Simultaneous Irradiation with UV-A, -B, and -C Lights Promotes Effective Decontamination of Planktonic and Sessile Bacteria: A Pilot Study.

Surfaces in highly anthropized environments are frequently contaminated by both harmless and pathogenic bacteria. Accidental contact between these contaminated surfaces and people could contribute to uncontrolled or even dangerous microbial diffusion. Among all possible solutions useful to achieve effective disinfection, ultraviolet irradiations (UV) emerge as one of the most "Green" technologies since they can inactivate microorganisms via the formation of DNA/RNA dimers, avoiding the environmental pollution associated with the use of chemical sanitizers. To date, mainly UV-C irradiation has been used for decontamination purposes, but in this study, we investigated the cytotoxic potential on contaminated surfaces of combined UV radiations spanning the UV-A, UV-B, and UV-C spectrums, obtained with an innovative UV lamp never conceived so far by analyzing its effect on a large panel of collection and environmental strains, further examining any possible adverse effects on eukaryotic cells. We found that this novel device shows a significant efficacy on different planktonic and sessile bacteria, and, in addition, it is compatible with eukaryotic skin cells for short exposure times. The collected data strongly suggest this new lamp as a useful device for fast and routine decontamination of different environments to ensure appropriate sterilization procedures.

Bioconversion of 4-hydroxyestradiol by extradiol ring-cleavage dioxygenases from Novosphingobium sp. PP1Y.

Livestock breeding activities and pharmaceutical wastes lead to considerable accumulation of steroid hormones and estrogens in wastewaters. Here estrogens act as pro-cancerogenic agents and endocrine disruptors interfering with the sexual development of aquatic animals and having toxic effects in humans. Environmental bacteria play a vital role in estrogens degradation. Their wide reservoir of enzymes, such as ring cleavage dioxygenases (RCDs), can degrade the steroid nucleus, catalyzing the meta-cleavage of A, B or D steroid rings. In this work, 4 extra-diol ring cleavage dioxygenases (ERCDs), PP28735, PP26077, PP00124 and PP00193, were isolated from the marine sphingomonad Novosphingobium sp. PP1Y and characterized. Enzymes kinetic parameters were determined on different synthetic catecholic substrates. Then, the bioconversion of catechol estrogens was evaluated. PP00124 showed to be an efficient catalyst for the degradation of 4-hydroxyestradiol (4-OHE2), a carcinogenic hydroxylated derivate of E2. 4-OHE2 complete cleavage was obtained using PP00124 both in soluble form and in whole recombinant E. coli cells. LC-MS/MS analyses confirmed the generation of a semialdehyde product, through A-ring meta cleavage. To the best of our knowledge, PP00124 is the first characterized enzyme able to directly degrade 4-OHE2 via meta cleavage. Moreover, the complete 4-OHE2 biodegradation using recombinant whole cells highlighted advantages for bioremediation purposes.

Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases.

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.

Environment-Sensitive Fluorescent Labelling of Peptides by Luciferin Analogues.

Environment-sensitive fluorophores are very valuable tools in the study of molecular and cellular processes. When used to label proteins and peptides, they allow for the monitoring of even small variations in the local microenvironment, thus acting as reporters of conformational variations and binding events. Luciferin and aminoluciferin, well known substrates of firefly luciferase, are environment-sensitive fluorophores with unusual and still-unexploited properties. Both fluorophores show strong solvatochromism. Moreover, luciferin fluorescence is influenced by pH and water abundance. These features allow to detect local variations of pH, solvent polarity and local water concentration, even when they occur simultaneously, by analyzing excitation and emission spectra. Here, we describe the characterization of (amino)luciferin-labeled derivatives of four bioactive peptides: the antimicrobial peptides GKY20 and ApoBL, the antitumor peptide p53pAnt and the integrin-binding peptide RGD. The two probes allowed for the study of the interaction of the peptides with model membranes, SDS micelles, lipopolysaccharide micelles and Escherichia coli cells. Kd values and binding stoichiometries for lipopolysaccharide were also determined. Aminoluciferin also proved to be very well-suited to confocal laser scanning microscopy. Overall, the characterization of the labeled peptides demonstrates that luciferin and aminoluciferin are previously neglected environment-sensitive labels with widespread potential applications in the study of proteins and peptides.

Cost-effective production of recombinant peptides in Escherichia coli.

Among bioactive peptides, cationic antimicrobial peptides (AMPs), also referred to as host defence peptides (HDPs), are valuable tools to treat infections, being able to kill a wide variety of microbes directly and/or modulate host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites. However, high manufacturing costs have greatly limited their development as drugs, thus highlighting the need to develop novel and competitive production strategies. Here, a cost-effective procedure was established to produce the high amounts of peptides required for basic and clinical research. Firstly, a novel culture medium was designed, which was found to support significantly higher cell densities and recombinant expression levels of peptides under test compared to conventional media. The procedure has been also efficiently scaled up by using a 5 L fermenter, while the costs have been lowered significantly by developing a successful auto-induction strategy, which has been found to support higher yields of target constructs and cell biomass compared to conventional strategies based on expression induction by IPTG. Interestingly, it was estimated that by increasing production scale from 100 to 1000 mg/batch, unit costs decreased strongly from 253 to 42 €/mg. These costs appear highly competitive when compared to chemical synthesis strategies. Altogether, the data indicate that the strategy represents an important starting point for the future development of large-scale manufacture of HDPs.

Structural and functional insights into RHA-P, a bacterial GH106 α-L-rhamnosidase from Novosphingobium sp. PP1Y.

α-L-Rhamnosidases (α-RHAs, EC 3.2.1.40) are glycosyl hydrolases (GHs) hydrolyzing terminal α-l-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rhamnose from natural flavonoids has boosted the use of these enzymes in several biotechnological applications over the past decades, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a rhamnosidase of the GH106 family has been described. In our previous work, an α-l-rhamnosidase belonging to this family, named RHA-P, was isolated from the marine microorganism Novosphingobium sp. PP1Y. The initial biochemical characterization highlighted the biotechnological potential of RHA-P for bioconversion applications. In this work, further functional and structural characterization of the enzyme is provided. The recombinant protein was obtained fused to a C-terminal His-tag and, starting from the periplasmic fractions of induced recombinant cells of E. coli strain BL21(DE3), was purified through a single step purification protocol. Homology modeling of RHA-P in combination with a site directed mutagenesis analysis confirmed the function of residues D503, E506, E644, likely located at the catalytic site of RHA-P. In addition, a kinetic characterization of the enzyme on natural flavonoids such as naringin, rutin, hesperidin and quercitrin was performed. RHA-P showed activity on all flavonoids tested, with a catalytic efficiency comparable or even higher than other bacterial α-RHAs described in literature. The results confirm that RHA-P is able to hydrolyze both α-1,2 and α-1,6 glycosidic linkages, and suggest that the enzyme may locate different polyphenolic aromatic moities in the active site.

Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue.

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoBL, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.

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.

Methylobacterium populi VP2: plant growth-promoting bacterium isolated from a highly polluted environment for polycyclic aromatic hydrocarbon (PAH) biodegradation.

The use of microorganisms to accelerate the natural detoxification processes of toxic substances in the soil represents an alternative ecofriendly and low-cost method of environmental remediation compared to harmful incineration and chemical treatments. Fourteen strains able to grow on minimal selective medium with a complex mixture of different classes of xenobiotic compounds as the sole carbon source were isolated from the soil of the ex-industrial site ACNA (Aziende Chimiche Nazionali Associate) in Cengio (Savona, Italy). The best putative degrading isolate, Methylobacterium populi VP2, was identified using a polyphasic approach on the basis of its phenotypic, biochemical, and molecular characterisation. Moreover, this strain also showed multiple plant growth promotion activities: it was able to produce indole-3-acetic acid (IAA) and siderophores, solubilise phosphate, and produce a biofilm in the presence of phenanthrene and alleviate phenanthrene stress in tomato seeds. This is the first report on the simultaneous occurrence of the PAH-degrading ability by Methylobacterium populi and its multiple plant growth-promoting activities. Therefore, the selected indigenous strain, which is naturally present in highly contaminated soils, is good candidate for plant growth promotion and is capable of biodegrading xenobiotic organic compounds to remediate contaminated soil alone and/or soil associated with plants.

The marine isolate Novosphingobium sp. PP1Y shows specific adaptation to use the aromatic fraction of fuels as the sole carbon and energy source.

Novosphingobium sp. PP1Y, isolated from a surface seawater sample collected from a closed bay in the harbour of Pozzuoli (Naples, Italy), uses fuels as its sole carbon and energy source. Like some other Sphingomonads, this strain can grow as either planktonic free cells or sessile-aggregated flocks. In addition, this strain was found to grow as biofilm on several types of solid and liquid hydrophobic surfaces including polystyrene, polypropylene and diesel oil. Strain PP1Y is not able to grow on pure alkanes or alkane mixtures but is able to grow on a surprisingly wide range of aromatic compounds including mono, bi, tri and tetracyclic aromatic hydrocarbons and heterocyclic compounds. During growth on diesel oil, the organic layer is emulsified resulting in the formation of small biofilm-coated drops, whereas during growth on aromatic hydrocarbons dissolved in paraffin the oil layer is emulsified but the drops are coated only if the mixtures contain selected aromatic compounds, like pyrene, propylbenzene, tetrahydronaphthalene and heterocyclic compounds. These peculiar characteristics suggest strain PP1Y has adapted to efficiently grow at the water/fuel interface using the aromatic fraction of fuels as the sole carbon and energy source.

Differential binding of human immunoagents and Herceptin to the ErbB2 receptor.

Overexpression of the ErbB2 receptor is associated with the progression of breast cancer, and is a sign of a poor prognosis. Herceptin, a humanized antibody directed to the ErbB2 receptor, has been proven to be effective in the immunotherapy of breast cancer. However, it can result in cardiotoxicity, and a large fraction of breast cancer patients are resistant to Herceptin treatment. We have engineered three novel, fully human, anti-ErbB2 immunoagents: Erbicin, a human single-chain antibody fragment; ERB-hRNase, a human immunoRNase composed of Erbicin fused to a human RNase; ERB-hcAb, a human 'compact' antibody in which two Erbicin molecules are fused to the Fc fragment of a human IgG1. Both ERB-hRNase and ERB-hcAb strongly inhibit the growth of ErbB2-positive cells in vivo. The interactions of the Erbicin-derived immunoagents and Herceptin with the extracellular domain of ErbB2 (ErbB2-ECD) were investigated for the first time by three different methods. Erbicin-derived immunoagents bind soluble extracellular domain with a lower affinity than that measured for the native antigen on tumour cells. Herceptin, by contrast, shows a higher affinity for soluble ErbB2-ECD. Accordingly, ErbB2-ECD abolished the in vitro antitumour activity of Herceptin, with no effect on that of Erbicin-derived immunoagents. These results suggest that the fraction of immunoagent neutralized by free extracellular domain shed into the bloodstream is much higher for Herceptin than for Erbicin-derived immunoagents, which therefore may be used at lower therapeutic doses than those employed for Herceptin.