targeting of dangerous pathogens, exemplified by staphylococci, by agents specifically disrupting their cell walls.

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.

Antimicrobial resistance poses a significant threat to humanity, and the development of new antibiotics is urgently needed. Our research has focused on thiopeptide antibiotics such as micrococcin P2 (MP2) and derivatives thereof as new anti-infective agents. Thiopeptides are sulfur-rich, structurally complex substances that exhibit potent activity against Gram-positive pathogens and Mycobacteria species, including clinically resistant strains. The clinical development of thiopeptides has been hampered by the lack of efficient synthetic platforms to conduct detailed structure-activity relationship studies of these natural products. The present contribution touches upon efficient synthetic routes to MP2 that laid the groundwork for clinical translation. The medicinal chemistry campaign on MP2 has been guided by computational molecular dynamic simulations and parallel investigations to improve drug-like properties, such as enhancing the aqueous solubility and optimizing antibacterial activity. Such endeavors have enabled identification of promising lead compounds, AJ-037 and AJ-206, against Mycobacterium avium complex (MAC). Extensive in vitro studies revealed that these compounds exert potent activity against MAC species, a subspecies of non-tuberculous mycobacteria (NTM) that proliferate inside macrophages. Two additional pre-clinical candidates have been identified: AJ-024, for the treatment of Clostridioides difficile infections, and AJ-147, for methicillin-resistant Staphylococcus aureus impetigo. Both compounds compare quite favorably with current first-line treatments. In particular, the ability of AJ-147 to downregulate pro-inflammatory cytokines adds a valuable dimension to its clinical use. In light of above, these new thiopeptide derivatives are well-poised for further clinical development., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; however, only those killing Gram-negative bacteria are currently known. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium Clostridioides difficile. Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with target envelope differences. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics., (© 2024. The Author(s).)

Strain Lactiplantibacillus plantarum D1 with bacteriocin producing ability was found in the intestine of Gambusia affinis. The bacteriocin was found to have high inhibitory activity against multiple Streptococcus species and several other Gram-positive and Gram-negative bacteria. Bacteriocin was purified from culture supernatant by ion-exchange chromatography, Sep-Pak C 18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectral analysis determined that purified bacteriocin has a molecular mass of 2,731 Da. A partial N-terminal sequence KRKKHKXQIYNNGM was obtained from the Edman analysis. The N-terminal sequence was employed to search against a translation of the draft genome of strain D1. The translated full amino acid sequence of the mature peptide is as follows: NH 2 - KRKKHKCQIYNNGMPTGQYRWC, which has a molecular weight of 2738 Da. A BLAST search revealed that this bacteriocin was most similar to bactofencin A but differed from it with three amino acid residues. No identical peptide or protein has been previously reported, and this peptide, termed bactofencin YH, was therefore considered to be a new bacteriocin produced by Lactiplantibacillus plantarum D1., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Bacteriocins are antimicrobial peptides produced by bacteria to prevent the growth of pathogens. Combining bacteriocins with metal nanoparticles, like silver nanoparticles (AgNPs), has developed into a viable strategy to get over bacteriocin limitations. In this study, bacteriocin BacZY05 was extracted from Bacillus subtilis ZY05 and purified using various techniques. The resulting purified bacteriocin was then combined with silver nanoparticles to form bacteriocin silver nanoconjugates (BacZY05-AgNPs). The physicochemical properties of the BacZY05-AgNPs were characterized using various analytical techniques. The mean diameter of the synthesized AgNPs was approximately 20-60 nm with an oval or spherical shape. The antimicrobial activity of the BacZY05-AgNPs was evaluated against several indicator strains by their zone of inhibition (ZOI), using the agar well diffusion method. Compared to bacteriocin (ZOI- 13 to 20 mm) and AgNPs (ZOI- 10-22 mm) alone, the antibacterial activity data demonstrated a 1.3-1.5-fold increase in the activity of bacteriocin-nanoconjugates (ZOI- 22 to 26 mm). For Staphylococcus aureus MTCC3103 and Klebsiella pneumoniae MTCC109, BacZY05-capped AgNPs exhibited the lowest minimum inhibitory concentration (MIC), measuring 10.93 µg/mL. For Salmonella typhi NCIM2501, the MIC was 28.75 µg/mL. The highest MIC value was 57.5 µg/mL for Escherichia coli DH5α and Vibrio cholerae MTCC3909. With BacZY05-capped AgNPs, the lowest minimum bactericidal concentration (MBC) of 28.75 µg/mL was observed for Staphylococcus aureus MTCC31003. In the cases of Salmonella typhi NCIM2501 and Klebsiella pneumoniae MTCC109 concentration was 57.5 µg/mL. Vibrio cholerae MTCC3909 and Escherichia coli DH5α had the highest MBC values at 115 µg/mL., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Enterococcus faecalis CAUM157 (KACC 81148BP), a Gram-positive bacteria isolated from raw cow's milk, was studied for its bacteriocin production. The antimicrobial activity of CAUM157 was attributed to a two-peptide class IIb bacteriocin with potent activity against food-borne pathogen Listeria monocytogenes and periodontal disease-causing pathogens (Prevotella intermedia KCTC 15693  T and Fusobacterium nucleatum KCTC 2488  T ). M157 bacteriocins exhibit high temperature and pH stability and resist hydrolytic enzyme degradation and detergent denaturation, potentially due to their structural conformation. Based on amino acid sequence, M157A and M157B were predicted to be 5.176 kDa and 5.182 kDa in size, respectively. However, purified bacteriocins and chemically synthesized N-formylated M157 peptides both showed 5.204 kDa (M157A) and 5.209 kDa (M157B) molecular mass, confirming the formylation of the N-terminal methionine of both peptides produced by strain CAUM157. Furthermore, the strain demonstrated favorable growth and fermentation with minimal bacteriocin production when cultured in whey-based media, whereas a 1.0% tryptone or soytone supplementation resulted in higher bacteriocin production. Although Ent. faecalis CAUM157 innately harbors genes for virulence factors and antimicrobial resistance (e.g., tetracycline and erythromycin), its bacteriocin production is valuable in circumventing the need for live microorganisms, particularly in food applications for pathogen control., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacteriocin producing strain Brevibacillus laterosporus TK3, was isolated from ' Tatwakhar '- a flour prepared from seeds of Indian Horse Chestnut ( Aesculus indica ). Bacterial strain Brevibacillus laterosporus TK3 identified by morphological, biochemical techniques followed by 16S rRNA gene sequencing. The 16S rRNA sequence of bacteriocin producer was deposited in NCBI GenBank under accession no. KP861913.1. Bacteriocin of Brevibacillus laterosporus TK3 showed strong antagonistic activity against food spoiling/pathogenic bacteria viz. Listeria monocytogenes, Staphylococcus aureus and Clostridium perfringens. Bacteriocin production by Brevibacillus laterosporus TK3 was enhanced by optimizing production time, pH of the medium, inoculum size and incubation temperature. Maximum bacteriocin activity (6000 AU/ml) was recorded/obtained in basal salt medium of pH 5.5 with an inoculum size of 1.5 OD at 10% and incubation period of 24h at 35  ° C. The bacteriocin was purified by single step gel exclusion chromatography. Molecular weight of active bacteriocin from Brevibacillus laterosporous TK3 was found to be 6 kDa according to SDS PAGE. The molecular mass of purified bacteriocin was confirmed as 5953.89 Da by MALDI TOF analysis. The purified bacteriocin was found desirable/suitable for food preservation as it showed wide spectrum of antimicrobial activity, resistance to high temperature, wide pH range and sensitivity to proteolytic enzymes thus, making it safe for human consumption.

MccY is a novel, structurally stable microcin with antibacterial activity against Enterobacteriaceae. However, the bioavailability of orally administrated MccY is unknown. This study evaluated the effects of MccY as a antimicrobial on pre-digestion in vitro and its intake, digestion and gut metabolism in vivo. The result of pre-digestion results that MccY maintained its biological activity and was resistant to decomposition. The study established a safe threshold of 4.46-9.92 mg/kg for the MccY dosage-body weight relationship in BALB/c mice. Mice fed with MccY demonstrated improved body weight and intestinal barrier function, accompanied with increased IgM immunogenicity and decreased levels of TNF-α, IL-6, and IL-10 in the intestine. MccY significantly facilitates the growth and activity of probiotics including Lactobacillus, Prevotella, and Bacteroides, and leading to the production of SCFAs and MCFAs during bacterial interactions. Furthermore, MccY effectively protects against the inflammatory response caused by Salmonella Typhimurium infection and effectively clears the Salmonella bacteria from the gut. In conclusion, MccY is seen as a promising new therapeutic target drug for enhancing the intestinal microbe-barrier axis and preventing enteritis., Competing Interests: Declaration of competing interest This study has no interest conflicts. This study was approved by South China Agricultural University Laboratory Animal Ethics Committee (No. 2021C084) and written consent for participation was obtained from the respondents through an online. All methods were performed in accordance with the relevant guidelines and regulations. This study was support by Guangdong Poultry Industry Technical System Disease control post expert (2023KJ128); National Broiler Industry Technical System (CARS-41) and Rural Revitalization Strategy of Guangdong Province (5500-F23015)., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cancer poses a severe threat to human health. Although conventional chemotherapy remains a cornerstone of cancer treatment, its significant side effects and the growing issue of drug resistance necessitate the urgent search for more efficient and less toxic anticancer drugs. In recent years, bacteriocins, antimicrobial peptides of microbial origin, have garnered significant attention due to their targeted antitumor activity. This unique activity is mainly attributed to their cationic and amphiphilic nature, which enables bacteriocins to specifically kill tumor cells without harming normal cells. When involving non-membrane-disrupting mechanisms, such as apoptosis induction, cell cycle blockade, and metastasis inhibition, the core mechanism of action is achieved by disrupting cell membranes, which endows bacteriocins with low drug resistance and high selectivity. However, the susceptibility of bacteriocins to hydrolysis and hemolysis in vivo limits their clinical application. To overcome these challenges, structural optimization of bacteriocins or their combination with nanotechnology is proposed for future development. This review aims to study the mechanism of action and current research status of bacteriocins as anticancer treatments, thus providing new insights for their clinical development and application.

Wild-type Lactococcus lactis strain LAC460 secretes prophage-encoded bacteriocin-like lysin LysL, which kills some Lactococcus strains, but has no lytic effect on the producer. LysL carries two N-terminal enzymatic active domains (EAD), and an unknown C-terminus without homology to known domains. This study aimed to determine whether the C-terminus of LysL carries a cell wall binding domain (CBD) for target specificity of LysL. The C-terminal putative CBD region of LysL was fused with His-tagged green fluorescent protein (HGFPuv). The HGFPuv_CBDlysL gene fusion was ligated into the pASG-IBA4 vector, and introduced into Escherichia coli. The fusion protein was produced and purified with affinity chromatography. To analyse the binding of HGFPuv_CBDLysL to Lactococcus cells, the protein was mixed with LysL-sensitive and LysL-resistant strains, including the LysL-producer LAC460, and the fluorescence of the cells was analysed. As seen in fluorescence microscope, HGFPuv_CBDLysL decorated the cell surface of LysL-sensitive L. cremoris MG1614 with green fluorescence, whereas the resistant L. lactis strains LM0230 and LAC460 remained unfluorescent. The fluorescence plate reader confirmed the microscopy results detecting fluorescence only from four tested LysL-sensitive strains but not from 11 tested LysL-resistant strains. Specific binding of HGFPuv_CBDLysL onto the LysL-sensitive cells but not onto the LysL-resistant strains indicates that the C-terminus of LysL contains specific CBD. In conclusion, this report presents experimental evidence of the presence of a CBD in a lactococcal phage lysin. Moreover, the inability of HGFPuv_CBDLysL to bind to the LysL producer LAC460 may partly explain the host's resistance to its own prophage lysin., (© 2024. The Author(s).)

Glycosylated bacteriocins, known as glycocins, were first discovered in 2011. These bioactive peptides are produced by bacteria to gain survival advantages. They exhibit diverse types of glycans and demonstrate varied antimicrobial activity. Currently, there are 13 experimentally known glycocins, with over 250 identified in silico across different bacterial phyla. Notably, glycocins are recognized for their glycan-mediated antimicrobial activity, proving effective against drug-resistant and foodborne pathogens. Many glycocins contain rare S-linked glycans. Glycosyltransferases (GTs), responsible for transferring sugar to glycocins and involved in glycocin biosynthesis, often cluster together in the producer's genome. This clustering makes them valuable for custom glycoengineering with diverse substrate specificities. Heterologous expression of glycocins has paved the way for the establishment of microbial factories for glycopeptide and glycoconjugate production across various industries. In this review, we emphasize the primary roles of fully and partially characterized glycocins and their glycosylating enzymes. Additionally, we explore how specific glycan structures facilitate these functions in antibacterial activities. Furthermore, we discuss newer approaches and increasing efforts aimed at exploiting bacterial glycobiology for the development of food preservatives and as replacements or complements to traditional antibiotics, particularly in the face of antibiotic-resistant pathogenic bacteria., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2024. Published by Elsevier Inc.)

In the postantibiotic era, the pathogenicity and resistance of pathogens have increased, leading to an increase in intestinal inflammatory disease. Bacterial infections remain the leading cause of animal mortality. With increasing resistance to antibiotics, there has been a significant decrease in resistance to both inflammation and disease in animals, thus decreasing production efficiency and increasing production costs. These side effects have serious consequences and have detracted from the development of China's pig industry. Microcin C7 (McC7) demonstrates potent antibacterial activity against a broad spectrum of pathogens, stable physicochemical properties, and low toxicity, reducing the likelihood of resistance development. Thus, McC7 has received increasing attention as a potential clinical antibacterial and immunomodulatory agent. McC7 has the potential to serve as a new generation of antibiotic substitutes; however, its commercial applications in the livestock and poultry industry have been limited. In this review, we summarize and discuss the biosynthesis, biochemical properties, structural characteristics, mechanism of action, and immune strategies of McC7. We also describe the ability of McC7 to improve intestinal health. Our aim in this study was to provide a theoretical basis for the application of McC7 as a new feed additive or new veterinary drug in the livestock and poultry breeding industry, thus providing a new strategy for alleviating resistance through feed and mitigating drug resistance. Furthermore, this review provides insight into the new functions and anti-infection mechanisms of bacteriocin peptides and proposes crucial ideas for the research, product development, and application of bacteriocin peptides in different fields, such as the food and medical industries.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase, and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, demonstrate complex formation between TclI, TclJ, and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core., Importance: Thiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin., Competing Interests: The authors declare no conflict of interest.

Shanxi aged vinegar microbiome encodes a wide variety of bacteriocins. The aim of this study was to mine, screen and characterize novel broad-spectrum bacteriocins from the large-scale microbiome data of Shanxi aged vinegar through machine learning, molecular simulation and activity validation. A total of 158 potential bacteriocins were innovatively mined from 117,552 representative genes based on metatranscriptomic information from the Shanxi aged vinegar microbiome using machine learning techniques and 12 microorganisms were identified to secrete bacteriocins at the genus level. Subsequently, employing AlphaFold2 structure prediction and molecular dynamics simulations, eight bacteriocins with high stability were further screened, and all of them were confirmed to have bacteriostatic activity by the Escherichia coli BL21 expression system. Then, gene_386319 (named LAB-3) and gene_403047 (named LAB-4) with the strongest antibacterial activities were purified by two-step methods and analyzed by mass spectrometry. The two bacteriocins have broad-spectrum antimicrobial activity with minimum inhibitory concentration values of 6.79 μg/mL-15.31 μg/mL against Staphylococcus aureus and Escherichia coli. Furthermore, molecular docking analysis indicated that LAB-3 and LAB-4 could interact with dihydrofolate reductase through hydrogen bonds, salt-bridge forces and hydrophobic forces. These findings suggested that the two bacteriocins could be considered as promising broad-spectrum antimicrobial agents., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Food preservation is a schematic and scientific procedure employed for the maintenance and improvement of food's quality, shelf life, and nutritional value. Although, on one hand, ancient conventional methods such as freezing, pasteurization, canning, and chemical methods have the potential to lengthen the shelf life of edible substances, but on the other hand, they can deteriorate its nutritional value as well. Present research focuses on the identification of promising bacteriocins against Pseudomonas fragi via subtractive proteomics pipeline as an alternative approach for food preservation. Bacteriocins are small peptides produced by certain microbes to naturally defend themselves by destroying other closely related bacteria residing in their neighborhood. P. fragi lies among the most notable microbes responsible for the elicitation of food spoilage. Due to increasing emergence and prevalence of multidrug resistance bacteria, there is a need to unravel novel drug targets, crucially involved in food decay process. Based on subtractive scrutinization, UDP-N-acetylglucosamine O-acyltransferase (LpxA) was chosen as promising therapeutic protein target that could play a significant role in progression of food spoilage. Subtilosin A, thuricin-CD, and mutacin B-NY266 were found as the most robust inhibitors of LpxA according to the molecular docking assay results. Molecular dynamic simulations and binding energy calculations via MM/PBSA method of LpxA and three top hit docked complexes, i.e., LpxA-subtilosin A, LpxA-thuricin-CD, and LpxA-mutacin B-NY266, revealed stability throughout simulations and ensured that shortlisted bacteriocins had strong affinity for LpxA., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nisin serves as the prototype within the lantibiotic group of antimicrobial peptides, exhibiting a broad-spectrum inhibition against Gram-positive bacteria, including important food-borne pathogens and clinically relevant antibiotic-resistant strains. The gene-encoded nature of nisin allows for gene-based bioengineering, enabling the generation of novel derivatives. It has been demonstrated that nisin mutants can be produced with improved functional properties. Here, we particularly focus on the uncommon amino acid residues dehydroalanine (Dha) and dehydrobutyrin (Dhb), whose functions are not yet fully elucidated. Prior to this study, we developed a new expression system that utilizes the nisin modification machinery NisBTC to advance expression, resulting in enhanced peptide dehydration efficiency. Through this approach, we discovered that the dehydrated amino acid Dhb at position 18 in the peptide rombocin, a short variant of nisin, displayed four times higher activity compared to the non-dehydrated peptide against the strain Lactococcus lactis. Furthermore, we observed that in the peptides nisin and rombocin, the dehydrated amino acid Dha at residue positon 18 exhibited superior activity compared to the dehydrated amino acid Dhb. Upon purifying the wild-type nisin and its variant nisinG18/Dha to homogeneity, the minimum inhibitory concentration (MIC) indicated that the variant exhibited activity similar to that of wild-type nisin in inhibiting the growth of Bacillus cereus but showed twice the MIC values against the other four tested Gram-positive strains. Further stability tests demonstrated that the dehydrated peptide exhibited properties similar to wild-type nisin under different temperatures but displayed higher resistance to proteolytic enzymes compared to wild-type nisin., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Strain ST3Ha, isolated from commercially available smoked salmon, was identified as Pediococcus pentosaceus based on biochemical and physiological tests and 16S rRNA sequencing. Strain ST3Ha produces a class IIa bacteriocin active against lactic acid bacteria, Listeria monocytogenes and Enterococcus faecalis. The antimicrobial peptide was inactivated by proteolytic enzymes, confirming his proteinaceous nature, but was not affected when treated with α-amylase, SDS, Tween 20, Tween 80, urea, and EDTA. No change in activity was recorded after 2 h at pH values between 2.0 and 9.0 and after treatment at 100 °C for 120 min or 121 °C for 15 min. The mode of action against Listeria ivanovii subsp. ivanovii ATCC 19119 and E. faecalis ATCC 19443 was bactericidal, resulting in cell lyses and enzyme leakage. The highest level of activity (1.6 × 10 6 AU/mL) was recorded when cells were grown at 37 °C or 30 °C in MRS broth (pH 6.5). Antimicrobial peptide ST3Ha adsorbs at high levels to the sensitive test organisms on strain-specific manner and depending on incubation temperature, environmental pH, and presence of supplemented chemicals. Based on PCR analysis, P. pentosaceus ST3Ha harbor a 1044-bp plasmid-associated fragment corresponding in size to that recorded for pediocin PA-1. Sequencing of the fragment revealed a gene identical to pedB, reported for pediocin PA-1. The combined application of the low levels (below MIC) of ciprofloxacin and bacteriocin ST3Ha results in the synergetic effect in the inhibition of L. ivanovii subsp. ivanovii ATCC 19119. Expressed by P. pentosaceus ST3Ha, bacteriocin was characterized as low cytotoxic, a characteristic relevant for its application in food industry and/or in human and veterinary medical practices., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Covering: 1992 up to 2023Since their discovery, lasso peptides went from peculiarities to be recognized as a major family of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that were shown to be spread throughout the bacterial kingdom. Microcin J25 was first described in 1992, making it one of the earliest known lasso peptides. No other lasso peptide has since then been studied to such an extent as microcin J25, yet, previous review articles merely skimmed over all the research done on this exceptional lasso peptide. Therefore, to commemorate the 30th anniversary of its first report, we give a comprehensive overview of all literature related to microcin J25. This review article spans the early work towards the discovery of microcin J25, its biosynthetic gene cluster, and the elucidation of its three-dimensional, threaded lasso structure. Furthermore, the current knowledge about the biosynthesis of microcin J25 and lasso peptides in general is summarized and a detailed overview is given on the biological activities associated with microcin J25, including means of self-immunity, uptake into target bacteria, inhibition of the Gram-negative RNA polymerase, and the effects of microcin J25 on mitochondria. The in vitro and in vivo models used to study the potential utility of microcin J25 in a (veterinary) medicine context are discussed and the efforts that went into employing the microcin J25 scaffold in bioengineering contexts are summed up.

The ability of antimicrobial peptides to efficiently kill their bacterial targets depends on the efficiency of their binding to the microbial membrane. In the case of enterocins, there is a three-part interaction: initial binding, unpacking of helices on the membrane surface, and permeation of the lipid bilayer. Helical unpacking is driven by disruption of the peptide hydrophobic core when in contact with membranes. Enterocin 7B is a leaderless enterocin antimicrobial peptide produced from Enterococcus faecalis that functions alone, or with its cognate partner enterocin 7A, to efficiently kill a wide variety of Gram-stain positive bacteria. To better characterize the role that tertiary structural plasticity plays in the ability of enterocin 7B to interact with the membranes, a series of arginine single-site mutants were constructed that destabilize the hydrophobic core to varying degrees. A series of experimental measures of structure, stability, and function, including CD spectra, far UV CD melting profiles, minimal inhibitory concentrations analysis, and release kinetics of calcein, show that decreased stabilization of the hydrophobic core is correlated with increased efficiency of a peptide to permeate membranes and in killing bacteria. Finally, using the computational technique of adaptive steered molecular dynamics, we found that the atomistic/energetic landscape of peptide mechanical unfolding leads to free energy differences between the wild type and its mutants, whose trends correlate well with our experiment.

Lasso peptides make up a class of natural products characterized by a threaded structure. Given their small size and stability, chemical synthesis would offer tremendous potential for the development of novel therapeutics. However, the accessibility of the pre-folded lasso architecture has limited this advance. To better understand the folding process de novo , simulations are used herein to characterize the folding propensity of microcin J25 (MccJ25), a lasso peptide known for its antimicrobial properties. New algorithms are developed to unambiguously distinguish threaded from nonthreaded precursors and determine handedness, a key feature in natural lasso peptides. We find that MccJ25 indeed forms right-handed pre-lassos, in contrast to past predictions but consistent with all natural lasso peptides. Additionally, the native pre-lasso structure is shown to be metastable prior to ring formation but to readily transition to entropically favored unfolded and nonthreaded structures, suggesting that de novo lasso folding is rare. However, by altering the ring forming residues and appending thiol and thioester functionalities, we are able to increase the stability of pre-lasso conformations. Furthermore, conditions leading to protonation of a histidine imidazole side chain further stabilize the modified pre-lasso ensemble. This work highlights the use of computational methods to characterize lasso folding and demonstrates that de novo access to lasso structures can be facilitated by optimizing sequence, unnatural modifications, and reaction conditions like pH.

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.

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.

Background: Colorectal cancer (CRC) treatment using time-saving and cost-effective targeted therapies with high selectivity and low toxicity drugs, is a great challenge. In primary investigations on Gallocin, as the most proposed factor in CRC pathogenesis caused by Streptococcus gallolyticus , it was surprisingly found that this bacteriocin has four α-helix structures and some anti-cancer sequences., Objective: The aim of this study was to determine the ability of Gallocin-based anticancer peptides (ACPs) against epidermal growth factor receptor (EGFR) and vascular epidermal growth factor receptor (VEGFR) and the evaluation of their pharmacokinetic properties using bioinformatic approaches., Methods: Support vector machine algorithm web-based tools were used for predicting ACPs. The physicochemical characteristics and the potential of anti-cancer activity of Gallocin-derived ACPs were determined by in silico tools. The 3D structure of predicted ACPs was modeled using modeling tools. The interactions between predicted ACPs and targets were investigated by molecular docking exercises. Then, the stability of ligand-receptor interactions was determined by molecular dynamic simulation. Finally, ADMET analysis was carried out to check the pharmacokinetic properties and toxicity of ACPs., Results: Four amino acid sequences with anti-cancer potential were selected. Through molecular docking, Pep2, and Pep3 gained the best scores, more binding affinity, and strong attachments by the formation of reasonable H-bonds with both EGFR and VEGFR. Molecular simulation confirmed the stability of Pep3- EGFR. According to pharmacokinetic analysis, the ACPs were safe and truthful., Conclusion: Designed peptides can be nominated as drugs for CRC treatment. However, different in-vitro and in-vivo assessments are required to approve this claim., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Bacteriocins are a kind of antimicrobial peptides that kill or inhibit the growth of bacterial strains. The purpose of this study was to investigate the antibacterial effect of Serratia marcescens on several pathogenic bacterial strains. Bacteriocin produced by S. marcescens was purified by chromatography with Sephadex G-75 column, and its antibacterial effect on gram-negative bacteria, including Escherichia coli ATCC 700928, Pseudomonas aeruginosa PTCC 1707, S. marcescens PTCC 1621, Vibrio fischeri PTCC 1693, and Vibrio harveyi PTCC 1755, were evaluated by the disk diffusion method. The structure of bacteriocin was determined by nuclear magnetic resonance spectroscopy. The interaction of bacteriocin with the antigen 43 (Ag43) of E. coli was evaluated by the molecular docking method. Bacteriocin extracted from bacterial isolates had antibacterial activity on E. coli strains but not on other studied strains. Bioinformatics analysis also showed bacteriocin docking with Ag43 with an energy of -159.968 kJ/mol. Natural compounds, such as bacteriocin, can be an alternative to common chemical compounds and antibiotics. To reach a definite conclusion in this regard, there is a need for further research and understanding of their mechanism of action., Competing Interests: The authors declare that they have no conflict of interest.

The rise of drug-resistant bacteria is a major threat to public health, highlighting the urgent need for new antimicrobial compounds and treatments. Bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by bacteria, hold promise as alternatives to conventional antibiotics. In this study, we identified and characterized a novel leaderless bacteriocin, bawcin, the first bacteriocin to be characterized from a Bacillus wiedmannii species. Chemically synthesized and purified bawcin was shown to be active against a broad range of Gram-positive bacteria, including foodborne pathogens Staphylococcus aureus , Bacillus cereus , and Listeria monocytogenes . Stability screening revealed that bawcin is stable over a wide range of pH (2.0-10.0), temperature conditions (25-100 °C), and against the proteases, papain and pepsin. Lastly, three-dimensional structure homology modeling suggests that bawcin contains a saposin-fold with amphipathic helices and a highly cationic surface that may be critical for membrane interaction and the subsequent cell death of its targets. This study provides the foundational understanding of the activity and properties of bawcin, offering valuable insights into its applications across different antimicrobial uses, including as a natural preservative in food and livestock industries.

Lactic acid bacteria (LAB) were screened from Lutjanus russellii (red sea bass), and their antimicrobial activities were evaluated against two Aeromonas species isolated from the Nile tilapia , namely, Aeromonas veronii (AV) and Aeromonas jandaei (AJ). Three LAB isolates, Enterococcus faecium MU8 (EF_8), Enterococcus faecalis MU2 (EFL_2), and E. faecalis MU9 (EFL_9), were found to inhibit both AV and AJ; however, their cell-free supernatant (CFS) did not do so. Interestingly, bacteriocin-like substances (BLS) induced by cocultures of EF_8 with AV exhibited the highest antimicrobial activity against both Aeromonas sp. The size of BLS was less than 1.0 kDa; the purified BLS were susceptible to proteinase K digestion, indicating that they are peptides. BLS contained 13 identified peptides derived from E. faecium, as determined by liquid chromatography-tandem mass spectrometry. Cocultures of Gram-positive-producing and -inducing LAB strains have been used to increase bacteriocin yields. To our knowledge, this is the first report describing inducible BLS produced by cocultures of Gram-positive-producing and Gram-negative-inducing strains.

Although multiple vaccines have been developed against infectious diseases, the rapid emergence of new pathogens develops an urgent need for novel strategies to combat infectious diseases. Antimicrobial peptides (AMPs) are excellent agents to fight against infectious diseases having unique multiple mechanisms of action against various pathogens. Apart from the direct applications, AMPs can also be developed as subunit vaccines or could be used as a highly immunogenic carrier protein with highly antigenic but non-immunogenic antigens. Here in the present study, we have identified a novel defensin-like bacteriocin, laterosporulin25 (LS25) upon genome mining of Brevibacillus laterosporus DSM25, a probiotic bacterial strain. By using immunoinformatic tools, we have studied the immunogenic and physiochemical properties of LS25. LS25 is characterized as defensin-like bacteriocin, having 51 amino acids and a molecular weight of 5862.7 Da. The modeled tertiary structure of LS25 is docked with TLR3 and TLR4-MD2 complex to confirm the facilitation of induced immune response that is further validated using molecular dynamics simulations and In-silico immune stimulations. Overall, detailed immunoinformatics analysis suggested LS25 as a potential candidate to be used as an adjuvant or carrier protein for subunit vaccine development, however, further in-vitro and in-vivo experiments are essential to validate its potential., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

To date, there have been no or just a few reports of successful cloning and expression to create biologically active ocins or bacteriocins. Cloning, expression, and production of class I ocins are problematic because of their structural arrangements, coordinated functions, size, and posttranslational modifications. Mass synthesis of these molecules is necessary for commercialization and to restrict the excessive use of conventional antibiotics, which encourages the development of antibiotic-resistant bacteria. In the case of class III ocins, there are no reports of obtaining biological active proteins to date. Being able to obtain biologically active proteins requires an understanding of mechanistic features due to their expanding importance and broad spectrum of activity. As a result, we intend to clone and express the class III type. The class I types that are devoid of posttranslational modifications were transformed into class III through fusion. Therefore, this construct resembles a class III type ocin. With the exception of Zoocin, expression of the proteins was found to be physiologically ineffective after cloning. But, few cell morphological changes such as elongation, aggregation, and the formation of terminal hyphae were observed. However, it was discovered that the target indicator had been altered to Vibrio spp. in a few. All the three ocins were subjected to in-silico structure prediction/analysis. Finally, we confirm the existence of unidentified additional intrinsic factors for successful expression to obtain biologically active protein., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Labyrinthopeptins constitute a class of ribosomal synthesized peptides belonging to the type III family of lantibiotics. They exist in different variants and display broad antiviral activities as well as show antiallodynic activity. Although their mechanism of action is not understood, it has been described that Labyrinthopeptins interact with membrane phospholipids modulating its biophysical properties and point out to membrane destabilization as its main point of action. We have used all-atom molecular dynamics to study the location of labyrinthopeptin A2 in a complex membrane as well as the existence of specific interactions with membrane lipids. Our results indicate that labyrinthopeptin A2, maintaining its globular structure, tends to be placed at the membrane interface, mainly between the phosphate atoms of the phospholipids and the oxygen atom of cholesterol modulating the biophysical properties of the membrane lipids. Outstandingly, we have found that labyrinthopeptin A2 tends to be preferentially surrounded by sphingomyelin while excluding cholesterol. The bioactive properties of labyrinthopeptin A2 could be attributed to the specific disorganization of raft domains in the membrane and the concomitant disruption of the overall membrane organization. These results support the improvement of Labyrinthopeptins as therapeutic molecules, opening up new opportunities for future medical advances., Competing Interests: Declaration of Competing Interest The author declares that there are no conflicts of interest., (Copyright © 2023 The Author. Published by Elsevier B.V. All rights reserved.)

Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.

Food safety is the primary concern of the food industry. The aim of the current research is to study the antimicrobial effects of cell-free supernatant of Lactobacillus pentosus against Bacillus cereus and Klebsiella pneumoniae. B. cereus and K. pneumoniae were isolated from infant formula milk product and meat sample, respectively. Their identification was performed through morphological characterization and biochemical testing. Molecular identification of K. pneumoniae was based on 16s ribotyping. A previously isolated and reported strain of L. pentosus was used for the isolation of CFS (Cellfree supernatants). Antimicrobial activity was studied through agar well diffusion assay. Inhibitory activity was recorded by measuring the zone of inhibition. CFS activity was evaluated for temperature and pH. The antimicrobial activity of CFS of L. pentosus produced at different temperatures and pH was investigated against B. cereus and K. pneumoniae. A clear zone of inhibition was observed against B. cereus while no ZOI was formed against K. pneumoniae. K. pneumoniae was found resistant to the CFS. Crude bacteriocin exhibited heat stability for a temperature of 121°C for 30 minutes and pH range of 3-7. The current study concluded that bacteriocin produced from L. pentosus can be used for the control of B. cereus. Its heat and pH stability allows its potential therapeutic use in the food industry as a food preservative and to control food poisoning cases due to B. cereus. K. pneumoniaeis was found resistant to the isolated bacteriocin, and therefore L. pentosus cannot be used for control against K. pneumoniae.

Cypemycin is a parent linaridin peptide known to contain nonproteinogenic dehydrobutyrine, N , N -dimethylalanine, and aminovinyl-cysteine residues. The enzymatic process by which this ribosomally synthesized peptide is formed remains elusive largely because of the deficiency of knowledge in post-translational modifications (PTMs) conducted by CypH and CypL, the two membrane-associated enzymes unique to linaridin biosynthesis. Based on heterologous reconstitution of the pathway in Streptomyces coelicolor , we report the detailed structural characterization of cypemycin as a previously unknown, d-amino acid-rich linaridin. In particular, the unprecedented family-determining activity of CypH and CypL was revealed, which, in addition to hydrolysis for removal of the N -terminal leader peptide, leads to transformation of the core peptide part of the precursor peptide through mechanistically related 16 reactions for residue epimerization (11 amino acids), dehydration (4 Thr), and dethiolation (Cys19). Subsequent functionalization for linaridin maturation includes CypD-involved aminovinyl-cysteine formation and N , N -dimethylation of the newly exposed N -terminal d-Ala residue that requires CypM activity. Genetic, chemical, biochemical, engineering, and modeling approaches were used to access the structure of cypemycin and the versatility of the CypH and CypL combination that is achieved in catalysis. This work furthers the appreciation of PTM chemistry and facilitates efforts for expanding linaridin structural diversity using synthetic biology methods.

Class III lanthipeptides are an emerging subclass of lanthipeptides, representing an underexplored trove of new natural products with potentially broad chemical diversity and important biological activity. Bioinformatic analysis of class III lanthipeptide biosynthetic gene cluster (BGC) distribution has revealed their high abundance in the phylum Firmicutes. Many of these clusters also feature methyltransferase (MT) genes, which likely encode uncommon class III lanthipeptides. However, two hurdles, silent BGCs and low-yielding pathways, have hindered the discovery of class III lanthipeptides from Firmicutes. Here, we report the design and construction of a biosynthetic pathway refactoring and heterologous overexpression strategy which seeks to overcome these hurdles, simultaneously activating and increasing the production of these Firmicutes class III lanthipeptides. Applying our strategy to MT-containing BGCs, we report the discovery of new class III lanthipeptides from Firmicutes bearing rare N , N -dimethylations. We reveal the importance of the first two amino acids in the N-terminus of the core peptide in controlling the MT dimethylation activity. Leveraging this feature, we engineer class III lanthipeptides to enable N , N -dimethylation, resulting in significantly increased antibacterial activity. Furthermore, the refactoring and heterologous overexpression strategy showcased in this study is potentially applicable to other ribosomally synthesized and post-translationally modified peptide BGCs from Firmicutes, unlocking the genetic potential of Firmicutes for producing peptide natural products.

Bacterial resistance towards antibiotics is a major global health issue. Very few novel antimicrobial agents and therapies have been made available for clinical use during the past decades, despite an increasing need. Antimicrobial peptides have been intensely studied, many of which have shown great promise in vitro. We have previously demonstrated that the bacteriocin Plantaricin NC8 αβ (PLNC8 αβ) from Lactobacillus plantarum effectively inhibits Staphylococcus spp., and shows little to no cytotoxicity towards human keratinocytes. However, due to its limitations in inhibiting gram-negative species, the aim of the present study was to identify novel antimicrobial peptidomimetic compounds with an enhanced spectrum of activity, derived from the β peptide of PLNC8 αβ. We have rationally designed and synthesized a small library of lipopeptides with significantly improved antimicrobial activity towards both gram-positive and gram-negative bacteria, including the ESKAPE pathogens. The lipopeptides consist of 16 amino acids with a terminal fatty acid chain and assemble into micelles that effectively inhibit and kill bacteria by permeabilizing their cell membranes. They demonstrate low hemolytic activity and liposome model systems further confirm selectivity for bacterial lipid membranes. The combination of lipopeptides with different antibiotics enhanced the effects in a synergistic or additive manner. Our data suggest that the novel lipopeptides are promising as future antimicrobial agents, however additional experiments using relevant animal models are necessary to further validate their in vivo efficacy., (© 2023. The Author(s).)

Lantibiotics are post-translationally modified antibiotic peptides with lanthionine thioether bridges that represent potential alternatives to conventional antibiotics. The lantibiotic pseudomycoicidin is produced by Bacillus pseudomycoides DSM 12442 and is effective against many Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. While prior work demonstrated that pseudomycoicidin possesses one disulfide bridge and four thioether bridges, the ring topology has so far remained unclear. Here, we analyzed several pseudomycoicidin analogues that are affected in ring formation via MALDI-TOF-MS and tandem mass spectrometry with regard to their dehydration and fragmentation patterns, respectively. As a result, we propose a bridging pattern involving Thr8 and Cys13, Thr10 and Cys16, Ser18 and Cys21, and Ser20 and Cys26, thus, forming two double ring systems. Additionally, we localized the disulfide bridge to connect Cys3 and Cys7 and, therefore, fully elucidated the bridging pattern of pseudomycoicidin., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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.

Microcins are a class of antimicrobial peptides produced by certain Gram-negative bacterial species to kill or inhibit the growth of competing bacteria. Only 10 unique, experimentally validated class II microcins have been identified, and the majority of these come from Escherichia coli. Although the current representation of microcins is sparse, they exhibit a diverse array of molecular functionalities, uptake mechanisms, and target specificities. This broad diversity from such a small representation suggests that microcins may have untapped potential for bioprospecting peptide antibiotics from genomic data sets. We used a systematic bioinformatics approach to search for verified and novel class II microcins in E. coli and other species within its family, Enterobacteriaceae . Nearly one-quarter of the E. coli genome assemblies contained one or more microcins, where the prevalence of hits to specific microcins varied by isolate phylogroup. E. coli isolates from human extraintestinal and poultry meat sources were enriched for microcins, while those from freshwater were depleted. Putative microcins were found in various abundances across all five distinct phylogenetic lineages of Enterobacteriaceae , with a particularly high prevalence in the "Klebsiella" clade. Representative genome assemblies from species across the Enterobacterales order, as well as a few outgroup species, also contained putative microcin sequences. This study suggests that microcins have a complicated evolutionary history, spanning far beyond our limited knowledge of the currently validated microcins. Efforts to functionally characterize these newly identified microcins have great potential to open a new field of peptide antibiotics and microbiome modulators and elucidate the ways in which bacteria compete with each other. IMPORTANCE Class II microcins are small bacteriocins produced by strains of Gram-negative bacteria in the Enterobacteriaceae . They are generally understood to play a role in interbacterial competition, although direct evidence of this is limited, and they could prove informative in developing new peptide antibiotics. However, few examples of verified class II microcins exist, and novel microcins are difficult to identify due to their sequence diversity, making it complicated to study them as a group. Here, we overcome this limitation by developing a bioinformatics pipeline to detect microcins in silico . Using this pipeline, we demonstrate that both verified and novel class II microcins are widespread within and outside the Enterobacteriaceae , which has not been systematically shown previously. The observed prevalence of class II microcins suggests that they are ecologically important, and the elucidation of novel microcins provides a resource that can be used to expand our knowledge of the structure and function of microcins as antibacterials.

Bacteriocins exhibited a wide spectrum of antibacterial activity against different pathogens. The aim of current study was to characterize the bacteriocins produced by Bifidobacterium spp. isolated from ruminants. The Bifidobacterium isolates were identified as B. longum, B. pseudolongum, B. bifidum, B. thermophilum, B. boum, B. merycicum and B. ruminantium. Bacteriocins were found to be pH stable, heat resistant, highly diffusible, NaCl tolerant and resistant to UV radiations. SDS, EDTA and urea induced 14%, 21% and 24% bacteriocins activity loss. Modified MRS broth (1% tryptone, 1% yeast extract and 2% glucose) was found to be the best nutrient medium for optimal production of bacteriocins. Minimum inhibitory concentration (MIC) values varied from 300 μl/ml to 500 μl/ml and minimum bactericidal concentration (MBC) values ranged from 500 μl/ml to >500 μl/ml for E. coli and S. aureus respectively. The highest protein concentration (29.0248 mg/ml) was recorded for Bifidobacteria bacteriocin produced by B. longum. Tricine-Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) revealed that molecular weight of isolated bifidobacterial bacteriocins was in the range of 3.6 kDa-30 kDa. Current study indicated that bifidobacterial bacteriocins have considerable potential to be used as biopreservative., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The abuse of traditional antibiotics has led to an increase in the resistance of bacteria and viruses. Similar to the function of antibacterial peptides, bacteriocins are more common as a kind of peptides produced by bacteria that have bactericidal or bacterial effects. More importantly, the marine environment is one of the most abundant resources for extracting marine microbial bacteriocins (MMBs). Identifying bacteriocins from marine microorganisms is a common goal for the development of new drugs. Effective use of MMBs will greatly alleviate the current antibiotic abuse problem. In this work, deep learning is used to identify meaningful MMBs. We propose a random multi-scale convolutional neural network method. In the scale setting, we set a random model to update the scale value randomly. The scale selection method can reduce the contingency caused by artificial setting under certain conditions, thereby making the method more extensive. The results show that the classification performance of the proposed method is better than the state-of-the-art classification methods. In addition, some potential MMBs are predicted, and some different sequence analyses are performed on these candidates. It is worth mentioning that after sequence analysis, the HNH endonucleases of different marine bacteria are considered as potential bacteriocins.

The current study aimed to screen bacteriocin producing LAB from different dairy products and evaluation of their biological properties. Initially, 12 (4-chess, 4-curd, and 4-yohurt) LAB species were isolated and only 4 isolates alone were selected based on their clear yellow halo zone around the colonies in the selective medium. The selected 4 isolates were identified based on their morphological and biochemical characteristics. Among them, the strain CH3 have showed better antimicrobial effects on selected human pathogens. The isolated strain CH3 were further identified as Lactococcus lactis strain CH3 (MZ636710) by SEM imaging and 16 s rRNA molecular sequencing. Bacteriocin was extracted from L. lactis strain CH3 and partially purified using 60 % ammonium sulphate and then completely purified by G-50 column chromatography. The purified bacteriocin showed a specific activity of 5859.37 AU/mg in 24.7 % of recovery and 10.9-fold purification. The molecular weight of bacteriocin was 3.5 kDa as observed in SDS-PAGE. The bacteriocin showed sensitivity to proteolytic enzymes and resistance to high temperature, wide range of pH, organic solvents and detergents. FT-IR spectral studies of bacteriocin detected the existence of OH/NH-stretching, CH, and COC and CO bonds. NMR spectrum showed one doublet and 4 various singlet peaks at different ppm, indicating the occurrence of six amino acids in the structure of purified bacteriocin. The purified bacteriocin have shown stronger antimicrobial and anti-biofilm activity against selected human pathogens at 100 μg/mL. SEM showed the evidence of structural deformation and loss of membrane integrity of bacterial cells treated with bacteriocin. Bacteriocin exhibited greater DPPH radical scavenging potential with an EC 50 value of 12.5 μg/mL. Bacteriocin have not shown significant toxicity on normal human dermal fibroblast (NHDF) cells (83.2 % at 100 μg/ mL). Furthermore, in silico studies using molecular modeling and docking were performed to know the proteins involved in antimicrobial action. The results suggests that bacteriocin could be an alternative to combat AMR pathogens and more suitable for food and dairy industries to preserve food without contamination., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The current study was carried out to investigate metabolic activities and main probiotic characteristics of two Latilactobacillus sakei strains (8.P1 and 28.P2) isolated from pastırma, a highly seasoned, air-dried cured beef. Both strains showed antimicrobial activity against important foodborne pathogens like Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa and so forth. For the characterization of antimicrobial activity, the effect of various enzymes, temperature and pH were tested. The results of the tests demonstrated that the antimicrobial activity of strains was based on the production of protein-structured compounds such as bacteriocin or bacteriocin like peptides. In metabolic activity studies, amounts of the lactic acid, proteolytic activity and hydrogen peroxide produced by the 8.P1 and 28.P2 were found to range between 16.09 and 17.32 mg/mL, 0.24 and 0.04 mg/mL and 0.98 and 0.04 µg/mL, respectively. It was also observed that neither strain could produce exopolysaccharide. Both strains were found susceptible to vancomycin, chloramphenicol, gentamicin, tetracycline, and netilmicin sulfate. When the strains are evaluated with respect to their probiotic potential, 28.P2 could tolerate acidic conditions, but 8.P1 showed sensitivity. The survival rate of the strains in the simulated gastric juice and their adhesion abilities were found suitable to stay alive in the gastrointestinal tract and to proliferate in the intestine. The evaluation of all the features of both strains demonstrated that both strains had the potential to be used as a protective culture. In addition, it was observed that 8.P1 and 28.P2 were more suitable as a starter culture and a probiotic candidate respectively., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Lanthipeptides are a class of cyclic peptides characterized by the presence of one or more lanthionine (Lan) or methyllanthionine (MeLan) thioether rings. These cross-links are produced by α,β-unsaturation of Ser or Thr residues in peptide substrates by dehydration, followed by a Michael-type conjugate addition of Cys residues onto the dehydroamino acids. Lanthipeptides may be broadly classified into at least five different classes, and the biosynthesis of classes I-IV lanthipeptides requires catalysis by LanC cyclases that control both the site-specificity and the stereochemistry of the conjugate addition. In contrast, there are no current examples of LanCs that occur in class V biosynthetic clusters, despite the presence of lanthionine rings in these compounds. In this work, bioinformatics-guided co-occurrence analysis identifies more than 240 putative class V lanthipeptide clusters that contain a LanC cyclase. Reconstitution studies demonstrate that the cyclase-catalyzed product is notably distinct from the product formed spontaneously. Stereochemical analysis shows that the cyclase diverts the final product to a configuration that is distinct from one that is energetically favored. Structural characterization of the final product by multi-dimensional NMR spectroscopy reveals that it forms a helical stapled peptide. Mutational analysis identified a plausible order for cyclization and suggests that enzymatic rerouting to the final structure is largely directed by the construction of the first lanthionine ring. These studies show that lanthipeptide cyclases are needed for the biosynthesis of some constrained peptides, the formations of which would otherwise be energetically unfavored.

Background: Due to the detrimental effects of chemical preservatives, there has been an increasing demand for safer, healthier and natural bio-preservatives. Bacteriocins have attracted increasing interest because of their potential as natural bio-preservatives., Results: We screened a large number of Bacillus thuringiensis strains and isolated one strain (B. thuringiensis P86) with antimicrobial activity against several foodborne pathogens. Three novel leaderless bacteriocins, including thucin A1, thucin A2 and thucin A3, were purified and identified from the culture supernatant of B. thuringiensis P86, whose molecular masses were 5552.02, 5578.07 and 5609.06 Da, respectively. Thucin A1 was then selected as a representative to be tested, and it exhibited potent inhibitory activity against all tested gram-positive bacteria. More importantly, thucin A1 showed stronger antimicrobial activity than nisin A against two important foodborne pathogens Bacillus cereus and Listeria monocytogenes. In addition, thucin A1 exhibited strong acid-base adaptability (pH 2-11), high endurance to heat, good stability to trypsin and pepsin, no hemolysis activity and cytotoxicity, and could effectively inhibit or eliminate Bacillus cereus and Listeria monocytogenes in skim milk., Conclusions: Our findings indicate that these novel leaderless bacteriocins are potentially promising food biopreservatives., (© 2022. The Author(s).)

Bacteriocins are a large family of peptides synthesized ribosomally by a variety of bacterial species. The genome of one of the thermophilic Gram-positive bacteria, Aeribacillus pallidus PI8, was found to possess an operon comprising five genes possibly involved in the production of a putative bacteriocin that was named pcnABCDE for the production of "pallidocyclicin." This study investigated the function of the pcn operon experimentally. The heterologous expression of the entire pcn operon from the plasmid was toxic to Escherichia coli but not to Bacillus subtilis. However, when the entire pcn operon was expressed constitutively, even the growth of B. subtilis was impaired, and at least pcnA was implied to serve as the precursor of pallidocyclicin. In addition, a strain of B. subtilis expressing the entire pcn operon from the plasmid showed toxicity to another thermophilic species, Geobacillus kaustophilus, at elevated temperatures, whereas another strain lacking pcnE alone from the pcn operon lost the toxicity, suggesting that pcnE might be involved in the biosynthesis of pallidocyclicin when it is produced in B. subtilis.

Traditionally fermented foods and beverages are still produced and consumed at a large scale in Romania. They are rich sources for novel lactic acid bacteria with functional properties and with potential application in food industry or health. Lactobacillus helveticus 34.9, isolated from a home-made fermented milk is able to inhibit the growth of other bacteria, such as other lactic acid bacteria, but also strains of Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Halobacillus hunanensis, a halobacterium isolated from the degraded wall of a Romanian monastery. L. helveticus 34.9 produces a large bacteriocin (35 KDa), active in a wide pH range, but inactivated by heat and proteinase K treatment. It shares about 20% sequence coverage with helveticin J, as determined by LC-MS analysis. Bacteriocin production was enhanced under stress conditions, especially when combined stresses were applied. Its mode of action and degree of inhibition depended on the concentration and on the indicator strain that was used; L. delbrueckii subsp. bulgaricus LMG 6901 T cells from a suspension were killed, but the viability of H. hunanensis 5Hum cells was only reduced to 60%, within 8 h. However, the bacteriocin was able to prevent the bacterial growth of both indicator strains when added to the cultivation medium prior inoculation. Scanning electron microscopy images revealed morphological changes induced by the bacteriocin treatment in both sensitive strains, but more severe in the case of L. delbrueckii subsp. bulgaricus. Due to the broad antibacterial spectrum and its production under various stress conditions, the bacteriocin or the producing strain may find application in health, food and non-food related fields, including in the restoration of historical buildings., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Plantaricin LD1 was purified to homogeneity using activity-guided chromatography. Enterococcus faecalis ATCC 29212 was found to be sensitive to plantaricin LD1 showing 13 ± 0·21 mm zone of growth inhibition. The minimum inhibitory concentration (MIC) was found to be 50 µg ml -1 against Ent. faecalis ATCC 29212. The in vitro biofilm formation by Ent. faecalis ATCC 29212 was observed, which was completely inhibited in the presence of bacteriocin. Similarly, biofilm formation was also observed on the teeth surface showing purple colour, whereas treated-teeth were clean and indicated no biofilm formation. Further, untreated cells of Ent. faecalis ATCC 29212 were found normal and plantaricin LD1-treated cells were ruptured when seen under light microscope, suggesting killing of target cells. These findings have proven the initial leads for antimicrobial and anti-biofilm activity of plantaricin LD1 against Ent. faecalis and its possible application for the treatment of endodontic diseases., (© 2022 The Society for Applied Microbiology.)