Explore the vast range of titles available.

Background The development of antimicrobial treatments as alternatives to antibiotics to combat antimicrobial-resistant (AMR) bacteria is a global priority. Antimicrobial peptides and proteins such as Host Defense Peptides (HDPs) and endolysins are one of the alternatives that are being explored. HDPs are small, cationic, and amphiphilic antimicrobial peptides derived from the innate immune system exhibiting a broad-spectrum antimicrobial activity. On the other hand, endolysins are enzymes produced by bacteriophages to hydrolyze the bacterial peptidoglycan layer, offering more specific antimicrobial activity than HDPs. While short peptides can be chemically synthesized, this approach presents several limitations, and recombinant production is also being used. Escherichia coli is the most used bacterial expression system for protein production. Alternative systems based on Generally Recognized as Safe (GRAS) microorganisms such as Lactic Acid Bacteria (LAB) have also been employed. However, so far, no comparative studies have evaluated the production and activity of antimicrobial proteins expressed in E. coli versus LAB and this study aims to address that gap. Results To evaluate potential differences in the production of antimicrobial proteins using E. coli and two LAB ( Lactococcus lactis and Lactiplantibacillus plantarum ) hosts, various proteins were evaluated. These included two HDPs fused to a GFP, two multidomain HDP-based proteins and one endolysin. The results revealed a clear influence of the expression system on the quality of HDP-based protein, including both GFP fusions and multidomain constructs. Protein yield was higher in E. coli and all HDP-based proteins exhibited higher antimicrobial activity when expressed in E. coli compared to L. lactis and L. plantarum . In contrast, endolysin activity was comparable when produced in E. coli and L. lactis . Conclusions These results demonstrate that the choice of bacterial expression host significantly affects not only the yield but, more importantly, the antimicrobial activity of HDP-based proteins. For these proteins, the antimicrobial activity was consistently higher when produced in E. coli . In contrast, endolysins exhibited similar characteristics regardless of whether they were expressed in E. coli or in L. lactis .

The growing threat of infectious diseases requires novel therapeutics with different mechanisms of action. Antimicrobial peptides (AMPs), which are crucial for innate immunity, are a promising research area. The medicinal leech (Hirudo medicinalis) is a potential source of bioactive AMPs that are vital while interacting with microorganisms. This study aims to investigate the antimicrobial properties of peptides found in the H. medicinalis genome using a novel high-throughput screening method based on the expression of recombinant AMP genes in Escherichia coli. This approach enables the direct detection of AMP activity within cells, skipping the synthesis and purification steps, while allowing the simultaneous analysis of multiple peptides. The application of this method to the first identified candidate AMPs from H. medicinalis resulted in the discovery of three novel peptides: LBrHM1, NrlHM1 and NrlHM2. These peptides, which belong to the lumbricin and macin families, exhibit significant activity against E. coli. Two fragments of the new LBrHM1 homologue were synthesised and studied: a unique N-terminal fragment (residues 1–23) and a fragment (residues 27–55) coinciding with the active site of lumbricin I. Both fragments exhibited antimicrobial activity in a liquid medium against Bacillus subtilis. Notably, the N-terminal fragment lacks homologues among previously described AMPs.

Multiple sclerosis (MS) is an autoimmune disease that affects the CNS, the pathophysiology of which remains unclear and for which there is no definitive cure. Antimicrobial peptides (AMPs) are immunomodulatory molecules expressed in various tissues, including the CNS. Here, we investigated whether the cathelicidin-related AMP (CRAMP) modulated the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We showed that, at an early stage, CNS-recruited neutrophils produced neutrophil extracellular traps (NETs) rich in CRAMP that were required for EAE initiation. NET-associated CRAMP stimulated IL-6 production by dendritic cells via the cGAS/STING pathway, thereby promoting encephalitogenic Th17 response. However, at a later disease stage, neurons also expressed CRAMP that reduced EAE severity. Camp knockdown in neurons led to disease exacerbation, while local injection of CRAMP1-39 at the peak of EAE promoted disease remission. In vitro, CRAMP1-39 regulated the activation of microglia and astrocytes through the formyl peptide receptor (FPR) 2. Finally, administration of butyrate, a gut microbiota-derived metabolite, stimulated the expression of neural CRAMP via the free fatty acids receptors 2/3 (FFAR2/3), and prevented EAE. This study shows that CRAMP produced by different cell types has opposing effects on neuroinflammation, offering therapeutic opportunities for MS and other neuroinflammatory disorders.

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.

Antimicrobial peptides (AMPs) are crucial in the humoral immunity aspect of invertebrates' innate immune systems. However, studies on AMP discovery in the Pacific white shrimp (Litopenaeus vannamei) using omics data have been limited. Addressing the growing concern of antibiotic resistance in aquaculture, this study focused on the identification and characterization of AMPs in L. vannamei using advanced genomic and transcriptomic techniques. The genome of L. vannamei was performed to predict and identify a total of 754 AMP-derived genes, distributed across most chromosomes and spanning 24 distinct AMP families, and further identified 236 AMP-derived genes at the mRNA level in hemocytes. A subset of 20 chemically synthesized peptides, derived from these genes, exhibited significant antimicrobial activity, with over 85% showing effectiveness against key bacterial strains such as Staphylococcus aureus and Vibrio parahaemolyticus. The expression patterns of these AMPs were also investigated in different shrimp tissues and at various infection stages, revealing dynamic responses to pathogenic challenges. These findings highlight the significant potential of AMPs in L. vannamei as novel, effective alternatives to traditional antibiotics in aquaculture, offering insights into their diverse structural properties and biological functions. Together, this comprehensive characterization of the AMP repertoire in L. vannamei demonstrates the efficacy of using omics data for AMP discovery and lays the groundwork for their potential applications.

Diet is among the most important factors contributing to intestinal homeostasis, and basic functions performed by the small intestine need to be tightly preserved to maintain health. Little is known about the direct impact of high-fat (HF) diet on small-intestinal mucosal defenses and spatial distribution of the microbiota during the early phase of its administration. We observed that only 30 d after HF diet initiation, the intervillous zone of the ileum-which is usually described as free of bacteria-became occupied by a dense microbiota. In addition to affecting its spatial distribution, HF diet also drastically affected microbiota composition with a profile characterized by the expansion of Firmicutes (appearance of Erysipelotrichi), Proteobacteria (Desulfovibrionales) and Verrucomicrobia, and decrease of Bacteroidetes (family S24-7) and Candidatus arthromitus A decrease in antimicrobial peptide expression was predominantly observed in the ileum where bacterial density appeared highest. In addition, HF diet increased intestinal permeability and decreased cystic fibrosis transmembrane conductance regulator (Cftr) and the Na-K-2Cl cotransporter 1 (Nkcc1) gene and protein expressions, leading to a decrease in ileal secretion of chloride, likely responsible for massive alteration in mucus phenotype. This complex phenotype triggered by HF diet at the interface between the microbiota and the mucosal surface was reversed when the diet was switched back to standard composition or when mice were treated for 1 wk with rosiglitazone, a specific agonist of peroxisome proliferator-activated receptor-γ (PPAR-γ). Moreover, weaker expression of antimicrobial peptide-encoding genes and intervillous bacterial colonization were observed in Ppar-γ-deficient mice, highlighting the major role of lipids in modulation of mucosal immune defenses.

Background Antimicrobial peptides (AMPs) have attracted extensive attention in various fields. Gloverin is a group of AMPs derived from Lepidoptera insects. In this study, a novel gloverin CpGlv was identified through sequence alignment. The open reading frame (ORF) of mature CpGlv, devoid of its signal peptide region, was recombinantly expressed in Escherichia coli . The antibacterial activity of the fusion protein CpGlv against Staphylococcus aureus ( S. aureus ) was then assessed by determining the minimum inhibitory concentration (MIC) and using scanning electron microscopy (SEM) to observe the ultrastructure of S. aureus cells. Furthermore, the effects of CpGlv on the cell wall and membrane permeability of S. aureus were evaluated by detecting the leakage of intracellular nucleic acids, proteins, alkaline phosphatase (ALP), and β-galactosidase, as well as through the propidium iodide (PI) staining experiment. Finally, the influence of CpGlv on the S. aureus biofilm formation was investigated using the crystal violet staining assay. Results The results showed that the ORF of CpGlv contained a nucleotide sequence of 591 bp. The expressed fusion protein CpGlv had a molecular weight of approximately 24 kDa. The purified CpGlv exhibited significant antibacterial activity against S. aureus , with a MIC of 40 µg/mL. 2×MIC of CpGlv caused S. aureus cells to exhibit adhesion, swelling, deformation, and even rupture. Further, CpGlv resulted in the leakage of intracellular nucleic acids, proteins, ALP, and β-galactosidase in S. aureus . After PI staining, there was intracellular fluorescence intensity in S. aureus treated by CpGlv. Additionally, 1/4×MIC of CpGlv significantly reduced the biofilm formation of S. aureus . Conclusion A novel gloverin CpGlv was identified, expressed, and purified, and the recombinant CpGlv exhibited antimicrobial activity against S. aureus by increasing the cell membrane and wall permeability, and inhibiting the biofilm formation, laying a foundation for in-depth research on the antibacterial mechanism and application of CpGlv.

Many antimicrobial peptides directly disrupt bacterial membranes yet can also damage mammalian membranes. It is therefore central to their therapeutic use that rules governing the membrane selectivity of antimicrobial peptides be deciphered. However, this is difficult even for short peptides owing to the large combinatorial space of amino acid sequences. Here we describe a method for measuring the loss or maintenance of antimicrobial-peptide activity for thousands of peptide-sequence variants simultaneously, and its application to Protegrin-1, a potent yet toxic antimicrobial peptide, to determine the positional importance and flexibility of residues across its sequence while identifying variants with changes in membrane selectivity. More bacterially selective variants maintained a membrane-bound secondary structure while avoiding aromatic residues and cysteine pairs. A machine-learning model trained with our datasets accurately predicted membrane-specific activities for over 5.7 million Protegrin-1 variants, and identified one variant that showed substantially reduced toxicity and retention of activity in a mouse model of intraperitoneal infection. The high-throughput methodology may help elucidate sequence–structure–function relationships in antimicrobial peptides and inform the design of peptide-based synthetic drugs. A high-throughput mutation-scanning method for interrogating sequence variation in antimicrobial peptides facilitates the understanding of sequence–structure–function relationships affecting the membrane selectivity of antimicrobial peptides.

Background: The overwhelming majority of the antimicrobial peptides (AMPs) studied in mussels (Mytilus spp.) so far are specifically expressed by hemocytes and display compact disulfide-stabilized structures. However, gill-specific myticalins play a role in mucosal immunity and are one of the very few examples of known molluscan AMPs lacking cysteine residues. Methods: We investigate the molecular evolution of myticalins, compiling a collection of sequences obtained by carefully annotating 169 genome assemblies of different Mytilus species. We determine the gene presence/absence patterns and gene expression profiles for the five myticalin subfamilies, including the newly reported myticalin E. Results: All sequences are deposited in MyticalinDB, a novel database that includes a total of 100 unique mature myticalin peptides encoded by 215 protein precursors, greatly enriching the compendium of these molecules from previous reports. Among the five subfamilies, myticalin A and C are the most widespread and highly expressed across all Mytilus species. Interestingly, structural prediction reveals a previously unreported strong amphipathic nature for some myticalins, which may be highly relevant for their biological activity. Conclusions: The results reported in this work support the role of myticalins in gill-associated mucosal immunity and highlight the importance of inter-individual molecular diversity in establishing an efficient response to microbial infections. The newly established MyticalinDB provides a valuable resource for investigating the evolution and extraordinary molecular diversity of this AMP family.

Background The liver-expressed antimicrobial peptide 2 (LEAP2) family is an important group of antimicrobial peptides (AMPs) involved in vertebrate defence against bacterial infections. However, research on LEAP2 in amphibians is still in its infancy. Results This study aimed to explore the role of LEAP2 in the Chinese spiny frog ( Quasipaa spinosa ). The cDNA of the LEAP2 gene ( QsLEAP2 ) was cloned from a Chinese spiny frog. The QsLEAP2 protein comprises a signal peptide, a prodomain, and a mature peptide. Sequence analysis indicated that QsLEAP2 is a member of the amphibian LEAP2 cluster and closely related to the LEAP2 of the African clawed frog ( Xenopus laevis ). Expression of QsLEAP2 was detected in various tissues, with the liver exhibiting the highest expression. Following infection with Aeromonas hydrophila , QsLEAP2 expression was significantly upregulated in the spleen, lungs, kidneys, liver, and gut. The synthetic mature peptide QsLEAP2 exhibited selective antimicrobial activity against several bacterial strains in vitro. It disrupted bacterial membrane integrity and hydrolysed bacterial genomic DNA, exhibiting bactericidal effects on specific bacterial species. Furthermore, QsLEAP2 induced chemotaxis in RAW264.7 murine leukemic monocytes/macrophages, enhancing their phagocytic activity and respiratory bursts. Docking simulations revealed an interaction between QsLEAP2 and QsMOSPD2. Conclusions These findings provide new insights into the role of LEAP2 in the amphibian immune system.