Papers by Alessandro Tossi
Infection and Immunity, Jun 1, 2010
Human -defensin 3 (hBD3) is a highly charged (؉11) cationic host defense peptide, produced by ep... more Human -defensin 3 (hBD3) is a highly charged (؉11) cationic host defense peptide, produced by epithelial cells and neutrophils. hBD3 retains antimicrobial activity against a broad range of pathogens, including multiresistant Staphylococcus aureus, even under high-salt conditions. Whereas antimicrobial host defense peptides are assumed to act by permeabilizing cell membranes, the transcriptional response pattern of hBD3-treated staphylococcal cells resembled that of vancomycin-treated cells (V. Sass, U. Pag, A. Tossi, G. Bierbaum, and H. G. Sahl, Int. J. Med. Microbiol. 298:619-633, 2008) and suggested that inhibition of cell wall biosynthesis is a major component of the killing process. hBD3-treated cells, inspected by transmission electron microscopy, showed localized protrusions of cytoplasmic contents, and analysis of the intracellular pool of nucleotide-activated cell wall precursors demonstrated accumulation of the final soluble precursor, UDP-MurNAc-pentapeptide. Accumulation is typically induced by antibiotics that inhibit membrane-bound steps of cell wall biosynthesis and also demonstrates that hBD3 does not impair the biosynthetic capacity of cells and does not cause gross leakage of small cytoplasmic compounds. In in vitro assays of individual membrane-associated cell wall biosynthesis reactions (MraY, MurG, FemX, and penicillin-binding protein 2 [PBP2]), hBD3 inhibited those enzymes which use the bactoprenol-bound cell wall building block lipid II as a substrate; quantitative analysis suggested that hBD3 may stoichiometrically bind to lipid II. We report that binding of hBD3 to defined, lipid II-rich sites of cell wall biosynthesis may lead to perturbation of the biosynthesis machinery, resulting in localized lesions in the cell wall as demonstrated by electron microscopy. The lesions may then allow for osmotic rupture of cells when defensins are tested under low-salt conditions.
Current Protein & Peptide Science, May 1, 2010
To defend themselves from attack by pathogens, plants can rely only on their innate defense syste... more To defend themselves from attack by pathogens, plants can rely only on their innate defense systems. Defensins are antimicrobial peptides that contribute to plant immunity by displaying a direct cidal activity against various pathogens, some of which are responsible for plant diseases. These determine a significant decrease in the quality and safety of agricultural products, especially among food crops, and cause significant economic losses. There is consequently an increasing interest for antimicrobial compounds such as the defensins, which might be applied in different ways to protect important food or bio-fuel crops. In this review we analyse the techniques that have been reported in the literature for the production of isolated plant defensins of adequate quality and sufficient quantity for potential use in plant protection. For research purposes, defensins have been heterogously expressed in diverse hosts, such as bacteria, yeasts, fungi and plants. Chemical synthesis is instead not commonly used for their production, due to structural characteristics that makes it difficult to obtain the correct protein folding. To consider the possibility of producing plant defensins in a large scale, cost-effective methods guaranteeing high quality product are required. Future studies may thus focus on the development of more stable compounds, as well as decreasing production costs by improving preparative syntheses or biotechnological procedures such as using transgenic crops as plant factories.
CABI eBooks, 2017
Cathelicidins are a ubiquitous family of host defence peptides (HDPs) in vertebrate animals. Unli... more Cathelicidins are a ubiquitous family of host defence peptides (HDPs) in vertebrate animals. Unlike other HDP families, they are defined by the common and relatively well conserved proregion rather than the mature active peptides, which are highly diverse and conform to at least five different structural groups. They seem to have fol-lowed a rather distinctive evolutionary path in their development. Cathelicidin-derived peptides play a relevant role in defending the host against microbial infection, by displaying both a broad-spectrum, direct antimicrobial activity and the capacity to modulate other host responses to infection and injury. Both types of effect depend on the structural type, which in turn affects the particular mode of action of each peptide. This chapter begins by briefly describing the discovery of cathelicidins before dis-cussing their molecular diversity and con-sidering their evolution. It then considers their expression and processing, the struc-ture-dependence of the distinct modes of action shown by different members, and briefly touches on th
Pharmaceutics, May 8, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Small, Apr 1, 2019
Nanomaterials with size scales of few tens of nanometers are ideal platforms to link physicochemi... more Nanomaterials with size scales of few tens of nanometers are ideal platforms to link physicochemical behavior at the nanoscale with targeted biological functions, where each Gold nanoparticles (AuNPs) covered with mixtures of immiscible ligands present potentially anisotropic surfaces that can modulate their interactions at complex nano-bio interfaces. Mixed, self-assembled, monolayer (SAM)protected AuNPs, prepared with incompatible hydrocarbon and fluorocarbon amphiphilic ligands, are used here to probe the molecular basis of surface phase separation and disclose the role of fluorinated ligands on the interaction with lipid model membranes and cells, by integrating in silico and experimental approaches. These results indicate that the presence of fluorinated amphiphilic ligands enhances the membrane binding ability and cellular uptake of gold nanoparticles with respect to those coated only with hydrogenated amphiphilic ligands. For mixed monolayers, computational results suggest that ligand phase separation occurs on the gold surface, and the resulting anisotropy affects the number of contacts and adhesion energies with a membrane bilayer. This reflects in a diverse membrane interaction for NPs with different surface morphologies, as determined by surface plasmon resonance, as well as differential effects on cells, as observed by flow cytometry and confocal microscopy. Overall, limited changes in monolayer features can significantly affect NP surface interfacial properties, which, in turn, affect the interaction of SAM-AuNPs with cellular membranes and subsequent effects on cells.
Biochemical Journal, Jan 22, 2015
The human cathelicidin peptide LL-37 is an important effector of our innate immune system and con... more The human cathelicidin peptide LL-37 is an important effector of our innate immune system and contributes to host defence with direct antimicrobial activity, immunomodulatory properties and by stimulating wound healing. Its sequence has evolved to confer specific structural characteristics that strongly affect these biological activities, and that differentiates it from orthologues of other primate species. Here we report a detailed study of the folding and self-assembly of this peptide in comparison to 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. Phe 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. FTIR 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.
Biochemical Journal, Aug 9, 2005
A novel method, based on the rational and systematic modulation of macroscopic structural charact... more A novel method, based on the rational and systematic modulation of macroscopic structural characteristics on a template originating from a large number of natural, cell-lytic, amphipathic α-helical peptides, was used to probe how the depths and shapes of hydrophobic and polar faces and the conformational stability affect antimicrobial activity and selectivity with respect to eukaryotic cells. A plausible mode of action explaining the peptides' behaviour in model membranes, bacteria and host cells is proposed. Cytotoxic activity, in general, correlated strongly with the hydrophobic sector depth, and required a majority of aliphatic residue side chains having more than two carbon atoms. It also correlated significantly with the size of polar sector residues, which determines the penetration depth of the peptide via the so-called snorkel effect. Both an oblique gradient of long to short aliphatic residues along the hydrophobic face and a stabilized helical structure increased activity against host cells but not against bacteria, as revealed by haemolysis, flow cytofluorimetric studies on lymphocytes and surface plasmon resonance studies with model phosphatidylcholine/cholesterol membranes. The mode of interaction changes radically for a peptide with a stable, preformed helical conformation compared with others that form a structure only on membrane binding. The close correlation between effects observed in biological and model systems suggests that the 'carpet model' correctly represents the type of peptides that are bacteriaselective, whereas the behaviour of those that lyse host cells is more complex.
Minerva Biotecnologica, 1996
Inhibitors of HIV-1 aspartic protease, an enzyme which is essential for viral processing and matu... more Inhibitors of HIV-1 aspartic protease, an enzyme which is essential for viral processing and maturation, represent an important new type of anti-AIDS drug. To be effective, these must be potent and selec tive, must present a suitable pharmacological profile with respect to drug uptake and clearance, and should also be synthesizeable in good yields. We have developed a design method based on computer aided modification of a reference compound for which the crystal structure of the complex with the enzyme is known. After relaxation of the new structures to an optimized geometry, the complexation energy is calculated, relative to that of the reference inhibitor, taking into account all aspects of the interaction, including solvation. This affords a numerical prediction of the putative effectiveness of the new structure as an inhibitor, relative to that of the reference structure, and thus allows us to rapidly evaluate modifications that could result in increased potency and reduce the number of compounds that it is actually necessary to synthesize so as to obtain useful lead compounds for drug development. In an initial study, we have determined the role of flanking residues in modulating inhibition, for hexapeptide mimetics with a central, reduced amide non-cleavable bond. The structure was based on that of the reference inhibitor MVT-101. We have found that by tuning the residues flanking the central bond, the complexation energy could be markedly improved, and have determined that the putatively optimal structure should contain an aromatic residue (Phe or Tyr) at positions p1 and p1', immediately flanking the central bond, a hydrophobic residue at P2, a glutamic acid residue at P2', and an aromatic residue (Phe, Tyr or Trp) at positions P3 and P3', most distant from the central bond. Synthesis of a series of inhibitors containing these modifications was carried out entirely in the solid phase, using Fmoc type chemistry, on a polyethylene glycol/polystyrene resin, and in vitro enzyme inhibition assays have confirmed the computer-based predictions. In particular, it was possible to obtain several inhibitors with potency in the low nanomolar range, an improvement of several orders of magnitude with respect to the parent compound
Letter to the Editor -Defensin 2 in the Rhesus Monkey (Macaca mulatta) and the Long-Tailed Macaq... more Letter to the Editor -Defensin 2 in the Rhesus Monkey (Macaca mulatta) and the Long-Tailed Macaque (M. fascicularis) Antimicrobial peptides are important components of natural immunity and have been described for and isolated from plants, insects, and mammals (3). They have been classified in several different families on the basis of their structural features, antimicrobial properties, and expression patterns (2). In mammals, defensins and cathelicidins can be considered the most important antimicrobial peptides, whose main but not only function is to provide a first line of defense against bacterial, fungal, and viral infections, both at epithelial surfaces and in phagocytic cells (4, 5). Animal models have been used to understand different aspects of innate immune responses in several human diseases (7). Recently, Bals et al. (1) suggested that the rhesus monkey (Macaca mulatta) could be a useful model to study innate immunity in human pathologies. In fact they have found a high homology between -defensins 1 and 2 and the cathelicidin LL-37/hCAP-18 in Homo sapiens and M. mulatta, which suggests that innate immune responses in nonhuman primates are very similar to those in humans. We have sequenced the two exons of -defensin 2 homologous in two different species of the genus Macaca: M. mulatta and M. fascicularis. Genomic DNAs were extracted from hairs by using the Chelex-100 method (8). Primers for PCR (5Ј-GA CCTTTATAAGGTGGAAGGCTT-3Ј [forward] and 5Ј-CTA CGCCATTCTTCCATTTGG-3Ј [reverse] for exon 1 and 5Ј-T GAGTTTTGAGTTCTTACACGCT-3Ј [forward] and 5Ј-GG AGAGCAGAAAAAGGTGTTT-3Ј [reverse] for exon 2) were designed on the basis of the published human -defensin 2 sequence (EMBL accession number AF071216). DNA sequencing of both DNA strands was performed using the Big-Dye Terminator Reaction kit (Applera). Ten wild-caught animals of each species were analyzed. No intraspecific variability in nucleotide sequences was observed. M. mulatta and M. fascicularis peptides showed the same nucleotide sequences for the coding region of -defensin 2; conversely, we observed a 90% homology between Macaca (both M. mulatta and M. fascicularis) and the H. sapiens coding regions (19 of 195 sites were found to be variable). This results in 14 (22%) of 65 amino acids being different in the Macaca and human peptides. Most of the amino acid replacements were localized in the active region of the protein (Fig. 1). Our results and the findings of Bals et al. (1) could be explained by hypothesizing that (i) M. mulatta and M. fascicularis have selected more -defensins than humans or (ii) other, unknown -defensins not yet isolated exist in humans. The first
Developmental and Comparative Immunology, Apr 1, 2020
This study reports the identification of four novel proline-rich antimicrobial peptides (PR-AMP) ... more This study reports the identification of four novel proline-rich antimicrobial peptides (PR-AMP) from the transcriptome of the red swamp crayfish Procambarus clarkii. The newly identified putative peptides (PcAst-1b,-1c, −2 and −3), which are related with the previously identified hemocyte-specific PR-AMP astacidin-1, are encoded by the multi-genic astacidin gene family. The screening of available and proprietary transcriptomes allowed to define the taxonomical range of distribution of this gene family to Astacoidea and Parastacoidea. The antimicrobial properties of three synthetic PcAst peptides (PcAst-1a,-1b/c and −2), were characterized against reference bacteria or multidrug resistant clinical isolates, and their cytotoxicity was evaluated towards human transformed cell lines. The antimicrobial activity ranged from potent and broad-spectrum, in low-salt medium, to poor, whereas it was generally low in full nutrient broth. No significant toxic effects were observed on cultured human cells. RNAseq data from 12 different tissues indicated a strong specificity for haemocytes under naïve physiological condition, with moderate expression (5-fold lower) in gills. Quantitative real time PCR revealed a rapid (within 2 h) and significant up-regulation of PcAst-1a (Astacidin 1) and PcAst-2 expression in response to LPS injection. Due to the variation in antimicrobial potency and inducibility, the roles of the other astacidins (PcAst-1b,-1c and −3) need to be further investigated to determine their significance to the immune responses of the red swamp crayfish.
International Journal of Molecular Sciences, Jun 6, 2018
Biofilms are complex systems produced by bacteria and constituted by macromolecular matrix embedd... more Biofilms are complex systems produced by bacteria and constituted by macromolecular matrix embedding cells. They provide advantages to bacteria including protection against antimicrobials. The protection given by biofilms produced by Klebsiella pneumoniae strains towards antimicrobial peptides of the innate immune system was investigated. In particular, the role of matrix bacterial exopolysaccharides was explored. Three clinical strains producing exopolysaccharides with different chemistry were selected and the interaction of purified biofilm polysaccharides with two bovine cathelicidins was studied by circular dichroism spectroscopy and microbiological assays to establish their influence on the peptide's antimicrobial activity. The spectroscopic data indicated a different extent of interaction with the two peptides, in a manner dependent on their sugar composition, and in particular the presence of rhamnose residues correlated with a lower interaction. The extent of interaction was then related to the protection towards antimicrobial peptides, conferred by the addition of the different exopolysaccharides, in minimum inhibitory concentration (MIC) assays against a reference Escherichia coli strain. Microbiological results were in very good agreement with spectroscopic data, confirming the active role of matrix polysaccharides in determining a biofilm's protective capacity and indicating lower protection levels afforded by rhamnose containing exopolysaccharides.
Cell chemical biology, May 1, 2018
Proprietà letteraria riservata. I diritti di traduzione, memorizzazione elettronica, di riproduzi... more Proprietà letteraria riservata. I diritti di traduzione, memorizzazione elettronica, di riproduzione e di adattamento totale e parziale di questapubblicazione, con qualsiasi mezzo (compresi i microfilm, le fotocopie e altro) sono riservati per tutti i paesi. Program Thursday, 25 August 12.00-14.00 Registration and light lunch at the Congress Venue 14.00-14.30 Welcome addresses Session 1 Molecular and Cell Biophysics Chairs: László Mátyus, Renato Gennaro 14.30-15.00 PL1 Kovačić Damir (University of Split, HR) A new auditory neuro-electronic interface: spiral ganglion neurons cultured on advanced micro-electrode array 15.00-15.25 IL1.1 Panyi György (University of Debrecen, H) Shaker-IR K + channel gating in heavy water: role of structural water molecules in inactivation 15.25-15.50 IL1.2 Zahradníková Alexandra (Slovak Academy of Sciences, SK) Calcium wave generation in cardiac myocytes 15.50-16.05 SO1.1 Mesarec Luka (University of Ljubljana, SLO) On the role of external force of actin filaments in the formation of tubular protrusions of closed membrane shapes with anisotropic membrane components 16.05-16.40 Coffee Break 16.40-16.55 SO1.4 Frecer Vladimir (Comenius University in Bratislava & ICARST, SK) Computational Design of Histone Deacetylase Inhibitors as Antitumor Agents 16.55-17.10 SO1.5 Božič Bojan (University of Ljubljana, SLO) The cellular effects induced by the pore-forming agent nystatin 19.00-23.30 Congress Dinner Friday, 26 August Session 2 Supramolecular Assemblies and Aggregation Chairs: Valeria Militello, Srđan Antić 09.00-09.30 PL2 Hianik Tibor (Comenius University Bratislava, SK) DNA-aptamers structure, physical properties and applications 09.30-09.55 IL2.1 Librizzi Fabio (CNR-Istitute of Biophysics, I) Amyloid formation and its inhibition by chaperones and chaperon-like molecules: the effect on different nucleation mechanisms 09.55-10.20 IL2.2 Sedlák Erik (Safarik University, SK) Role of cardiolipin for stability of cytochrome c oxidase
Nature Precedings, Apr 17, 2009
New antibiotics against multidrug-resistant bacteria are urgently needed, but rapid acquisition o... more New antibiotics against multidrug-resistant bacteria are urgently needed, but rapid acquisition of resistance limits their usefulness. Endogenous antimicrobial peptides (AMPs) with moderate selectivity, but multimodal mechanism of action, have remained effective against bacteria for millions of years. Their therapeutic application, however, requires optimizing the balance between antibacterial activity and selectivity, so that rational design methods for increasing selectivity are highly desirable. We have created training (n=36) and testing (n=37) sets from frog-derived AMPs with determined therapeutic index (TI). The 'sequence moments' concept then enabled us to find a oneparameter linear model resulting in a good correlation between measured and predicted TI (r 2 =0.83 and 0.64 for each set, respectively). The concept was then used in the AMP-Designer algorithm to propose primary structures for highly selective AMPs against Gram-negative bacteria. Testing the activity of one such peptide produced a TI>200 as compared to the best AMP in the database , with TI=125.
Frontiers in Microbiology, Dec 17, 2021
of AgNPs may have effects beyond just modulation of direct antibacterial activity. In addition, s... more of AgNPs may have effects beyond just modulation of direct antibacterial activity. In addition, some conjugated nanoparticles are selectively toxic to tumor cells. However, caution is required as not all modulatory effects are necessarily beneficial to normal host cells.
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Papers by Alessandro Tossi