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In the field of tissue engineering, the use of core-shell fibers represents an advantageous approach to protect and finely tune the release of bioactive compounds with the aim to regulate their efficacy. In this work, core-shell electrospun polycaprolactone nanofiber-based membranes, loaded with rifampicin and coated with silver nanoparticles, were developed and characterized. The membranes are composed by randomly oriented nanofibers with a homogeneous diameter, as demonstrated by scanning electron microscopy (SEM). An air-plasma treatment was applied to increase the hydrophilicity of the membranes as confirmed by contact angle measurements. The rifampicin release from untreated and air-plasma treated membranes, evaluated by UV spectrophotometry, displayed a similar and constant over-time release profile, demonstrating that the air-plasma treatment does not degrade the rifampicin, loaded in the core region of the nanofibers. The presence and the distribution of silver nanoparticles on the nanofiber surface were investigated by SEM and Energy Dispersive Spectroscopy. Moreover, SEM imaging demonstrated that the produced membranes possess a good stability over time, in terms of structure maintenance. The developed membranes showed a good biocompatibility towards murine fibroblasts, human osteosarcoma cells and urotheliocytes, reveling the absence of cytotoxic effects. Moreover, doble-functionalized membranes inhibit the growth of E. coli and S. aureus. Thanks to the possibilities offered by the coaxial electrospinning, the membranes here proposed are promising for several tissue engineering applications., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

The paenilamicins are a group of hybrid non-ribosomal peptide-polyketide compounds produced by the honey bee pathogen Paenibacillus larvae that display activity against Gram-positive pathogens, such as Staphylococcus aureus . While paenilamicins have been shown to inhibit protein synthesis, their mechanism of action has remained unclear. Here, we have determined structures of the paenilamicin PamB2 stalled ribosomes, revealing a unique binding site on the small 30S subunit located between the A- and P-site tRNAs. In addition to providing a precise description of interactions of PamB2 with the ribosome, the structures also rationalize the resistance mechanisms utilized by P. larvae . We could further demonstrate that PamB2 interferes with the translocation of mRNA and tRNAs through the ribosome during translation elongation, and that this inhibitory activity is influenced by the presence of modifications at position 37 of the A-site tRNA. Collectively, our study defines the paenilamicins as a new class of context-specific translocation inhibitors., Competing Interests: Competing interests The authors declare no competing interests.

The optimized proline-rich antimicrobial peptide B7-005 was loaded on bone scaffolds based on polysaccharides and hydroxyapatite. Alginate was firstly chosen in order to exploit its negative charges, which allowed an efficient B7-005 loading but hindered its release, due to the strong interactions with the positive charged peptide. Hence, alginate was substituted with agarose which allowed to prepare scaffolds with similar structure, porosity, and mechanical performance than the ones prepared with alginate and hydroxyapatite. Moreover, agarose scaffolds could release B7-005 within the first 24 h of immersion in aqueous environment. The peptide did not impaired MG-63 cell adhesion and proliferation in the scaffold, and a positive cell proliferation trend was observed up to two weeks. The released B7-005 was effective against the pathogens E. coli, K. pneumoniae , and A. baumannii , but not against S. aureus and P. aeruginosa , thus requiring further tuning of the system to improve its antimicrobial activity., (© 2023 IOP Publishing Ltd.)

Cathelicidins, a family of host defence peptides in vertebrates, play an important role in the innate immune response, exhibiting antimicrobial activity against many bacteria, as well as viruses and fungi. This work describes the design and synthesis of shortened analogues of porcine cathelicidin PMAP-36, which contain structural changes to improve the pharmacokinetic properties. In particular, 20-mers based on PMAP-36 (residues 12-31) and 13-mers (residues 12-24) with modification of amino acid residues at critical positions and introduction of lipid moieties of different lengths were studied to identify the physical parameters, including hydrophobicity, charge, and helical structure, required to optimise their antibacterial activity. Extensive conformational analysis, performed by CD and NMR, revealed that the substitution of Pro25-Pro26 with Ala25-Lys26 increased the α-helix content of the 20-mer peptides, resulting in broad-spectrum antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus epidermidis strains. Interestingly, shortening to just 13 residues resulted in only a slight decrease in antibacterial activity. Furthermore, two sequences, a 13-mer and a 20-mer, did not show cytotoxicity against HaCat cells up to 64 µM, indicating that both derivatives are not only effective but also selective antimicrobial peptides. In the short peptide, the introduction of the helicogenic α-aminoisobutyric acid forced the helix toward a prevailing 3 10 structure, allowing the antimicrobial activity to be maintained. Preliminary tests of resistance to Ser protease chymotrypsin indicated that this modification resulted in a peptide with an increased in vivo lifespan. Thus, some of the PMAP-36 derivatives studied in this work show a good balance between chain length, antibacterial activity, and selectivity, so they represent a good starting point for the development of even more effective and proteolysis-resistant active peptides., (© 2023. Springer Nature Limited.)

Resistance to aminoglycoside antibiotics is a serious problem, typically arising from inactivating enzymes, reduced uptake, or increased efflux in the important pathogens for which they are used as treatment. Conjugating aminoglycosides to proline-rich antimicrobial peptides (PrAMPs), which also target ribosomes and have a distinct bacterial uptake mechanism, might mutually benefit their individual activities. To this aim we have developed a strategy for noninvasively modifying tobramycin to link it to a Cys residue and through this covalently link it to a Cys-modified PrAMP by formation of a disulfide bond. Reduction of this bridge in the bacterial cytosol should release the individual antimicrobial moieties. We found that the conjugation of tobramycin to the well-characterized N-terminal PrAMP fragment Bac7(1-35) resulted in a potent antimicrobial capable of inactivating not only tobramycin-resistant bacterial strains but also those less susceptible to the PrAMP. To a certain extent, this activity also extends to the shorter and otherwise poorly active fragment Bac7(1-15). Although the mechanism that allows the conjugate to act when its individual components do not is as yet unclear, results are very promising and suggest this may be a way of resensitizing pathogens that have developed resistance to the antibiotic.

Mussels ( Mytilus spp.) tolerate infections much better than other species living in the same marine coastal environment thanks to a highly efficient innate immune system, which exploits a remarkable diversification of effector molecules involved in mucosal and humoral responses. Among these, antimicrobial peptides (AMPs) are subjected to massive gene presence/absence variation (PAV), endowing each individual with a potentially unique repertoire of defense molecules. The unavailability of a chromosome-scale assembly has so far prevented a comprehensive evaluation of the genomic arrangement of AMP-encoding loci, preventing an accurate ascertainment of the orthology/paralogy relationships among sequence variants. Here, we characterized the CRP-I gene cluster in the blue mussel Mytilus edulis , which includes about 50 paralogous genes and pseudogenes, mostly packed in a small genomic region within chromosome 5. We further reported the occurrence of widespread PAV within this family in the Mytilus species complex and provided evidence that CRP-I peptides likely adopt a knottin fold. We functionally characterized the synthetic peptide sCRP-I H1, assessing the presence of biological activities consistent with other knottins, revealing that mussel CRP-I peptides are unlikely to act as antimicrobial agents or protease inhibitors, even though they may be used as defense molecules against infections from eukaryotic parasites.

Cinnamic acids are an important class of phenolic compounds, which have many beneficial effects on human health but are also interesting synthetic intermediates thanks to the presence of several reactive sites. While studying the reactivity of cinnamic acids with diazonium salts from aromatic amines, an unexpected reactivity has been discovered, leading to the formation of 1,2-diaza-1,3-dienes instead of traditional diazo-coupling products. The new compounds have been fully characterized by mono and bidimensional NMR spectroscopy and mass spectrometry. Preliminary studies on the biological activity of the compounds have been carried out testing both their antibacterial and antitumor activity, leading to promising results., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

An infecting and propagating parasite relies on its innate defense system to evade the host's immune response and to survive challenges from commensal bacteria. More so for the nematode Anisakis, a marine parasite that during its life cycle encounters both vertebrate and invertebrate hosts and their highly diverse microbiotas. Although much is still unknown about how the nematode mitigates the effects of these microbiota, its antimicrobial peptides likely play an important role in its survival. We identified anisaxins, the first cecropin-like helical antimicrobial peptides originating from a marine parasite, by mining available genomic and transcriptomic data for Anisakis spp. These peptides are potent bactericidal agents in vitro, selectively active against Gram-negative bacteria, including multi-drug resistant strains, at sub-micromolar concentrations. Their interaction with bacterial membranes was confirmed by solid state NMR (ssNMR) and is highly dependent on the peptide concentration as well as peptide to lipid ratio, as evidenced by molecular dynamics (MD) simulations. MD results indicated that an initial step in the membranolytic mode of action involves membrane bulging and lipid extraction; a novel mechanism which may underline the peptides' potency. Subsequent steps include membrane permeabilization leading to leakage of molecules and eventually cell death, but without visible macroscopic damage, as shown by atomic force microscopy and flow cytometry. This membranolytic antibacterial activity does not translate to cytotoxicity towards human peripheral blood mononuclear cells (HPBMCs), which was minimal at well above bactericidal concentrations, making anisaxins promising candidates for further drug development. STATEMENT OF SIGNIFICANCE: Witnessing the rapid spread of antibiotic resistance resulting in millions of infected and dozens of thousands dying worldwide every year, we identified anisaxins, antimicrobial peptides (AMPs) from marine parasites, Anisakis spp., with potent bactericidal activity and selectivity towards multi-drug resistant Gram-negative bacteria. Anisaxins are membrane-active peptides, whose activity, very sensitive to local peptide concentrations, involves membrane bulging and lipid extraction, leading to membrane permeabilization and bacterial cell death. At the same time, their toxicity towards host cells is negligible, which is often not the case for membrane-active AMPs, therefore making them suitable drug candidates. Membrane bulging and lipid extraction are novel concepts that broaden our understanding of peptide interactions with bacterial functional structures, essential for future design of such biomaterials., 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 © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

As bioactive small proteins with antimicrobial and immunomodulatory activities that are naturally produced by all living organisms, antimicrobial peptides (AMPs) have a marked potential as next-generation antibiotics. However, their development as antibacterial agents is limited by low stability and cytotoxicity. D-BMAP18, a membrane-permeabilizing antimicrobial peptide composed of D-amino acids, has shown good antibacterial and anti-inflammatory activities but also a non-negligible cytotoxicity against eukaryotic cell lines. In this study, a prodrug has been developed that extends the peptide with a negatively charged, inactivating sequence containing the cleavage site for neutrophil elastase (NE). The ultimate goal was to allow the activation of D-BMAP18 by endogenous elastase only at the site of infection/inflammation, enabling a slow and targeted release of the pharmacologically active peptide. In vitro activation of Pro-D-BMAP18 was confirmed using purified NE. Its antimicrobial and cytotoxic activities were tested in the presence and absence of elastase and compared to those of the parental form. The prodrug had minimal activity in the absence of elastase, while its proteolysis product retained an appreciable antimicrobial activity but lower cytotoxicity. Moreover, Pro-D-BMAP18 was found to be correctly converted to D-BMAP18 in the presence of CF sputum as a model of the lung environment and showed good antimicrobial activity under these conditions.

The 3D structure and surface characteristics of proteins and peptides are crucial for interactions with receptors or ligands and can be modified to some extent to modulate their biological roles and pharmacological activities. The introduction of halogen atoms on the side-chains of amino acids is a powerful tool for effecting this type of tuning, influencing both the physico-chemical and structural properties of the modified polypeptides, helping to first dissect and then rationally modify features that affect their mode of action. This review provides examples of the influence of different types of halogenation in amino acids that replace native residues in proteins and peptides. Examples of synthetic strategies for obtaining halogenated amino acids are also provided, focusing on some representative compounds and their biological effects. The role of halogenation in native and designed antimicrobial peptides (AMPs) and their mimetics is then discussed. These are in the spotlight for the development of new antimicrobial drugs to counter the rise of antibiotic-resistant pathogens. AMPs represent an interesting model to study the role that natural halogenation has on their mode of action and also to understand how artificially halogenated residues can be used to rationally modify and optimize AMPs for pharmaceutical purposes.

Bivalve mollusks are continuously exposed to potentially pathogenic microorganisms living in the marine environment. Not surprisingly, these filter-feeders developed a robust innate immunity to protect themselves, which includes a broad panel of antimicrobial peptides. Among these, myticalins represent a recently discovered family of linear cationic peptides expressed in the gills of Mytilus galloprovincialis. Even though myticalins and insect and mammalian proline-rich antimicrobial peptides (PrAMPs) share a similar amino acid composition, we here show that none of the tested mussel peptides use a non-lytic mode of action relying on the bacterial transporter SbmA. On the other hand, all the tested myticalins perturbed and permeabilized the membranes of E. coli BW25113, as shown by flow-cytometry and atomic force microscopy. Circular dichroism spectra revealed that most myticalins did not adopt recognizable secondary structures in the presence of amphipathic environments, such as biological membranes. To explore possible uses of myticalins for biotech, we assessed their biocompatibility with a human cell line. Non-negligible cytotoxic effects displayed by myticalins indicate that their optimization would be required before their further use as lead compounds in the development of new antibiotics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Natural and de novo designed peptides are gaining an ever-growing interest as drugs against several diseases. Their use is however limited by the intrinsic low bioavailability and poor stability. To overcome these issues retro-inverso analogues have been investigated for decades as more stable surrogates of peptides composed of natural amino acids. Retro-inverso peptides possess reversed sequences and chirality compared to the parent molecules maintaining at the same time an identical array of side chains and in some cases similar structure. The inverted chirality renders them less prone to degradation by endogenous proteases conferring enhanced half-lives and an increased potential as new drugs. However, given their general incapability to adopt the 3D structure of the parent peptides their application should be careful evaluated and investigated case by case. Here, we review the application of retro-inverso peptides in anticancer therapies, in immunology, in neurodegenerative diseases, and as antimicrobials, analyzing pros and cons of this interesting subclass of molecules.

The emergence of multidrug-resistant bacteria is a worldwide health problem. Antimicrobial peptides have been recognized as potential alternatives to conventional antibiotics, but still require optimization. The proline-rich antimicrobial peptide Bac7(1-16) is active against only a limited number of Gram-negative bacteria. It kills bacteria by inhibiting protein synthesis after its internalization, which is mainly supported by the bacterial transporter SbmA. In this study, we tested two different lipidated forms of Bac7(1-16) with the aim of extending its activity against those bacterial species that lack SbmA. We linked a C12-alkyl chain or an ultrashort cationic lipopeptide Lp-I to the C-terminus of Bac7(1-16). Both the lipidated Bac-C12 and Bac-Lp-I forms acquired activity at low micromolar MIC values against several Gram-positive and Gram-negative bacteria. Moreover, unlike Bac7(1-16), Bac-C12, and Bac-Lp-I did not select resistant mutants in E. coli after 14 times of exposure to sub-MIC concentrations of the respective peptide. We demonstrated that the extended spectrum of activity and absence of de novo resistance are likely related to the acquired capability of the peptides to permeabilize cell membranes. These results indicate that C-terminal lipidation of a short proline-rich peptide profoundly alters its function and mode of action and provides useful insights into the design of novel broad-spectrum antibacterial agents.

Electrospun polycaprolactone (PCL) membranes have been widely explored in the literature as a solution for several applications in tissue engineering and regenerative medicine. PCL hydrophobicity and its lack of bioactivity drastically limit its use in the medical field. To overcome these drawbacks, many promising strategies have been developed and proposed in the literature. In order to increase the bioactivity of electrospun PCL membranes designed for guided bone and tissue regeneration purposes, in the present work, the membranes were functionalized with a coating of bioactive lactose-modified chitosan (CTL). Since CTL can be used for the synthesis and stabilization of silver nanoparticles, a coating of this compound was employed here to provide antibacterial properties to the membranes. Scanning electron microscopy imaging revealed that the electrospinning process adopted here allowed us to obtain membranes with homogeneous fibers and without defects. Also, PCL membranes retained their mechanical properties after several weeks of aging in simulated body fluid, representing a valid support for cell growth and tissue development. CTL adsorption on membranes was investigated by fluorescence microscopy using fluorescein-labeled CTL, resulting in a homogeneous and slow release over time. Inductively coupled plasma-mass spectrometry was used to analyze the release of silver, which was shown to be stably bonded to the CTL coating and to be slowly released over time. The CTL coating improved MG63 osteoblast adhesion and proliferation on membranes. On the other hand, the presence of silver nanoparticles discouraged biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus without being cytotoxic. Overall, the stability and the biological and antibacterial properties make these membranes a valid and versatile material for applications in guided tissue regeneration and in other biomedical fields like wound healing.

The aim of this work was to assess the antimicrobial efficacy on Pseudomonas aeruginosa of nanomicelles loaded with curcumin (CUR) alone and activated by blue laser light in an antimicrobial photodynamic therapy (APDT) approach. First, free CUR in liquid suspension and loaded in three amphiphilic nanomicelles (CUR-DAPMA, CUR-SPD and CUR-SPM) were tested both on bacteria and keratinocytes. While free CUR exerted limited efficacy showing moderate cytotoxicity, a strong inhibition of bacterial growth was obtained using all three nanosystems without toxicity on eukaryotic cells. CUR-SPM emerged as the most effective, and was therefore employed in APDT experiments. Among the three sublethal blue laser (λ 445 nm) protocols tested, the ones characterized by a fluence of 18 and 30 J/cm 2 further decreased the antimicrobial concentration to 50 nM. The combination of blue laser APDT with CUR-SPM nanomicelles results in an effective synergistic activity that represents a promising novel therapeutic approach on resistant species., (© 2020 Wiley-VCH GmbH.)

Proline-rich antimicrobial peptides (PrAMPs) may be a valuable weapon against multi-drug resistant pathogens, combining potent antimicrobial activity with low cytotoxicity. We have identified novel PrAMPs from five cetacean species (cePrAMPs), and characterized their potency, mechanism of action and in vitro cytotoxicity. Despite the homology between the N-terminal of cePrAMPs and the bovine PrAMP Bac7, some differences emerged in their sequence, activity spectrum and mode of action. CePrAMPs with the highest similarity with the Bac7(1-35) fragment inhibited bacterial protein synthesis without membrane permeabilization, while a second subgroup of cePrAMPs was more membrane-active but less efficient at inhibiting bacterial translation. Such differences may be ascribable to differences in presence and positioning of Trp residues and of a conserved motif seemingly required for translation inhibition. Unlike Bac7(1-35), which requires the peptide transporter SbmA for its uptake, the activity of cePrAMPs was mostly independent of SbmA, regardless of their mechanism of action. Two peptides displayed a promisingly broad spectrum of activity, with minimal inhibiting concentration MIC ≤ 4 µM against several bacteria of the ESKAPE group, including Pseudomonas aeruginosa and Enterococcus faecium . Our approach has led us to discover several new peptides; correlating their sequences and mechanism of action will provide useful insights for designing optimized future peptide-based antibiotics.

Most Cystic Fibrosis (CF) patients succumb to airway inflammation and pulmonary infections due to Pseudomonas aeruginosa . D -BMAP18, a membrane-permeabilizing antimicrobial peptide composed of D-amino acids, was evaluated as a possible antibacterial aimed to address this issue. The antipseudomonal activity of D -BMAP18 was tested in a pathophysiological context. The peptide displayed activity against CF isolates of Pseudomonas aeruginosa in the presence of CF sputum when combined with sodium chloride and DNase I. In combination with DNase I, D -BMAP18 discouraged the deposition of new biofilm and eradicated preformed biofilms of some P. aeruginosa strains. In addition, D -BMAP18 down regulated the production of TNF-α, IL1-β, and TGF-β in LPS-stimulated or IFN-γ macrophages derived from THP-1 cells indicating an anti-inflammatory activity. The biocompatibility of D -BMAP18 was assessed using four different cell lines, showing that residual cell-specific cytotoxicity at bactericidal concentrations could be abolished by the presence of CF sputum. Overall, this study suggests that D -BMAP18 may be an interesting molecule as a starting point to develop a novel therapeutic agent to simultaneously contrast lung infections and inflammation in CF patients.

Proline-rich antimicrobial peptides (PrAMPs) are promising lead compounds for developing new antimicrobials; however, their narrow spectrum of action is limiting. PrAMPs kill bacteria binding to their ribosomes and inhibiting protein synthesis. In this study, 133 derivatives of the PrAMP Bac7(1-16) were synthesized to identify the crucial residues for ribosome inactivation and antimicrobial activity. Then, five new Bac7(1-16) derivatives were conceived and characterized by antibacterial and membrane permeabilization assays, X-ray crystallography, and molecular dynamics simulations. Some derivatives displayed broad spectrum activity, encompassing Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumanii , Pseudomonas aeruginosa , and Staphylococcus aureus . Two peptides out of five acquired a weak membrane-perturbing activity while maintaining the ability to inhibit protein synthesis. These derivatives became independent of the SbmA transporter, commonly used by native PrAMPs, suggesting that they obtained a novel route to enter bacterial cells. PrAMP-derived compounds could become new-generation antimicrobials to combat antibiotic-resistant pathogens.

Proline-rich antimicrobial peptides (PrAMPs) are promising agents to combat multi-drug resistant pathogens due to a high antimicrobial activity, yet low cytotoxicity. A library of derivatives of the PrAMP Bac5(1-17) was synthesized and screened to identify which residues are relevant for its activity. In this way, we discovered that two central motifs -PIRXP- cannot be modified, while residues at N- and C- termini tolerated some variations. We found five Bac5(1-17) derivatives bearing 1-5 substitutions, with an increased number of arginine and/or tryptophan residues, exhibiting improved antimicrobial activity and broader spectrum of activity while retaining low cytotoxicity toward eukaryotic cells. Transcription/translation and bacterial membrane permeabilization assays showed that these new derivatives still retained the ability to strongly inhibit bacterial protein synthesis, but also acquired permeabilizing activity to different degrees. These new Bac5(1-17) derivatives therefore show a dual mode of action which could hinder the selection of bacterial resistance against these molecules., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The spread of antibiotic-resistant pathogens has boosted the search for new antimicrobial drugs. Proline-rich antimicrobial peptides are promising lead compounds for the development of next-generation antibiotics, given their very low cytotoxicity and their good antimicrobial activity targeting the bacterial ribosome. Bac5(1-25) is an N-terminal fragment of the bovine proline-rich antimicrobial peptide Bac5, whose mode of action has been recently described. In this work we tested a number of Bac5(1-25) fragments, and we characterized their antimicrobial activity against Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa. We evaluated their cytotoxicity toward human cells and their efficacy in inhibiting bacterial protein synthesis. This allowed us to identify some shorter fragments of Bac5(1-25) with a good balance between antibacterial efficacy, protein synthesis inhibition, and ease/cost-effectiveness of synthesis, suitable as lead compounds to develop new antibacterials., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlike most antimicrobial peptides (AMPs), the main mode of action of the subclass of proline-rich antimicrobial peptides (PrAMPs) is not based on disruption of the bacterial membrane. Instead, PrAMPs exploit the inner membrane transporters SbmA and YjiL/MdtM to pass through the bacterial membrane and enter the cytosol of specific Gram-negative bacteria, where they exert an inhibitory effect on protein synthesis. Despite sharing a high proline and arginine content with other characterized PrAMPs, the PrAMP Bac5 has a low sequence identity with them. Here we investigated the mode of action of three N-terminal Bac5 fragments, Bac5(1-15), Bac5(1-25), and Bac5(1-31). We show that Bac5(1-25) and Bac5(1-31) retained excellent antimicrobial activity toward Escherichia coli and low toxicity toward eukaryotic cells, whereas Bac5(1-15) was inactive. Bac5(1-25) and Bac5(1-31) inhibited bacterial protein synthesis in vitro and in vivo Competition assays suggested that the binding site of Bac5 is within the ribosomal tunnel, where it prevents the transition from the initiation to the elongation phase of translation, as reported for other PrAMPs, such as the bovine PrAMP Bac7. Surprisingly, unlike Bac7, Bac5(1-25) exhibited species-specific inhibition, being an excellent inhibitor of protein synthesis on E. coli ribosomes but a poor inhibitor on Thermus thermophilus ribosomes. This indicates that while Bac5 most likely has an overlapping binding site with Bac7, the mode of interaction is distinct, suggesting that Bac5 fragments may be interesting alternative lead compounds for the development of new antimicrobial agents., (Copyright © 2018 American Society for Microbiology.)

Cathelicidins are an important family of antimicrobial peptide effectors of innate immunity in vertebrates. Two members of this group, CATH-1 and CATH-2, have been identified and characterized in teleosts (ray-finned fish). In this study, we investigated the expression of these genes in different tissues of rainbow trout challenged with 4 different inactivated pathogens. By using qPCR, we detected a strong induction of both cath-1 and cath-2 genes within 24 hours after intraperitoneal inoculation with Lactococcus garvieae, Yersinia ruckeri, Aeromonas salmonicida, or Flavobacterium psychrophilum cells. Up to 700-fold induction of cath-2 was observed in the spleen of animals challenged with Y. ruckeri. Moreover, we found differences in the intensity and timing of gene up-regulation in the analyzed tissues. The overall results highlight the importance of cathelicidins in the immune response mechanisms of salmonids., (Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.)

Proline-rich antimicrobial peptides (PrAMPs) internalize into susceptible bacteria using specific transporters and interfere with protein synthesis and folding. To date, mammalian PrAMPs have so far been identified only in artiodactyls. Since cetaceans are co-phyletic with artiodactyls, we mined the genome of the bottlenose dolphin Tursiops truncatus, leading to the identification of two PrAMPs, Tur1A and Tur1B. Tur1A, which is orthologous to the bovine PrAMP Bac7, is internalized into Escherichia coli, without damaging the membranes, using the inner membrane transporters SbmA and YjiL/MdM. Furthermore, like Bac7, Tur1A also inhibits bacterial protein synthesis by binding to the ribosome and blocking the transition from the initiation to the elongation phase. By contrast, Tur1B is a poor inhibitor of protein synthesis and may utilize another mechanism of action. An X-ray structure of Tur1A bound within the ribosomal exit tunnel provides a basis to develop these peptides as novel antimicrobial agents., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The application of high-throughput sequencing technologies to non-model organisms has brought new opportunities for the identification of bioactive peptides from genomes and transcriptomes. From this point of view, marine invertebrates represent a potentially rich, yet largely unexplored resource for de novo discovery due to their adaptation to diverse challenging habitats. Bioinformatics analyses of available genomic and transcriptomic data allowed us to identify myticalins, a novel family of antimicrobial peptides (AMPs) from the mussel Mytilus galloprovincialis , and a similar family of AMPs from Modiolus spp., named modiocalins. Their coding sequence encompasses two conserved N-terminal (signal peptide) and C-terminal (propeptide) regions and a hypervariable central cationic region corresponding to the mature peptide. Myticalins are taxonomically restricted to Mytiloida and they can be classified into four subfamilies. These AMPs are subject to considerable interindividual sequence variability and possibly to presence/absence variation. Functional assays performed on selected members of this family indicate a remarkable tissue-specific expression (in gills) and broad spectrum of activity against both Gram-positive and Gram-negative bacteria. Overall, we present the first linear AMPs ever described in marine mussels and confirm the great potential of bioinformatics tools for the de novo discovery of bioactive peptides in non-model organisms., Competing Interests: The authors declare no conflicts of interest.

Covering: up to 2017The innate immune system employs a broad array of antimicrobial peptides (AMPs) to attack invading microorganisms. While most AMPs act by permeabilizing the bacterial membrane, specific subclasses of AMPs have been identified that pass through membranes and inhibit bacterial growth by targeting fundamental intracellular processes. One such subclass is the proline-rich antimicrobial peptides (PrAMPs) that bind to the ribosome and interfere with the process of protein synthesis. A diverse range of PrAMPs have been identified in insects, such as bees, wasps and beetles, and crustaceans, such as crabs, as well as in mammals, such as cows, sheep, goats and pigs. Mechanistically, the best-characterized PrAMPs are the insect oncocins, such as Onc112, and bovine bactenecins, such as Bac7. Biochemical and structural studies have revealed that these PrAMPs bind within the ribosomal exit tunnel with a reverse orientation compared to a nascent polypeptide chain. The PrAMPs allow initiation but prevent the transition into the elongation phase of translation. Insight into the interactions of PrAMPs with their ribosomal target provides the opportunity to further develop these peptides as novel antimicrobial agents.

The spread of antibiotic resistant-pathogens is driving the search for new antimicrobial compounds. Pulmonary infections experienced by cystic fibrosis (CF) patients are a dramatic example of this health-care emergency. Antimicrobial peptides could answer the need for new antibiotics but translating them from basic research to the clinic is a challenge. We have previously evaluated the potential of the small membranolytic peptide BMAP-18 to treat CF-related infections, discovering that while this molecule had a good activity in vitro it was not active in vivo because of its rapid degradation by pulmonary proteases. In this study, we synthesized and tested the proteases-resistant all- D enantiomer. In spite of a good antimicrobial activity against Pseudomonas aeruginosa and Stenotrophomonas maltophilia clinical isolates and of a tolerable cytotoxicity in vitro, D -BMAP18 was ineffective to treat P. aeruginosa pulmonary infection in mice, in comparison to tobramycin. We observed that different factors other than peptide degradation hampered its efficacy for pulmonary application. These results indicate that D -BMAP18 needs further optimization before being suitable for clinical application and this approach may represent a guide for optimization of other anti-infective peptides eligible for the treatment of pulmonary infections.

A distinct group of antimicrobial peptides kills bacteria by interfering with internal cellular functions and without concurrent lytic effects on cell membranes. Here we describe some methods to investigate the mechanisms of action of these antimicrobial peptides. They include assays to detect the possible temporal separation between membrane permeabilization and bacterial killing events, to assess the capacity of antimicrobial peptides to cross the bacterial membranes and reside in the cytoplasm, and later to inhibit vital cell functions such as DNA transcription and protein translation.