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The rapid emergence of antibiotic-resistant strains of Staphylococcus aureus presents a substantial challenge to global public health, underscoring the urgent need for novel antibiotics with diverse mechanisms of action. In this study, we conducted mutagenesis on the C -terminal region of the lantibiotic ripcin C to enhance its antimicrobial efficacy against S. aureus . The resulting optimized variant, ripcin C P23A , demonstrated potent and selective antimicrobial activity, with a minimal inhibitory concentration of 2-4 mg/L against S. aureus . Beyond its strong antimicrobial properties, ripcin C P23A exhibited significant antibiofilm activity against methicillin-resistant S. aureus (MRSA). Mechanistic studies revealed that, in addition to targeting lipid II, ripcin C P23A disrupts bacterial membranes, a capability absent in ripcin C, which may contribute to its superior antimicrobial and antibiofilm effects. Moreover, ripcin C P23A displayed favorable biosafety and plasma stability profiles. Notably, in a mouse model of MRSA-induced mastitis, ripcin C P23A effectively reduced bacterial load, alleviated inflammation, and preserved the normal histomorphology of mammary glands. This study introduces ripcin C P23A as a promising antibiotic candidate for the treatment of MRSA-related infections.

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., 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., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Class II microcins are antimicrobial peptides that have shown some potential as novel antibiotics. However, to date, only 10 class II microcins have been described, and the discovery of novel microcins has been hampered by their short length and high sequence divergence. Here, we ask if we can use numerical embeddings generated by protein large language models to detect microcins in bacterial genome assemblies and whether this method can outperform sequence-based methods such as BLAST. We find that embeddings detect known class II microcins much more reliably than does BLAST and that any two microcins tend to have a small distance in embedding space even though they typically are highly diverged at the sequence level. In data sets of Escherichia coli , Klebsiella spp., and Enterobacter spp. genomes, we further find novel putative microcins that were previously missed by sequence-based search methods., Importance: Antibiotic resistance is becoming an increasingly serious problem in modern medicine, but the development pipeline for conventional antibiotics is not promising. Therefore, alternative approaches to combat bacterial infections are urgently needed. One such approach may be to employ naturally occurring antibacterial peptides produced by bacteria to kill competing bacteria. A promising class of such peptides are class II microcins. However, only a small number of class II microcins have been discovered to date, and the discovery of further such microcins has been hampered by their high sequence divergence and short length, which can cause sequence-based search methods to fail. Here, we demonstrate that a more robust method for microcin discovery can be built on the basis of a protein large language model, and we use this method to identify several putative novel class II microcins., Competing Interests: The authors declare no conflict of interest.

Antimicrobial peptides (AMPs) are short, usually cationic peptides with an amphiphilic structure, which allows them to easily bind and interact with the cellular membranes of viruses, bacteria, fungi, and other pathogens. Bacterial AMPs, or bacteriocins, can be produced from Gram-negative and Gram-positive bacteria via ribosomal synthesis to eliminate competing organisms. Bacterial AMPs are vital in addressing the increasing antibiotic resistance of various pathogens, potentially serving as an alternative to ineffective antibiotics. Bacteriocins have a narrow spectrum of action, making them highly specific antibacterial compounds that target particular bacterial pathogens. This review covers the two main groups of bacteriocins produced by Gram-negative and Gram-positive bacteria, their modes of action, classification, sources of positive effects they can play on the human body, and their limitations and future perspectives as an alternative to antibiotics.

Drug-resistant bacteria such as Escherichia coli and Staphylococcus aureus represent a global health problem that requires priority attention. Due to the current situation, there is an urgent need to develop new, more effective and safe antimicrobial agents. Biotechnological approaches can provide a possible alternative control through the production of new generation antimicrobial agents, such as silver nanoparticles (AgNPs) and bacteriocins. AgNPs stand out for their antimicrobial potential by employing several mechanisms of action that can act simultaneously on the target cell such as the production of reactive oxygen species and cell wall rupture. On the other hand, bacteriocins are natural peptides synthesized ribosomally that have antimicrobial activity and are produced, among others, by lactic acid bacteria (LAB), whose main mechanism of action is to produce pores at the level of the cell membrane of bacterial cells. However, these agents have disadvantages. Nanoparticles also have limitations such as the tendency to form aggregates, which decreases their antibacterial activity and possible cytotoxic effects, and bacteriocins have a narrow spectrum of action, require high doses to be effective, and can be degraded by proteases. Given these limitations, nanoconjugates of these two agents have been developed that can act synergistically in the control of pathogenic bacteria resistant to antibiotics. This review focuses on knowing relevant aspects of the antibiotic resistance of E. coli and S. aureus, the characteristics of these new generation antibacterial agents, and their effect alone or forming nanoconjugates that are more effective against the multiresistant mentioned bacteria., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Modification of the N- and C-termini of peptides enhances their stability against degradation by exopeptidases. The biosynthetic pathways of many peptidic natural products feature enzymatic modification of their termini, and these enzymes may represent a valuable pool of biocatalysts. The lantibiotic cacaoidin carries an N , N -dimethylated N-terminal amine group. Its biosynthetic gene cluster encodes the putative methyltransferase Cao4. In this work, we present reconstitution of the activity of the enzyme, which we termed CaoS C following standardized lanthipeptide nomenclature, using a heterologously produced peptide as the model substrate. In vitro methylation of diverse lanthipeptides revealed the substrate requirements of CaoS C . The enzyme accepts peptides of varying lengths and C-terminal sequences but requires dehydroalanine or dehydrobutyrine at the second position. CaoS C -mediated dimethylation of natural lantibiotics resulted in modestly enhanced antimicrobial activity of the lantibiotic haloduracin compared to that of the native compound. Improved activity and/or metabolic stability as a result of methylation illustrates the potential future application of CaoS C in the bioengineering of therapeutic peptides.

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., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.)

Haloarchaea are a group of moderate and extreme halophilic microorganisms, belonging to the Archaea domain, that constitute relevant microbial communities in salty environments like coastal and inland salted ponds, marshes, salty lagoons, etc. They can survive in stress conditions such as high salinity and, therefore, high ionic strength, high doses of ultraviolet radiation (UV), high temperature, and extreme pH values. Consequently, most of the species can be considered polyextremophiles owing to their ability to respond to the multiple extreme conditions characterizing their natural habitats. They cope with those stresses thanks to several molecular and metabolic adaptations. Thus, some of the molecules produced by haloarchaea show significantly different biological activities and physicochemical properties compared to their bacterial counterparts. Recent studies have revealed promising applications in biotechnology and medicine for these biomolecules. Among haloarchaeal biomolecules, rare natural pigments (C 50 carotenoids) and small peptides called halocins and microhalocins have attracted attention worldwide due to their effects on animal and human commercial tumoral cells, apart from the role as antibiotics described for halocins or the immunomodulatory activity reported from C 50 carotenoids like bacterioruberin. This review summarizes recent knowledge on these two types of biomolecules in connection with cancer to shed new light on the design of drugs and new therapies based on natural compounds.

Dysbiosis of the human gut microbiota is linked to numerous diseases. Understanding the molecular mechanisms by which microbes interact and compete with one another is required for developing successful strategies to modulate the microbiome. The natural product Microcin M (MccM) consists of a 77-residue bioactive peptide conjugated to a siderophore and is a class II microcin involved in microbial competition with an enigmatic mode-of-action. In this work, we investigated the basis for MccM activity and leveraged bioinformatics to expand the known chemical diversity of class II microcins. We applied automated fast-flow solid phase peptide synthesis coupled with chemoenzymatic chemistry to acquire MccM and demonstrated that its activity was bacteriostatic. We then used our synthetic molecule to ascertain that catecholate siderophore transporters in Escherichia coli K-12 are necessary for MccM import. Once inside the cell, we found that MccM treatment decreased the levels of intracellular ATP and interfered with gene expression. These effects were ameliorated in genetic mutants lacking ATP synthase or in conditions that support substrate-level phosphorylation. Further, we showed that MccM elevated the levels of reactive oxygen species within the target cell. We propose that MccM effects its bacteriostatic activity by decreasing the total energy level of the cell through inhibition of oxidative phosphorylation. Lastly, using genome mining, we bioinformatically identified 171 novel putative class II microcins. Our investigation sheds light on the natural processes involved in microbial competition and provides inspiration, in the form of new molecules, for future therapeutic endeavors.

Microcin J25 (MccJ25), a lasso peptide antibiotic with a unique structure that resembles the lariat knot, has been a topic of intense interest since its discovery in 1992. The precursor (McjA) contains a leader and a core segment. McjB is a protease activated upon binding to the leader, and McjC converts the core segment into the mature MccJ25. Previous studies suggested that these biosynthetic steps likely proceed in a (nearly) concerted fashion; however, there is only limited information regarding the structural and molecular intricacies of MccJ25 biosynthesis. To close this knowledge gap, we used AlphaFold2 to predict the structure of the precursor (McjA) in complex with its biosynthetic enzymes (McjB and McjC) and queried the critical predicted features by protein engineering. Based on the predicted structure, we designed protein variants to show that McjB can still be functional and form a proficient biosynthetic complex with McjC when its recognition and protease domains were circularly permutated or split into separate proteins. Specific residues important for McjA recognition were also identified, which permitted us to pinpoint a compensatory mutation (McjB M108T ) to restore McjA/McjB interaction that rescued an otherwise nearly nonproductive precursor variant (McjA T-2M ). Studies of McjA, McjB, and McjC have long been mired by them being extremely difficult to handle experimentally, and our results suggest that the AF2 predicted ternary complex structure may serve as a reasonable starting point for understanding MccJ25 biosynthesis. The prediction-validation workflow presented herein combined artificial intelligence and laboratory experiments constructively to gain new insights.

Within class II bacteriocins, we assume the presence of a separate subfamily of antimicrobial peptides possessing a broad spectrum of antimicrobial activity. Although these peptides are structurally related to the subclass IIa (pediocin-like) bacteriocins, they have significant differences in biological activities and, probably, a mechanism of their antimicrobial action. A representative of this subfamily is acidocin A from Lactobacillus acidophilus TK9201. We discovered the similarity between acidocin A and acidocin 8912 from Lactobacillus acidophilus TK8912 when analyzing plasmids from lactic acid bacteria and suggested the presence of a single evolutionary predecessor of these peptides. We obtained the C -terminally extended homolog of acidocin 8912, named acidocin 8912A, a possible intermediate form in the evolution of the former. The study of secondary structures and biological activities of these peptides showed their structural similarity to acidocin A; however, the antimicrobial activities of acidocin 8912 and acidocin 8912A were lower than that of acidocin A. In addition, these peptides demonstrated stronger cytotoxic and membranotropic effects. Building upon what we previously discovered about the immunomodulatory properties of acidocin A, we studied its proteolytic stability under conditions simulating those in the digestive tract and also assessed its ability to permeate intestinal epithelium using the Caco-2 cells monolayer model. In addition, we found a pronounced effect of acidocin A against fungi of the genus Candida, which might also expand the therapeutic potential of this bacterial antimicrobial peptide.

Circular bacteriocins are known for their structural stability and effective antimicrobial properties, positioning them as potential natural food preservatives. However, their widespread application is impeded by restricted availability. This research developed a total biosynthesis platform for circular bacteriocins, with a focus on AS-48 by involving recombinant production of the linear precursor in Escherichia coli , followed by enzymatic cyclization of the precursor into cyclic AS-48 using the ligase butelase-1 in vitro . An important discovery is that, aside from fusion tags, the C-terminal motif LE and LEKKK also could affect the expression yield of the precursor. This biosynthesis platform is both versatile and high-yielding, achieving yields of 10-20 mg/L of AS-48. Importantly, the biosynthetic AS-48 exhibited a secondary structure and antimicrobial activities comparable to those of the native molecules. As such, this work proposes an effective synthetic approach for circular bacteriocins, facilitating their advancement and application in the food industry.

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.

E50-52, a class IIa-peptidic bacteriocin produced by a strain of Enterococcus faecium , has broad-spectrum antimicrobial activity against various foodborne pathogens. However, effective utilization of the E50-52 has been limited by low production yields and challenges associated with separation and purification of this 39-amino acid antimicrobial peptide. In this study, we have successfully produced a biologically active recombinant form of E50-52 by fusing it with the 16-kDa catalytic domain of lysostaphin-class III bacteriocin (LssCAT), which resulted in high-yield production. Initially, the LssCAT-E50-52 chimeric protein was insoluble upon over-expression in Escherichia coli , but it became soluble using phosphate buffer (pH 7.4) supplemented with 8 M urea. Purification using immobilized-Ni 2+ affinity chromatography under urea denaturing conditions resulted in consistent production a homogenous products (LssCAT-E50-52) with >95% purity. The purified protein was refolded using an optimized stepwise dialysis process. The resulting refolded LssCAT-E50-52 protein exhibited dose-dependent inhibitory activity against Helicobacter pylori , a Gram-negative, flagellated, helical bacterium that is associated with gastric cancer. Overall, the optimized protocol described in this study effectively produced large quantities of high-purity recombinant LssCAT-E50-52 protein, yielding approximately 100 mg per liter of culture. To the best of our knowledge, this is the first report on the impact of LssCAT-E50-52 on H. pylori . This finding could pave the way for further research into bactericidal mechanism and potential applications of this bacteriocin in biomedical industry.

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.)

This research examines the impact of bacteriocin derived from Lactobacillus plantarum PTCC 1745 on the biofilm formations of A. baumannii isolates. Bacteriocin derived from L. plantarum PTCC 1745 was obtained through ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Testing for bacteriocin susceptibility has been conducted using the broth dilution method. The anti-biofilm activity of bacteriocin was evaluated using a microtiter plate method. Quantitative real-time PCR assay evaluated bap gene expression in bacteriocin-treated cells. According to SDS-PAGE, bacteriocin from L. plantarum has a 25-kDa apparent molecular weight. The MICs of bacteriocin ranged from 30 to 120 μg/mL, while the MBCs varied between 60 and 120 μg/mL. Compared to the non-treated group, strains bacteriocin-treated isolates had 59 % less ability to form biofilm. The mean relative expression of the bap gene among the MDR A. baumannii isolates decreased by 52 % compared to the untreated control. This study demonstrated that bacteriocin derived from L. plantarum PTCC 1745 had antibacterial and antibiofilm activity against MDR A. baumannii isolates., 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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).)

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.)

Here, we describe hitherto unknown shape-function of S/O-HexNActransferase SvGT (ORF AQF52_3101) instrumental in glycosylation of bacteriocin SvC (ORF AQF52_3099) in Streptomyces venezuelae ATCC 15439. Data from gel filtration, mass spectrometry, analytical ultracentrifugation, and Small Angle X-ray Scattering (SAXS), experiments confirmed elongated dimeric shape in solution for SvGT protein. Enzyme assays confirmed the dependence of SvGT on the availability of Mg 2+ ions to be functionally activated. SAXS data analysis provided that apo and Mg 2+ -activated protein adopt a shape characterized by a radius of gyration and maximum linear dimension of 5.2 and 17.0 nm, and 5.3 and 17.8 nm, respectively. Alphafold2 server was used to model the monomeric chain of this protein which was docked on self to obtain different poses of the dimeric entity. Experimental SAXS data was used to select and refine the structure of SvGT dimer. Results showed that Mg 2+ ions induce reorientation of the GT domain of one chain leading to a dimer with C2 symmetry, and the C-terminal portion entangles with each other in all states. Mutation-rendered alteration in activity profiles confirmed the role of conserved residues around catalytic motif. Global structure analysis puts forth the need to understand the role of constitutionally diverse C-terminal portion in regulating substrate selectivity.Communicated by Ramaswamy H. Sarma.

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.