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The urgent need for antibiotic alternatives has driven the search for antimicrobial peptides (AMPs) from many different sources, yet parasite-derived AMPs remain underexplored. In this study, three novel potential AMP precursors (mesco-1, -2 and -3) were identified in the parasitic flatworm Mesocestoides corti, via a genome-wide mining approach, and the most promising one, mesco-2, was synthesized and comprehensively characterized. It showed potent broad-spectrum antibacterial activity at submicromolar range against E. coli and K. pneumoniae and low micromolar activity against A. baumannii, P. aeruginosa and S. aureus. Mechanistic studies indicated a membrane-related mechanism of action, and circular dichroism spectroscopy confirmed that mesco-2 is unstructured in water but forms stable helical structures on contact with anionic model membranes, indicating strong interactions and helix stacking. It is, however, unaffected by neutral membranes, suggesting selective antimicrobial activity. Structure prediction combined with molecular dynamics simulations suggested that mesco-2 adopts an unusual bent helix conformation with the N-terminal sequence, when bound to anionic membranes, driven by a central GRGIGRG motif. This study highlights mesco-2 as a promising antibacterial agent and emphasizes the importance of structural motifs in modulating AMP function. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Treatment of cystic fibrosis-associated lung infections is hampered by the presence of multi-drug resistant pathogens, many of which are also strong biofilm producers. Antimicrobial peptides, essential components of innate immunity in humans and animals, exhibit relevant in vitro antimicrobial activity although they tend not to select for resistant strains. Results Three α-helical antimicrobial peptides, BMAP-27 and BMAP-28 of bovine origin, and the artificial P19(9/B) peptide were tested, comparatively to Tobramycin, for their in vitro antibacterial and anti-biofilm activity against 15 Staphylococcus aureus, 25 Pseudomonas aeruginosa, and 27 Stenotrophomonas maltophilia strains from cystic fibrosis patients. All assays were carried out in physical-chemical experimental conditions simulating a cystic fibrosis lung. All peptides showed a potent and rapid bactericidal activity against most P. aeruginosa, S. maltophilia and S. aureus strains tested, at levels generally higher than those exhibited by Tobramycin and significantly reduced biofilm formation of all the bacterial species tested, although less effectively than Tobramycin did. On the contrary, the viability-reducing activity of antimicrobial peptides against preformed P. aeruginosa biofilms was comparable to and, in some cases, higher than that showed by Tobramycin. Conclusions The activity shown by α-helical peptides against planktonic and biofilm cells makes them promising “lead compounds” for future development of novel drugs for therapeutic treatment of cystic fibrosis lung disease.

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/cm2 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Covering: up to 2017 The 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]