Molecular cloning and chemical synthesis of a novel antibacterial peptide derived from pig myeloid cells

A group of myeloid precursors of defense peptides has recently been shown to have highly homologous N-terminal regions. Using a strategy based on this homology, a novel cDNA was cloned from pig bone marrow RNA and found to encode a 153-residue polypeptide. This comprises a highly conserved region encompassing a 29-residue signal peptide and a 101-residue prosequence, followed by a unique, 23-residue, cationic, C-terminal sequence. A peptide corresponding to this C-terminal sequence was chemically synthesized and shown to exert antimicrobial activity against both Gram positive and negative bacteria at concentrations of 2-16 microM. The activity of this potent and structurally novel antibacterial peptide appears to be mediated by its ability to damage bacterial membranes, as shown by the rapid permeabilization of the inner membrane of Escherichia coli

Arabica coffee extract shows antibacterial activity against Staphylococcus epidermidis and Enterococcus faecalis and low toxicity towards a human cell line

The antimicrobial activity of a regular and decaffeinated Arabica coffee extract was evaluated against three different Gram-positive bacteria and two Gram-negatives, including pathogenic Staphylococci strains. The antimicrobial activity was shown to be independent from caffeine content and was more pronounced against the Gram-positive strains. The regular coffee extract exhibited a significant bacteriostatic effect against Staphylococcus aureus and Staphylococcus epidermidis at short exposure times and became bactericidal after prolonged exposure. The potential cytotoxicity of the regular coffee extract was also evaluated towards breast adenocarcinoma MCF7 cells, showing to become significant only after 24h exposure and at a higher concentration than that producing the antibacterial effect. These results highlight the potential of coffee extracts as a naturally active and non-toxic antibacterial compound suitable for biomedical applications

The human cathelicidin LL-37 - A pore-forming antibacterial peptide and host-cell modulator

The human cathelicidin hCAP18/LL-37 has become a paradigm for the pleiotropic roles of peptides in host defence. It has a remarkably wide functional repertoire that includes direct antimicrobial activities against various types of microorganisms, the role of \u2018alarmin\u2019 that helps to orchestrate the immune response to infection, the capacity to locally modulate inflammation both enhancing it to aid in combating infection and limiting it to prevent damage to infected tissues, the promotion of angiogenesis and wound healing, and possibly also the elimination of abnormal cells. LL-37 manages to carry out all its reported activities with a small and simple, amphipathic, helical structure. In this review we consider how different aspects of its primary and secondary structures, as well as its marked tendency to form oligomers under physiological solution conditions and then bind to molecular surfaces as such, explain some of its cytotoxic and immunomodulatory effects. We consider its modes of interaction with bacterial membranes and capacity to act as a pore-forming toxin directed by our organism against bacterial cells, contrasting this with the mode of action of related peptides from other species. We also consider its different membrane-dependent effects on our own cells, which underlie many of its other activities in host defence

Effect of size and N-terminal residue characteristics on bacterial cell penetration and antibacterial activity of the proline-rich peptide Bac7

Bac7 is a proline-rich antimicrobial peptide, selective for Gram-negative bacteria, which acts intracellularly after membrane translocation. Progressively shortened fragments of Bac7 allowed determining the minimal sequence required for entry and antimicrobial activity as a 16-residue, N-terminal fragment, while further shortening led to a marked decrease in both functions. Furthermore, two N-terminal arginine residues were required for efficient translocation and activity. Analogues in which these residues were omitted, or where the side chain steric or physicochemical characteristics were systematically altered, were tested on different Escherichia coli strains, including a mutant with a destabilized outer membrane and one lacking the relevant SbmA membrane transport protein. H-bonding capacity, stereochemistry, and charge, in that order, played a determining role for efficient transit through both the outer and cytoplasmic membranes. Our studies allowed building a more detailed model for the mode-of-action of Bac7, and confirming its potential as an anti-infective agent, also suggesting it may be a vehicle for internalization of other antibiotic cargo

The mechanism of killing by the proline-rich peptide Bac7(1-35) against clinical strains of Pseudomonas aeruginosa differs from that against other gram-negative bacteria

Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1\u201335) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa. By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1\u201335) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli. The results evidenced a new mechanism of action for PRAMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process

Novel synthesis of 1,2-diaza-1,3-dienes with potential biological activity from cinnamic acids and diazonium salts of anilines

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

The proline-rich peptide Bac7(1-35) reduces mortality from Salmonella typhimurium in a mouse model of infection

<p>Abstract</p> <p>Background</p> <p>Bac7 is a proline-rich peptide with a potent <it>in vitro </it>antimicrobial activity against Gram-negative bacteria. Here we investigated its activity in biological fluids and <it>in vivo </it>using a mouse model of <it>S. typhimurium </it>infection.</p> <p>Results</p> <p>The efficacy of the active 1-35 fragment of Bac7 was assayed in serum and plasma, and its stability in biological fluids analyzed by Western blot and mass spectrometry. The ability of the peptide to protect mice against <it>Salmonella </it>was assayed in a typhoid fever model of infection by determination of survival rates and bacterial load in liver and spleen of infected animals. In addition, the peptide's biodistribution was evaluated by using time-domain optical imaging. Bac7(1-35) retained a substantial <it>in vivo </it>activity showing a very low toxicity. The peptide increased significantly the number of survivors and the mean survival times of treated mice reducing the bacterial load in their organs despite its rapid clearance.</p> <p>Conclusions</p> <p>Our results provide a first indication for a potential development of Bac7-based drugs in the treatment of salmonellosis and, eventually, other Gram-negative infections. The <it>in vivo </it>activity for this peptide might be substantially enhanced by decreasing its excretion rate or modifying the treatment schedule.</p

Bovine Neutrophil Antibiotic Peptides and Their Precursors: Structure and Role in Innate Immunity

Four peptides were characterized in extracts of bovine neutrophil granules: an Arg-rich dodecapeptide, maintained in a cyclic structure by a disulfide bridge; a Trp-rich tridecapeptide named indo- licidin; and two 43- and 59 amino acids long peptides, named Bac5 and Bac7, with frequent repeats of the triplets Arg-Pro-Pro and Pro-Arg-Pro, respectively. The full length cDNA of the first three of these peptides was characterized recently. Sequence analysis showed that the prosequences of the predicted precursors of all the three peptides are highly identical and exhibited also a remarkable similarity to cathelin, a porcine inhibitor of cathepsin L. Purified proBacö actually proved in in vitro assays to inhibit cathepsin L, but not other cysteine proteinases such as cathepsin B. Unlike proBacö, proBac7 is selectively chemotactic to monocytes. Several fragments of Bac5 and Bac7 (from 6 to 35 residues) were synthesized by the Fmoc method. The results of antibacterial assays show that the N-terminal portion, the most cationic one in both Bac5 and Bac7, is essential for the antimicrobial activity and that the minimal length necessary to arrest the growth of susceptible bacteria is 18-20 residues

Structure-activity relationships of the antimicrobial peptide arasin 1 - and mode of action studies of the N terminal, proline-rich region

Arasin 1 is a 37 amino acid long proline-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. In this work the active region of arasin 1 was identified through structure-activity studies using different peptide fragments derived from the arasin 1 sequence. The pharmacophore was found to be located in the proline/arginine-rich NH2 terminus of the peptide and the fragment arasin 1(1–23) was almost equally active to the full length peptide. Arasin 1 and its active fragment arasin 1(1–23) were shown to be non-toxic to human red blood cells and arasin 1(1–23) was able to bind chitin, a component of fungal cell walls and the crustacean shell. The mode of action of the fully active N-terminal arasin 1(1–23) was explored through killing kinetic and membrane permeabilization studies. At the minimal inhibitory concentration (MIC), arasin 1(1–23) was not bactericidal and had no membrane disruptive effect. In contrast, at concentrations of 5×MIC and above it was bactericidal and interfered with membrane integrity. We conclude that arasin 1(1–23) has a different mode of action than lytic peptides, like cecropin P1. Thus, we suggest a dual mode of action for arasin 1(1–23) involving membrane disruption at peptide concentrations above MIC, and an alternative mechanism of action, possibly involving intracellular targets, at MIC

Fragments of the nonlytic proline-rich antimicrobial peptide Bac5 kill Escherichia coli cells by inhibiting protein synthesis

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 speciesspecific 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