Unveiling mechanisms of antimicrobial peptide: Actions beyond the membranes disruption.

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

Antibiotics-free compounds for managing carbapenem-resistant bacteria; a narrative review.

Carbapenem-resistant (CR) Gram-negative bacteria have become a significant public health problem in the last decade. In recent years, the prevalence of CR bacteria has increased. The resistance to carbapenems could result from different mechanisms such as loss of porin, penicillin-binding protein alteration, carbapenemase, efflux pump, and biofilm community. Additionally, genetic variations like insertion, deletion, mutation, and post-transcriptional modification of corresponding coding genes could decrease the susceptibility of bacteria to carbapenems. In this regard, scientists are looking for new approaches to inhibit CR bacteria. Using bacteriophages, natural products, nanoparticles, disulfiram, N-acetylcysteine, and antimicrobial peptides showed promising inhibitory effects against CR bacteria. Additionally, the mentioned compounds could destroy the biofilm community of CR bacteria. Using them in combination with conventional antibiotics increases the efficacy of antibiotics, decreases their dosage and toxicity, and resensitizes CR bacteria to antibiotics. Therefore, in the present review article, we have discussed different aspects of non-antibiotic approaches for managing and inhibiting the CR bacteria and various methods and procedures used as an alternative for carbapenems against these bacteria.

Synthesis and characterization of new antimicrobial peptides derived from Temporin F.

Antimicrobial peptides (AMPs) are a promising source of new compounds against resistant bacteria. Temporins are a class of AMPs found on the amphibian Rana temporaria and show activity against Gram-positive and Gram-negative bacteria. There are few studies on how these antimicrobials have been used, but new Temporin-F derivatives were engineered with Lys-substitutions to assess the impact of the net charge on antimicrobial activity and toxicity. We demonstrated through some assays that it is possible to increase the antibacterial activity while maintaining a reduced peptide hemolytic activity with specific substitutions. Our lead synthetic peptide, G6K-Temporin F, has shown higher antimicrobial activity against Gram-negative and Gram-positive bacteria in vitro (MIC range 2 to 32 µmol L-1), with low hemolytic activity maintained, resulting in an increase in the therapeutic window (TW), of 12.5. Also, it showed more resistant to enzymatic degradation. On the other hand, more significant increases in net charges, such as in P3K-G11K-Temporin F, result in a severe increase in toxicity with lower gains in antimicrobial activity (TW of 0.65). In conclusion, we demonstrated that a moderate increase in net charge can lead to a more active analog and G6K-Temporin F is revealed to be promising as a candidate for new AMP therapeutics.

The humoral immune response of the lepidopteran model insect, silkworm Bombyx mori L., to microbial pathogens.

Insects are valuable models for studying innate immunity and its role in combating infections. The silkworm Bombyx mori L., a well-studied insect model, is susceptible to a range of pathogens, including bacteria, fungi, viruses, and microsporidia. Their susceptibility makes it a suitable model for investigating host-pathogen interactions and immune responses against infections and diseases. This review focuses on the humoral immune response and the production of antimicrobial peptides (AMPs), the phenoloxidase (PO) system, and other soluble factors that constitute the primary defense of silkworms against microbial pathogens. The innate immune system of silkworms relies on pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs), which then activate various immune pathways including Imd, Toll, JAK/STAT, and RNA interference (RNAi). Their activation triggers the secretion of AMPs, enzymatic defenses (lysozyme and PO), and the generation of reactive oxygen species (ROS). Collectively, these pathways work together to neutralize and eliminate pathogens, thereby contributing to the defense mechanism of silkworms. Understanding the innate immunity of silkworms can uncover conserved molecular pathways and key immune components shared between insects and vertebrates. Additionally, it can provide valuable insights for improving sericulture practices, developing strategies to control diseases affecting silk production, and providing a theoretical foundation for developing pest control measures.

The antimicrobial protein RNase 7 directly restricts herpes simplex virus infection of human keratinocytes.

Approximately 22% of moderately to severely affected atopic dermatitis (AD) patients have a history of eczema herpeticum, a disseminated rash primarily caused by herpes simplex virus type 1 (HSV-1). Reduced activity of antimicrobial peptides may contribute to the increased susceptibility of AD patients to HSV-1. We previously demonstrated that the antimicrobial protein RNase 7 limits HSV-1 infection of human keratinocytes by promoting self-DNA sensing. Here, we addressed whether RNase 7 has any effect on HSV-1 infection when infecting keratinocytes without exogenously added costimulatory DNA, and which step(s) of the infection cycle RNase 7 interferes with. We quantified viral gene expression by RT-qPCR and flow cytometry, viral genome replication by qPCR, virucidal effects by plaque titration, and plaque formation and the subcellular localization of incoming HSV-1 particles by microscopy. Recombinant RNase 7 restricted HSV-1 gene expression, genome replication, and plaque formation in human keratinocytes. It decreased HSV-1 immediate-early transcripts independently of the induction of interferon-stimulated genes. Its main effect was on intracellular infection processes and not on extracellular virions or virus binding to cells. RNase 7 reduced the amount of cell-associated capsids and the HSV-1 envelope glycoprotein D at 3 but not at 0.5 h postinfection. Our data show that RNase 7 directly restricts HSV-1 infection of human keratinocytes, possibly by promoting the degradation of incoming HSV-1 particles. This suggests that RNase 7 may limit HSV-1 spread in the skin and that mechanisms that reduce its activity in the lesional skin of AD patients may increase their susceptibility to eczema herpeticum.

Salivary assessment of the immune/inflammatory responses and oxidative stress in older adults vaccinated with CoronaVac or ChadOx-1.

BACKGROUND: Although important information concerning COVID-19 vaccination is available, the effects of the CoronaVac and ChadOx-1 vaccines on immunity and the redox balance in the upper airway mucosa of the aged population are not fully understood. Therefore, the aim of this study was to investigate the impacts of two doses of the CoronaVac or ChadOx-1 vaccine on immune/inflammatory responses and oxidative stress in the airway mucosa of older adults. METHODS: Seventy-six older adults of both sexes, with a mean age of 75.1 ± 6.4 years, were separated according to vaccination status into the CoronaVac (n = 52) and ChadOx-1 (n = 24) groups. Saliva samples were collected before (pre) and 30 days after (post) the administration of the second dose of the CoronaVac or ChadOx-1 vaccine to assess the levels of antibodies (sIgA and IgG), antimicrobial peptides, cytokines, and oxidant/antioxidant agents. RESULTS: The immunogenicity in the ChadOx-1 group was 37.5% for sIgA and 25% for IgG, while that in the CoronaVac group was 18.9% for sIgA and 13.2% for IgG. Intergroup analysis revealed that (1) lower levels of IFN-α, IFN-γ, and IL-10 and a greater IFN-γ/IL-10 ratio, in addition to a greater IL-6/IL-10 ratio, were found in both the pre- and postvaccination periods, and (2) lower levels of total sIgA, IL-12p70, IL-17A, TNF-α, and the IL-12p70/IL-10 ratio, in addition to higher levels of specific sIgA for SARS-CoV-2 antigens and lysozyme, were observed only in the postvaccination period in the ChadOx-1 group than in the CoronaVac group. Intragroup analysis revealed (1) a significant increase in the salivary levels of total peroxides in the postvaccination period compared to those in the prevaccination period in both volunteer groups; (2) a decrease in the levels of lysozyme and the ratio between total antioxidant capacity (TAC) and total peroxides in the postvaccination period in the CoronaVac group compared with those in the prevaccination period; and (3) decreases in the TNF-α, IL-6, and IL-12p70 levels, and the IL-12p70/IL-10 ratio in the ChadoX-1 group, as well as a higher lactoferrin concentration in the postvaccination period than in the prevaccination period. Several positive and negative correlations between the parameters assessed here were found. CONCLUSIONS: In general, the ChadOx-1 group exhibited improvements in both immune/inflammatory responses and redox balance and greater immunogenicity than did the CoronaVac group.

Antibacterial Activity of Recombinant Liver-Expressed Antimicrobial Peptide-2 Derived from Olive Flounder, Paralichthys olivaceus.

Liver-expressed antimicrobial peptide-2 (LEAP-2) is a cysteine-rich peptide that plays a crucial role in the innate immune system of fish. To investigate the molecular function of LEAP-2 from olive flounder, Paralichthys olivaceus, we cloned the gene encoding LEAP-2 using PCR and expressed it in Escherichia coli. Analysis of LEAP-2 expression revealed predominant transcripts in the liver and lower levels in the intestine of olive flounder, whereas their expression levels in the liver and head kidney increased, during the initial stage of infection with the aquapathogenic bacterium Edwadrsiella piscicida. Recombinant LEAP-2 (rOfLEAP-2) purified from E. coli exhibited antimicrobial activity, as demonstrated by the ultrasensitive radial diffusion assay, against both Gram-positive (Bacillus subtilis, Streptococcus parauberis, and Lactococcus garvieae) and Gram-negative (Vibrio harveyi and E. coli) bacteria, with minimum inhibitory concentrations ranging from 25 to 100 µg/mL depending on the species tested. The antibacterial activity of rOfLEAP-2 was attributed to its ability to disrupt bacterial membranes, validated by the N-phenylnaphthalen-1-amine uptake assays and scanning electron microscope analysis against E. coli, V. harveyi, B. subtilis, and L. garvieae treated with rOfLEAP-2. Furthermore, a synergistic enhancement of antibacterial activity was observed when rOfLEAP-2 was combined with ampicillin or synthetic LEAP-1 peptide, suggesting a distinct mechanism of action from those of other antimicrobial agents. These findings provide evidence for the antibacterial efficacy of LEAP-2 from olive flounder, highlighting its potential therapeutic application against pathogenic bacteria.

Application of antimicrobial peptides in the poultry industry.

Poultry meat production and exportation contribute significantly to the global economy. However, various infections affect poultry production and consequently affect the economy. Nowadays, antibiotics are widely used in infection treatment and prevention. Antibiotic overuse is problematic because may cause antimicrobial resistance, which can be transferred to humans directly or indirectly, affecting public health. In addition, since antibiotics for animal growth stimulation are banned, it is important to search for new molecules to overcome these difficulties. As an alternative, antimicrobial peptides (AMPs) can show immunomodulatory, antimicrobial, and growth stimulation, which makes these molecules interesting as alternatives to antibiotic use. Studying AMPs can provide new ideas for treating the most important infections that affect poultry. Besides, this can assist in reducing the resistance problem. This review aims to examine recent studies about AMPs used against pathogens that can affect the poultry industry.

Trehalose prevents IL-4/IL-13-induced skin barrier impairment by suppressing IL-33 expression and increasing Nrf2 activation in human keratinocytes in vitro.

Skin barrier dysfunction initiates or deteriorates various cutaneous problems, such as atopic dermatitis (AD). At high concentrations, the nonreducing disaccharide α-d-glucopyranosyl α-d-glucopyranoside (trehalose) induces a transient senescence-like state in fibroblasts and promotes wound repair. Here, we investigated the effect of trehalose on normal human keratinocytes (KCs) and demonstrated its specific role in the skin barrier. RNA-seq analysis revealed that trehalose regulates the expression of many skin-barrier-associated genes. T helper 2 (Th2) cytokines interleukin (IL)-4/IL-13 were observed to downregulate several differentiation markers (FLG, LOR, K1, and K10) and epidermal antimicrobial proteins in monolayer-cultured KCs and living skin equivalents (LSE), and impaired skin barrier function in LSE, all of which were significantly upregulated or restored by trehalose. Trehalose inhibited IL-33 expression and reduced nuclear IL-33 levels by activating MEK5-extracellular signal-regulated kinase 5 (ERK5) and suppressing MEK1/2-ERK pathway. It also increased nuclear factor erythroid 2-related factor 2 (Nrf2) activation to trigger antioxidant enzyme production via c-Jun N-terminal kinase (JNK), thus, neutralizing IL-4/IL-13-mediated oxidative stress. Trehalose prevented IL-4/IL-13-mediated signal transducer and activator of transcription (STAT)3/STAT6 activation and restored IL-4/IL-13-suppressed skin barrier molecules via IL-33 downregulation and Nrf2 activation. This study demonstrated that trehalose may play a role in skin barrier repair in AD.

Antimicrobial activity of novel symmetrical antimicrobial peptides centered on a hydrophilic motif against resistant clinical isolates: in vitro and in vivo analyses.

Antibiotic resistance poses a significant public health threat worldwide. The rise in antibiotic resistance and the sharp decline in effective antibiotics necessitate the development of innovative antibacterial agents. Based on the central symmetric structure of glycine-serine-glycine, combined with tryptophan and arginine, we designed a range of antimicrobial peptides (AMPs) that exhibited broad-spectrum antibacterial activity. Notably, AMP W5 demonstrated a rapid and effective sterilization against methicillin-resistant Staphylococcus aureus (MRSA), displaying both a minimum inhibitory concentration and a minimum bactericidal concentration of 8 µM. Mechanistic studies revealed that AMP W5 killed bacterial cells by disrupting the cytoplasmic membrane integrity, triggering leakage of cell contents. AMP W5 also exhibited excellent biocompatibility in both in vitro and in vivo safety evaluations. AMP W5 treatment significantly reduced skin bacterial load in our murine skin infection model. In conclusion, we designed a novel centrosymmetric AMP representing a promising medical alternative to conventional antibiotics for treating MRSA infections. IMPORTANCE: Increasing antibiotic resistance and the paucity of effective antibiotics necessitate innovative antibacterial agents. Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen causing bacterial infections with high incidence and mortality rates, showing increasing resistance to clinical drugs. Antimicrobial peptides (AMPs) exhibit significant potential as alternatives to traditional antibiotics. This study designed a novel series of AMPs, characterized by a glycine-serine-glycine-centered symmetrical structure, and our results indicated that AMP W5 exhibited a rapid and effective bactericidal effect against MRSA. AMP W5 also demonstrated excellent biocompatibility and a bactericidal mechanism that disrupted membrane integrity, leading to leakage of cellular contents. The notable reduction in skin bacterial load observed in mouse models reinforced the clinical applicability of AMP W5. This study provides a promising solution for addressing the increasing threat of antibiotic-resistant bacteria and heralds new prospects for clinical applications.

Segment-Based Peptide Design Reveals the Importance of N-Terminal High Cationicity for Antimicrobial Activity Against Gram-Negative Pathogens.

Host defense antimicrobial peptides (AMPs) are recognized candidates to develop a new generation of peptide antibiotics. While high hydrophobicity can be deployed in peptides for eliminating Gram-positive bacteria, high cationicity is usually observed in AMPs against Gram-negative pathogen. This study investigates how the sequence distribution of basic amino acids affects peptide activity. For this purpose, we utilized human cathelicidin LL-37 as a template and designed four highly selective ultrashort peptides with similar length, net charge, and hydrophobic content. LL-10 + , RK-9 + , KR-8 + , and RIK-10 + showed similar activity against methicillin-resistant Staphylococcus aureus in vitro and comparable antibiofilm efficacy in a murine wound model. However, these peptides showed clear activity differences against Gram-negative pathogens with RIK-10 + (i.e., LL-37mini2) being the strongest and LL-10 + the weakest. To understand this activity difference, we characterized peptide toxicity; the effects of salts, pH, and serum on peptide activity; and the mechanism of action and determined the membrane-bound helical structure for RIK-10 + by two-dimensional NMR spectroscopy. By writing an R program, we generated charge density plots for these peptides and uncovered the importance of the N-terminal high-density basic charges for antimicrobial potency. To validate this finding, we reversed the sequences of two peptides. Interestingly, sequence reversal weakened the activity of RIK-10 + but increased the activity of LL-10 + especially against Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Those more active peptides with high cationicity at the N-terminus are also more hydrophobic based on HPLC retention times. A database search found numerous natural sequences that arrange basic amino acids primarily at the N-terminus. Combined, this study not only obtained novel peptide leads but also discovered one useful strategy for designing novel antimicrobials to control drug-resistant Gram-negative pathogens.

The modulation of intestinal commensal bacteria possibly contributes to the growth and immunity promotion in Epinephelus akaara after feeding the antimicrobial peptide Scy-hepc.

BACKGROUND: Our previous study revealed that feeding the antimicrobial peptide (AMP) product Scy-hepc significantly enhances the growth of mariculture fish through the activation of the GH-Jak2-STAT5-IGF1 axis. However, the contribution of gut microbiota to this growth enhancement remains unclear. This study aimed to elucidate the potential mechanism involved in intestinal absorption and modulation of gut microbiota in Epinephelus akaara following Scy-hepc feeding. RESULTS: The results showed that a 35 day regimen of Scy-hpec markedly promoted the growth of E. akaara compared to groups supplemented with either florfenicol, B. subtilis, or a vector. The growth enhancement is likely attributed to alterations in microbiota colonization in the foregut and midgut, characterized by an increasing abundance of potential probiotics (Rhizobiaceae and Lysobacter) and a decreased abundance of opportunistic pathogens (Psychrobacter and Brevundimonas) as determined by 16S rRNA analysis. Additionally, similar to the effect of florfenicol feeding, Scy-hepc significantly improved host survival rate by over 20% in response to a lethal dose challenge with Edwardsiella tarda. Further investigations demonstrated that Scy-hepc is absorbed by the fish foregut (20-40 min) and midgut (20-30 min) as confirmed by Western blot, ELISA, and Immunofluorescence. The absorption of Scy-hepc affected the swimming, swarming and surfing motility of Vibrio harveyi and Bacillus thuringiensis isolated from E. akaara's gut. Moreover, Scy-hepc induced the downregulation of 40 assembly genes and the upregulation expression of 5, with the most significant divergence in gene expression between opportunistic pathogens and probiotics concentrated in their motility genes (PomA/B, MotA/B). CONCLUSIONS: In summary, this study shows that feeding AMP Scy-hepc can promote growth and bolster immunity in E. akaara. These beneficial effects are likely due to the absorption of Scy-hepc in the fish's foregut and midgut, which modulates the colonization and motility of commensal bacteria, leading to favorable changes in the composition of the foregut and midgut microbiota. Therefore, a profound understanding of the mechanisms by which antimicrobial peptides affect host gut microbiota will contribute to a comprehensive assessment of their advantages and potential application prospects as substitutes for antibiotics in fish health and improving aquaculture practices.

Antifungal Efficacy of Ultrashort ß-Peptides against Candida Species: Mechanistic Understanding and Therapeutic Implications.

Candidiasis, a condition spurred by the unchecked proliferation of Candida species, poses a formidable global health threat, particularly in immunocompromised individuals. The emergence of drug-resistant strains complicates management strategies, necessitating novel therapeutic avenues. Antimicrobial peptides (AMPs) have garnered attention for their potent antifungal properties and broad-spectrum activity against Candida species. This study assessed the antifungal effectiveness of ultrashort ß-peptides against Candida strains, with a specific focus on peptide P3 (LAU-ß3,3-Pip-ß2,2-Ac6c-PEA). Our findings showed P3's remarkable fungistatic and fungicidal activities against Candida albicans, exhibiting an MIC of 4 µg/mL, comparable to those of standard antifungal drugs. The MIC value remained unchanged in the presence of ADC and BSA, indicating that serum albumin does not diminish the activity of P3. P3 demonstrates synergistic effects when combined with Fluconazole (FLU), Itraconazole (ITR), and Nystatin (NYS) to the extent that it becomes effective at 0.125, 0.125, and 0.03125 µg/mL, respectively. Concentration versus time-kill kinetics showed its time-dependent activity up to the first 12 h against C. albicans, and later concentration also played a role; indeed, at 24 h the whole culture was sterilized at 8× MIC. Post-antifungal effect assays confirmed prolonged suppression of pathogen growth after the removal of P3 from the media for significant durations. More importantly, P3 inhibits hyphae formation and biofilm development of Candida, outperforming Fluconazole with respect to these properties. Mechanistic insights display P3's potential to disrupt fungal cell membrane integrity and dose-dependent inhibition of ergosterol biosynthesis, essential for fungal cell wall integrity. Using the Bradford assay, it was observed that extracellular protein concentrations increased with higher doses of the compound, thereby validating the effect of P3 on membrane integrity. A comparative gene analysis using RT-PCR showed that P3 downregulates ERG3, ERG11, and HWP1, which are crucial for the survival and pathogenicity of C. albicans. The impact of P3 on ERG11 and ERG3 is more effective than that of Fluconazole. Molecular docking studies revealed strong binding of P3 to various isoforms of lanosterol 14-α-demethylase, a key enzyme in ergosterol synthesis. Furthermore, molecular dynamic simulations validated the stability of the most promising docking complex. Overall, our findings underscore P3's potential as a leading candidate for the development of innovative antifungal therapies, warranting further investigation and optimization.

Multispectral bioactivity studies of N-terminal fatty acid modified antimicrobial peptide Andricin B.

A series of Andricin B derivatives were designed and synthesized using fatty acid modification at N-terminus of the antimicrobial peptides. The hydrophobicity of Andricin B was altered through fatty acid modification, and the bioactivity was investigated. The interaction between Andricin B and its derivatives with DNA was measured using multi-spectroscopy. Spectroscopic analysis revealed that Andricin B and its derivatives can interact with ct-DNA and G-quadruplexes DNA, and the interaction related with the length of fatty acid chain. Antimicrobial activity tests showed a significant increase using peptides with 8-10 carbons fatty acid chain. C10-Andricin B exhibited the highest antimicrobial activity, with up to a 16-fold enhancement compared to the original peptide Andricin B. Meanwhile, the protease hydrolysis stability test showed that fatty acid modification improved the stability of Andricin B against protease. Scanning electron microscopy results distinctly showed that C8-Andricin B could rupture the cell wall of bacteria. All results indicated that fatty acid modification peptides are an effective strategy for enhancing activity and stability of antimicrobial peptides. This research provides valuable insights for further research on antimicrobial peptides.

Discovery of Antimicrobial Peptides in Urinary Tract Infections.

Antimicrobial peptides (AMPs) play a pivotal role in the innate immune system as a frontline defense against microbial threats. AMPs can serve as biomarkers and alternative antibiotics, overcoming mortality related to multidrug-resistant pathogens in urinary tract infections (UTIs). While the relevance of AMPs in UTIs has been validated and AMP drugs approved by the US Food and Drug Administration are in clinical use, information about their modification status, regulation, and mechanism of action remains sparse. Only a small fraction of sequences with potential AMP activity, predicted on the basis of known AMP characteristics, have been validated. Elucidation of the global profile of AMPs in the bladder, kidney, and urine under UTI conditions would facilitate an in-depth, disease-specific understanding of the innate immune system and the development of tailored AMP biomarkers and antibiotics. This mini-review focuses on a comprehensive strategy for global profiling and validation of AMPs in UTIs that incorporates AMP data repositories, prediction algorithms, and proteomics for healthy individuals and UTI patients. PATIENT SUMMARY: Short protein molecules called peptides that have antimicrobial activity show promise for the treatment of urinary tract infections. More research and testing of naturally occurring and synthetic peptides with this activity are needed to fully understand how they can help in patient care.

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

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

S100A12 inhibits Streptococcus pneumoniae and aids in wound healing of corneal epithelial cells both in vitro and in vivo.

Streptococcus pneumoniae, a leading cause of corneal infections worldwide, are extremely aggressive despite antibiotic sensitivity and exhibit increased resistance towards antibiotics. Antimicrobial peptides are often considered as potent alternatives against antibiotic resistance and here we have investigated the possible roles of S100A12, a host defense peptide, in wound healing and S. pneumoniae infection. S100A12 significantly inhibited growth of S. pneumoniae by disruption of membrane integrity along with increased generation of reactive oxygen species. Additionally, S100A12 accelerated cell migration and wound closure in human corneal epithelial cells and in a murine corneal wound model by activation of EGFR and MAPK signaling pathways.

The antimicrobial peptide Temporin-L induces vesicle formation and reduces the virulence in S. aureus.

The evolution of methicillin-resistant Staphylococcus aureus (MRSA) has required the development of new antimicrobial agents and new approaches to prevent and overcome drug resistance. AntiMicrobial Peptides (AMPs) represent promising alternatives due to their rapid bactericidal activity and their broad-spectrum of action against a wide range of microorganisms. The amphibian Temporins constitute a well-known family of AMPs with high antibacterial properties against both Gram-positive and Gram-negative bacteria. In this paper, we evaluated the in vivo effect of Temp-L on S. aureus performing morphological studies using Transmission Electron Microscopy (TEM) that revealed the occurrence of protrusions from the cell surface. The formation of vesicle-like structure was confirmed by Dynamic Light Scattering (DLS). The global effect of Temp-L on Staphylococcus aureus (S. aureus) was deeply investigated by differential proteomics leading to the identification of up-regulated proteins involved in the synthesis of the cell membrane and fatty acids, and down-regulated virulence factors. GC-MS analysis suggested a possible protective response mechanism implemented by the bacterium after treatment with Temp-L, as the synthesis of fatty acids was increased. Adhesion and invasion assays on eukaryotic cells confirmed a reduced virulence of S. aureus following treatment with Temp-L. These results suggested the targeting of virulence factors as novel strategy to replace traditional antimicrobial agents that can be used to treat infections, especially infections caused by the resistant pathogen S. aureus.

Identification of a Novel ß-Defensin Gene in Gilthead Seabream (Sparus aurata).

The excessive use of antibiotics in aquaculture favors the natural selection of multidrug-resistant bacteria, and antimicrobial peptides (AMPs) could be a promising alternative to this problem. The most studied AMPs in teleost fish are piscidins, hepcidins, and ß-defensins. In this work, we have found a new gene (defb2) encoding a type 2 ß-defensin in the genome of gilthead seabream, a species chosen for its economic interest in aquaculture. Its open reading frame (192 bp) encodes a protein (71 amino acids) that undergoes proteolytic cleavage to obtain the functional mature peptide (42 amino acids). The genetic structure in three exons and two introns and the six characteristic cysteines are conserved as the main signature of this protein family. In the evolutionary analysis, synteny shows a preservation of chromosomal localization and the phylogenetic tree constructed exposes the differences between both types of ß-defensin as well as the similarities between seabream and European seabass. In relation to its basal expression, ß-defensin 2 is mostly expressed in the intestine, thymus, skin, and gonads of the gilthead seabream (Sparus aurata). In head kidney leucoytes (HKLs), the expression was very low and did not change significantly when stimulated with various immunocompetent agents. However, the expression was significantly down-regulated in the liver, head-kidney, and blood 4 h post-injection with the fish pathogen Vibrio harveyi. When infected with nodavirus, the expression was downregulated in brain at 7 days post-infection. These results denote a possible complementarity between the expression patterns of ß-defensins and hepcidins. Further studies are needed to analyze gene duplications and expression patterns of ß-defensins and describe their mechanism of action in seabream and other teleost fish.

Inflammation promotes stomach epithelial defense by stimulating the secretion of antimicrobial peptides in the mucus.

The mucus serves as a protective barrier in the gastrointestinal tract against microbial attacks. While its role extends beyond merely being a physical barrier, the extent of its active bactericidal properties remains unclear, and the mechanisms regulating these properties are not yet understood. We propose that inflammation induces epithelial cells to secrete antimicrobial peptides, transforming mucus into an active bactericidal agent. To investigate the properties of mucus, we previously developed mucosoid culture models that mimic the healthy human stomach epithelium. Similar to organoids, mucosoids are stem cell-driven cultures; however, the cells are cultivated on transwells at air-liquid interface. The epithelial cells of mucosoids form a polarized monolayer, allowing differentiation into all stomach lineages, including mucus-secreting cells. This setup facilitates the secretion and accumulation of mucus on the apical side of the mucosoids, enabling analysis of its bactericidal effects and protein composition, including antimicrobial peptides. Our findings show that TNFα, IL1ß, and IFNγ induce the secretion of antimicrobials such as lactotransferrin, lipocalin2, complement component 3, and CXCL9 into the mucus. This antimicrobial-enriched mucus can partially eliminate Helicobacter pylori, a key stomach pathogen. The bactericidal activity depends on the concentration of each antimicrobial and their gene expression is higher in patients with inflammation and H.pylori-associated chronic gastritis. However, we also find that H. pylori infection can reduce the expression of antimicrobial encoding genes promoted by inflammation. These findings suggest that controlling antimicrobial secretion in the mucus is a critical component of epithelial immunity. However, pathogens like H. pylori can overcome these defenses and survive in the mucosa.