New aspects of the structure and mode of action of the human cathelicidin LL-37 revealed by the intrinsic probe p-cyanophenylalanine

The human cathelicidin peptide LL-37 is an important effector of our innate immune system and contributes to host defence with direct antimicrobial activity and immunomodulatory properties, and by stimulating wound healing. Its sequence has evolved to confer specific structural characteristics that strongly affect these biological activities, and differentiate it from orthologues of other primate species. In the present paper we report a detailed study of the folding and self-assembly of this peptide in comparison with rhesus monkey peptide RL-37, taking into account the different stages of its trajectory from bulk solution to contact with, and insertion into, biological membranes. Phenylalanine residues in different positions throughout the native sequences of LL-37 and RL-37 were systematically replaced with the non-invasive fluorescent and IR probe p-cyanophenylalanine. Steady-state and time-resolved fluorescence studies showed that LL-37, in contrast to RL-37, forms oligomers with a loose hydrophobic core in physiological solutions, which persist in the presence of biological membranes. Fourier transform IR and surface plasmon resonance studies also indicated different modes of interaction for LL-37 and RL-37 with anionic and neutral membranes. This correlated with a distinctly different mode of bacterial membrane permeabilization, as determined using a flow cytometric method involving impermeant fluorescent dyes linked to polymers of defined sizes

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

apoptotic pathways of u937 leukemic monocytes investigated by infrared microspectroscopy and flow cytometry

Infrared microspectroscopy and flow cytometry were used to study apoptosis in starved and CCCP-treated U937 monocyte cells

Gold nanoparticles with\ua0patterned surface monolayers for\ua0nanomedicine: current perspectives

Molecular self-assembly is a topic attracting intense scientific interest. Various strategies have been developed for construction of molecular aggregates with rationally designed properties, geometries, and dimensions that promise to provide solutions to both theoretical and practical problems in areassuch as drug delivery, medical diagnostics, and biosensors, to name but a few. In this respect, gold nanoparticles covered with self-assembled monolayers presenting nanoscale surface patterns\u2014typically patched, striped or Janus-like domains\u2014represent an emerging field. These systems are particularly intriguing for use in bio-nanotechnology applications, as presence of such monolayers with three-dimensional (3D) morphology providesnanoparticles with surface-dependent properties that, in turn, affect their biological behavior. Comprehensive understanding of the physicochemical interactions occurring at the interface between these versatile nanomaterials and biological systems is therefore crucial to fully exploit their potential. This review aims to explore the current state of development of such patterned, self-assembled monolayer-protected gold nanoparticles, through step-by-step analysis of their conceptual design, synthetic procedures, predicted and determined surface characteristics, interactions with and performance in biological environments, and experimental and computational methods currently employed for their investigation

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

D-BMAP18 antimicrobial peptide is active In Vitro, resists to pulmonary proteases but loses its activity in a murine model of Pseudomonas aeruginosa lung infection

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

Rapid and Reliable Detection of Antimicrobial Peptide Penetration into Gram-Negative Bacteria Based on Fluorescence Quenching▿

In this paper, we describe a rapid flow cytometry method to identify antimicrobial peptides that are internalized into bacterial cells and differentiate them from those that are membrane active. The method was applied to fluorescently labeled Bac71-35 and polymyxin B, whose mechanisms of action are, respectively, based on cell penetration and on membrane binding and permeabilization. Identification of peptides with the former mechanism is of considerable interest for the intracellular delivery of membrane-impermeant drugs

Effects on APC antigen presenting cells of short-term interaction with the human human host defense peptide beta-defensin 2

International audiencebeta-Defensins are antimicrobial peptides that exert their host-defense functions at the interface between the host and microbial biota. They display a direct, salt and medium sensitive cidal activity, in vitro, against a broad spectrum of bacteria and fungi and there is increasing evidence that they also play a role in alerting and enhancing cellular components of innate and adaptive immunity. Their interaction with biological membranes plays a central role in both these types of activities. In this study we have investigated the interaction of fluorescently labelled human beta defensin 2 (hBD2) with monocytes, macrophages and immature dendritic cells, observing a differential capacity to be rapidly internalised into these cells. Complementary microscopy techniques (TEM, optical and infrared) were used to explore the functional and biological implications of these interactions on immature dendritic cells. Short-term exposure to the peptide resulted in significant alterations in membrane composition and re-organization of the endomembrane system, with induction of degranulation. These events may be associated with the antigen-presenting activities or the chemotaxis of iDC, which appears to occur via both CCR6-dependent and independent mechanisms

Elastase-Activated Antimicrobial Peptide for a Safer Pulmonary Treatment of Cystic Fibrosis Infections

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