Anna Zanfardino

Anti-biofilm activity of an exopolysaccharide from a sponge-associated strain of Bacillus licheniformis

BackgroundSecondary metabolites ranging from furanone to exo-polysaccharides have been suggested to have anti-biofilm activity in various recent studies. Among these, Escherichia coli group II capsular polysaccharides were shown to inhibit biofilm formation of a wide range of organisms and more recently marine Vibrio sp. were found to secrete complex exopolysaccharides having the potential for broad-spectrum biofilm inhibition and disruption.ResultsIn this study we report that a newly identified ca. 1800 kDa polysaccharide having simple monomeric units of α-D-galactopyranosyl-(1→2)-glycerol-phosphate exerts an anti-biofilm activity against a number of both pathogenic and non-pathogenic strains without bactericidal effects. This polysaccharide was extracted from a Bacillus licheniformis strain associated with the marine organism Spongia officinalis. The mechanism of action of this compound is most likely independent from quorum sensing, as its structure is unrelated to any of the so far known quorum sensing molecules. In our experiments we also found that treatment of abiotic surfaces with our polysaccharide reduced the initial adhesion and biofilm development of strains such as Escherichia coli PHL628 and Pseudomonas fluorescens.ConclusionThe polysaccharide isolated from sponge-associated B. licheniformis has several features that provide a tool for better exploration of novel anti-biofilm compounds. Inhibiting biofilm formation of a wide range of bacteria without affecting their growth appears to represent a special feature of the polysaccharide described in this report. Further research on such surface-active compounds might help developing new classes of anti-biofilm molecules with broad spectrum activity and more in general will allow exploring of new functions for bacterial polysaccharides in the environment.

Ribonucleases with angiogenic and bactericidal activities from the Atlantic salmon

The importance of fish in vertebrate evolution has been better recognized in recent years after the intense work carried out on fish genomics. The recent discovery that fish genomes comprise homologs of ribonucleases, studied before only in tetrapods, and the isolation of ribonucleases from zebrafish have suggested an experimental model for studying fish and vertebrate evolution. Thus, the cDNAs encoding the RNases from the Atlantic salmon were expressed, and the recombinant RNases (Ss‐RNase‐1 and Ss‐RNase‐2) were isolated and characterized as both proteins and for their biological activities. Salmon RNases are less active than RNase A in degrading RNA, but are both sensitive to the action of the human cytosolic RNase inhibitor. The two enzymes possess both angiogenic and bactericidal activities. However, catalytically inactivated Ss‐RNases do not exert any angiogenic activity, but preserve their full bactericidal activity, which is surprisingly preserved even when the enzyme proteins are fully denatured. Analyses of the conformational stability of the two RNases has revealed that they are as stable as typical RNases of the superfamily, and Ss‐RNase‐2, the most active as an enzyme, is also the most resistant to thermal and chemical denaturation. The implications of these findings in terms of the evolution of early RNases, in particular of the physiological significance of the angiogenic and bactericidal activities of fish RNases, are analyzed and discussed.

A new cryptic cationic antimicrobial peptide from human apolipoprotein E with antibacterial activity and immunomodulatory effects on human cells

Cationic antimicrobial peptides (AMPs) possess fast and broad‐spectrum activity against both Gram‐negative and Gram‐positive bacteria, as well as fungi. It has become increasingly evident that many AMPs, including those that derive from fragments of host proteins, are multifunctional and able to mediate various immunomodulatory functions and angiogenesis. Among these, synthetic apolipoprotein‐derived peptides are safe and well tolerated in humans and have emerged as promising candidates in the treatment of various inflammatory conditions. Here, we report the characterization of a new AMP corresponding to residues 133–150 of human apolipoprotein E. Our results show that this peptide, produced either by chemical synthesis or by recombinant techniques in Escherichia coli, possesses a broad‐spectrum antibacterial activity. As shown for several other AMPs, ApoE (133–150) is structured in the presence of TFE and of membrane‐mimicking agents, like SDS, or bacterial surface lipopolysaccharide (LPS), and an anionic polysaccharide, alginate, which mimics anionic capsular exo‐polysaccharides of several pathogenic microorganisms. Noteworthy, ApoE (133–150) is not toxic toward several human cell lines and triggers a significant innate immune response, assessed either as decreased expression levels of proinflammatory cytokines in differentiated THP‐1 monocytic cells or by the induction of chemokines released from PBMCs. This novel bioactive AMP also showed a significant anti‐inflammatory effect on human keratinocytes, suggesting its potential use as a model for designing new immunomodulatory therapeutics.

Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods.

The bactericidal action on Escherichia coli of ZF‐RNase‐3 is triggered by the suicidal action of the bacterium OmpT protease

ZF‐RNase‐3 is one of the RNases from zebrafish (Danio rerio) with special (i.e. noncatalytic) properties. These include angiogenic and bactericidal activities. Given the interest of fish RNases as host‐defense effectors, we studied the mechanism of the bactericidal action of ZF‐RNase‐3 on Escherichia coli as a model Gram‐negative bacterium. The results obtained indicate that the bactericidal activity of ZF‐RNase‐3 is not lost when its catalytic RNase activity is obliterated. On the other hand, fully denatured ZF‐RNase‐3 conserves its bactericidal activity. When ZF‐RNase‐3 is added to E. coli cultures, it is cleaved at a specific Arg‐Arg peptide bond, thus engendering two peptide fragments. The larger fragment (residues 31–124), produced by proteolysis and reduction of a disulfide, is recognized as the actual bactericidal agent. The protease responsible for the proteolytic attack has been identified with OmpT, an outer membrane E. coli omptin protease. However, the most remarkable result obtained in the present study is the finding that the microbicidal action of ZF‐RNase‐3 can be achieved only with the suicidal cooperation of the bacterium itself.

Small azobenzene derivatives active against bacteria and fungi.

ATP synthase and protein kinase (PKs) are prime targets for drug discovery in a variety of diseases. It is well known that numerous stilbenes are capable to interact and inhibit ATP synthase and PKs. This work focuses on a series of azobenzene based molecules having high structural similarity with antimicrobial stilbenes. An investigation was carried out analyzing the potential toxicity of a large set of molecules by means of computational analysis. A small selection of potential low toxic molecules have been therefore synthesized, characterized and finally microbiologically tested. The synthesized compounds show potent bactericidal activity against Gram+ and a fungus, and are capable of inhibiting biofilm formation. Finally, the compounds demonstrated a thermal stability that makes them potential candidates for incorporation in polymer matrix for application as biomedical devices and food packaging.

Novel promising linezolid analogues: Rational design, synthesis and biological evaluation

A new series of 5-substituted oxazolidinones derived from linezolid, having urea and thiourea moieties at the C-5 side chain of the oxazolidinone ring, were prepared and their in vitro antibacterial activity was evaluated. The compound 10f demonstrated high antimicrobial activity, comparable to that of linezolid against Staphylococcus aureus.

Antimicrobial activity of eumelanin-based hybrids: The role of TiO2 in modulating the structure and biological performance

Eco-friendly hybrid Eumelanin-TiO2 nanostructures, recently obtained through in situ methodology based on hydrothermal route, have shown a striking antimicrobial activity, after exposure to oxidative environment, even under visible light induction condition. Nevertheless, the role of each component in defining the efficacy of these biological properties is far from being clearly defined. Furthermore, the effect of oxidative step on hybrids structure has not yet addressed. This study aims at elucidating the role of the ratio between eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and TiO2, for its polymerization in defining morphology and structural organization of TiO2-melanin nanostructures. Furthermore, tests on a Gram-negative Escherichia coli DH5α strain under UV irradiation and even visible light allowed to assess the contribution of each component, as well as of the TiO2-DHICA charge transfer complex to overall biological performance. Finally, results of biocide characterization were combined with spectroscopic evidences to prove that oxidative treatment induces a marked structural modification in melanin thus enhancing overall antimicrobial efficacy.

Titania as a driving agent for DHICA polymerization: a novel strategy for the design of bioinspired antimicrobial nanomaterials

Organic materials are widely employed to tune surface chemistry and/or as structuring agents of inorganic materials. Here, we propose a novel synthesis approach whereby TiO2 not only promotes 5,6-dihydroxyindole-2-carboxylic acid (DHICA) polymerization but also acts as a templating agent for the formation itself. Hybrid TiO2-DHICA melanin nanostructures have been produced, showing biocide activity even under visible light conditions (activation). Hybrid nanostructures have been analyzed and characterized by multiple techniques, proving that both organic and inorganic phases strongly affect each other during in situ formation, as far as it concerns both morphology and microstructure, conferring unique biocide properties to the resulting nanomaterials. This strategy ensures much more far-reaching implementation in the synthesis of hybrid nanosystems, opening new perspectives in the design of multifunctional materials.

The identification of a novel Sulfolobus islandicus CAMP-like peptide points to archaeal microorganisms as cell factories for the production of antimicrobial molecules

AbstractBackgroundPathogenic bacteria easily develop resistance to c onventional antibiotics so that even relatively new molecules are quickly losing efficacy. This strongly encourages the quest of new antimicrobials especially for the treatment of chronic infections. Cationic antimicrobial peptides (CAMPs) are small positively charged peptides with an amphipathic structure, active against Gram-positive and Gram-negative bacteria, fungi, as well as protozoa.ResultsA novel (CAMP)-like peptide (VLL-28) was identified in the primary structure of a transcription factor, Stf76, encoded by pSSVx, a hybrid plasmid–virus from the archaeon Sulfolobus islandicus. VLL-28 displays chemical, physical and functional properties typical of CAMPs. Indeed, it has a broad-spectrum antibacterial activity and acquires a defined structure in the presence of membrane mimetics. Furthermore, it exhibits selective leakage and fusogenic capability on vesicles with a lipid composition similar to that of bacterial membranes. VLL-28 localizes not only on the cell membrane but also in the cytoplasm of Escherichia coli and retains the ability to bind nucleic acids. These findings suggest that this CAMP-like peptide could exert its antimicrobial activity both on membrane and intra cellular targets.ConclusionsVLL-28 is the first CAMP-like peptide identified in the archaeal kingdom, thus pointing to archaeal microorganisms as cell factories to produce antimicrobial molecules of biotechnological interest. Furthermore, results from this work show that DNA/RNA-binding proteins could be used as sources of CAMPs.