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101. Proteomics Characterization of Outer Membrane Vesicles from the Extraintestinal Pathogenic Escherichia coli tolR IHE3034 Mutant

Author

Berlanda Scorza, Francesco, Doro, Francesco, Rodríguez-Ortega, Manuel José, Stella, Maria, Liberatori, Sabrina, Taddei, Anna Rita, Serino, Laura, Gomes Moriel, Danilo, Nesta, Barbara, Fontana, Maria Rita, Spagnuolo, Angela, Pizza, Mariagrazia, Norais, Nathalie, and Grandi, Guido

Abstract

Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a tolR mutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant. The vesicles were analyzed by two-dimensional electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which are localized to the outer membrane and periplasmic compartments. Interestingly based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that outer membrane vesicles represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis.

Published
2008

102. Proteomics Characterization of Outer Membrane Vesicles from the Extraintestinal Pathogenic Escherichia coliΔtolRIHE3034 Mutant*

Author

Berlanda Scorza, Francesco, Doro, Francesco, Rodríguez-Ortega, Manuel José, Stella, Maria, Liberatori, Sabrina, Taddei, Anna Rita, Serino, Laura, Gomes Moriel, Danilo, Nesta, Barbara, Fontana, Maria Rita, Spagnuolo, Angela, Pizza, Mariagrazia, Norais, Nathalie, and Grandi, Guido

Abstract

Extraintestinal pathogenic Escherichia coliare the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a ΔtolRmutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant. The vesicles were analyzed by two-dimensional electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which are localized to the outer membrane and periplasmic compartments. Interestingly based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coliand enteric bacteria and therefore are potential targets for vaccine and drug development. Finally we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that outer membrane vesicles represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis.

Published
2008
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103. In VitroPollen Tube Growth Reveals the Cytotoxic Potential of the Flavonols, Quercetin and Rutin

Author

Antognoni, Fabiana, Ovidi, Elisa, Taddei, Anna Rita, Gambellini, Gabriella, and Speranza, Anna

Abstract

Flavonols are phytochemicals widely found in commonly consumed foods. In spite of their beneficial effects on human health, however, cytotoxicity and even suspected genotoxicity have also been reported for the flavonol, quercetin. This points to the need for preventive studies to identify any cytotoxic effects associated with pure flavonol intake. This work was performed with the aim of verifying whether a plant-based in vitrosystem, the pollen tube, could be used to evaluate the cytotoxic potential of exogenous flavonols. Increasing concentrations of the aglycone, quercetin, and its glycoside, rutin, were assayed with regard to tube growth of kiwifruit pollen, determined by applying the pollen tube growth test protocol. This test, based on the photometric quantification of pollen tube mass production in suspension cultures, has already been applied in the sensitive and reliable toxicological evaluation of a wide range of chemicals. Whereas 60–800µM rutin promoted kiwifruit pollen tube elongation, 10–50µM quercetin strongly inhibited growth, and also produced irreversible malformations, such as screw-like tube growth, abnormal vacuolation, alteration of organelle streaming, and nuclear positioning. Thus, the cytotoxic potentials of the two flavonols have been confirmed to differ. Pollen tubes seem to afford a promising test system for a preventive, rapid in vitrobiosafety assessment of antioxidant nutritional supplements, without using laboratory animals.

Published
2004
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104. Sperm quality and reproductive traits in male offspring of female rabbits exposed to Lindane (<formula notation="TeX">$\gamma$</formula>-HCH) during pregnancy and lactation

Author

Fausto, Anna Maria, Morera, Patrizia, Margarit, Rita, and Taddei, Anna Rita

Abstract

Fifteen Grimaud female hybrid rabbits, 135 days old and weighting an average of $3.74\pm 0.01$ kg each, were administered an oral dose of 1 mg$\cdot$kg$^{-1}$ body weight of Lindane during gestation and lactation period. Fertility rate, libido, volume of ejaculate, concentration and morphology of spermatozoa were investigated to test the effects of the treatment on reproductive traits of first generation male rabbits. Ultrastructure of abnormal spermatozoa was described by Transmission Electron Microscopy and the different abnormalities were quantified. The results obtained indicate that low dose exposure of Lindane has effects on spermatozoa ultrastructure that proved to be susceptible to the treatment with the pesticide (cytoplasmic droplets: 5.3% in control group and 10.3% in Lindane group, $P \leq 0.05$; coiled tails: 1.3% in control group and 4.3% in Lindane group, $P\leq 0.05$) and could be utilised as a good marker of toxicity.

Published
2001

105. Interazioni cellula-matrice: culture cellulari in supporti a base di collagene

Author

Laghezza Masci, Valentina, Fausto, Anna Maria, and Taddei, Anna Rita

Subjects
Matrici in collagene, Microvescicole di membrana, Fibroblasti, Colture in vitro, BIO/05, Collagen matrix, Electron microscopy, In vitro culture, Fibroblasts, Microscopia elettronica, Microvesicles
Abstract

The primary aim of tissue engineering is to develop bio-functional matrices that mimic native tissues in vitro and hep to stimulate healing under normal or chronic situations. Collagen based scaffolds are widely used as temporary or permanent coverings to help dermal wound healing. Under natural conditions, wound healing is affected by many factors, including different cell types, growth factors and several components of the extracellular matrix. Due to the complexity of the cell-to-matrix interaction, many cell-based mechanisms are not yet properly known. The aim of this work was to study the ultrastructure of equine collagen type I scaffolds that are used in tissue regeneration. Furthermore, to simulate the healing process in vitro the mechanisms by which a stabilized cell line of the connective tissue interacts with such supports were also carefully studied. Scanning (SEM) and transmission electron microscopy (TEM) techniques were used to assess the structural features that this collagen scaffold should have to support cell migration and interaction with the host tissue. This study has clearly demonstrated that fibroblasts NIH3T3 can actually migrate through the collagen matrix by embracing collagen fibers with long filopodia and fold them back onto the cell surface to form large intracellular vacuoles. Gelatin Zymography and Western Blot techniques were also used to verify what role gelatinase B or MMP-9 play in the elaboration of the collagen matrix. Although the activation of this matrix-metalloproteinase is not conditioned by the presence of the collagen scaffold, the extracellular release of MMP-9 and its diffusion onto the collagen matrix was found to be highly correlated with shedding of the microvesicles from the cell membrane. This finding suggests that the MMP-9 activation and the following degradation could be triggered by the release of microvesicles. Their interaction with the prosthetic collagen is probably a precondition for fibroblasts to laying new extracellular matrix during wound healing. 1 Several comparative studies were carried out to verify the extent by which native equine collagen is structurally modified if combined with other elements (active substances or nanoparticles). Data have shown that, even under these conditions, collagen maintain enough structural stability to sustain cellular migration and proliferation. This finding opens the way to develop innovative dermal substitutes by making collagen bio-functionalized with probes specifically developed to address it toward certain target tissues. On the whole, the morpho-functional analysis carried out in this study demonstrates that equine type I collagen-based matrices provide conditions favorable for mimicking wound healing in vitro. In spite of the complexity of the process in vivo, and the variety of factors actually involved in wound healing, it is conceivable that the type of cell-to-matrix interactions – as due to migration, adhesion and proliferation – envisaged in this study may represent the first step in a cascade of reactions leading to tissue remodeling. Given this possibility, the experimental conditions worked out in this thesis for the establishment of a 3D culture in a collagen matrix may constitute a first pivotal attempt for testing additional cell parameters under conditions that could not be adequately controlled in vivo. L’ingegneria tissulare attualmente è volta allo sviluppo di matrici bio-funzionali in grado di mimare il tessuto nativo e di stimolare i processi di guarigione in situazioni normali o croniche. Matrici a base di collagene sono ampiamente usate come coperture temporanee o permanenti per aiutare la guarigione di ferite cutanee. In condizioni normali, il processo di guarigione è influenzato da molti fattori tra cui diversi tipi di cellule, fattori di crescita e diversi componenti della matrice extracellulare. Data la complessità della interazione cellula-matrice, molti meccanismi cellulari di base non sono ancora propriamente noti. Lo scopo di questo lavoro è stato studiare la morfologia e l’ultrastruttura di supporti a base di collagene equino di tipo I, usati nella rigenerazione dei tessuti. Inoltre, per simulare il processo di guarigione in vitro, sono stati ampiamente studiati i meccanismi attraverso i quali linee cellulari stabilizzate del tessuto connettivo interagiscono con tali supporti. Tecniche di Microscopia Elettronica a Scansione (SEM) e a Trasmissione (TEM) sono state utilizzate per valutare le caratteristiche strutturali che questa matrice di collagene deve avere per supportare la migrazione delle cellule e l'interazione con il tessuto ospite. Questo studio ha chiaramente dimostrato che fibroblasti NIH3T3 migrano attraverso la matrice avvolgendo le fibre di collagene mediante lunghi fillopodi che si ripiegano sulla superficie cellulare a formare ampi vacuoli intracellulari. Tecniche di Gelatin Zymography e Western Blot sono stati utilizzate per verificare quale sia il ruolo della gelatinasi B o MMP-9 nella elaborazione della matrice di collagene. Sebbene l'attivazione di questa metalloproteinasi di matrice non è condizionata dalla presenza di collagene protesico, il rilascio extracellulare di MMP-9 e la sua diffusione sulla matrice è risultato fortemente correlato allo spargimento di microvescicole di membrana. Questo risultato suggerisce che l'attivazione della MMP-9 e la sua successiva degradazione potrebbero essere innescati dal rilascio di microvescicole. La loro interazione con il collagene protesico è presumibilmente presupposto, nei fibroblasti, per la deposizione di nuova matrice extracellulare nel corso della guarigione delle ferite. Diversi studi comparativi sono stati effettuati per verificare la misura in cui il collagene nativo equino è strutturalmente modificato se combinato con altri elementi (sostanze attive o nanoparticelle). I dati hanno dimostrato che, anche in queste condizioni, il collagene mantiene sufficiente stabilità strutturale per sostenere la migrazione e la proliferazione cellulare. La possibilità di bio-funzionalizzare questo tipo di collagene, senza alterarne le caratteristiche, offre la possibilità di sviluppare sostituti dermici sempre più innovativi in grado di supportare tessuti bersaglio differenti. Nel complesso, l'analisi morfo-funzionale effettuato in questo studio dimostra che le matrici a base di collagene equino tipo I forniscono condizioni favorevoli per simulare la guarigione delle ferite in vitro. Nonostante la complessità del processo in vivo e la varietà dei fattori effettivamente coinvolti nella guarigione delle ferite, è concepibile che il tipo di interazioni cellula-matrice - come migrazione, adesione e proliferazione - previste in questo studio possano rappresentare il primo passo in una cascata di reazioni che portano al rimodellamento tissutale. Tenuto conto di questa eventualità, le condizioni sperimentali elaborate in questa tesi per la creazione di una cultura in 3D in una matrice di collagene, può costituire un primo tentativo fondamentale per testare parametri cellulari supplementari in condizioni che non possono essere adeguatamente controllate in vivo. Dottorato di ricerca in Evoluzione biologica e biochimica

Published
2015

106. Membrane alteration, anti-virulence properties and metabolomic perturbation of a chionodracine-derived antimicrobial peptide, KHS-Cnd, on two bacteria models.

Author

Imperlini E, Massaro F, Grifoni A, Maiurano F, Taddei AR, Borocci S, Buonocore F, and Porcelli F

Abstract

Antarctic fishes, living in an extreme environment and normally exposed to pathogens, are a promising source of antimicrobial peptides (AMPs). These are emerging as next-generation drugs due to their activity against multidrug resistant (MDR) bacteria. To infect hosts, beyond intrinsic/acquired resistance, MDR species also use virulence factors such as protease secretion. Hence, AMPs targeting virulence factors could represent a novel strategy to counteract the antimicrobial resistance (AMR). In this paper, we focused on a mutant peptide, named KHS-Cnd, that was obtained from the scaffold of the chionodracine (Cnd), a natural peptide identified in the icefish Chionodraco hamatus. We studied different effects caused by the peptide interaction with the cell membrane of two model bacteria, E. coli and B. cereus. First, we investigated its membranolytic activity revealing that the peptide action is more evident on E. coli, with a 69 % uptake of the used dye at 3 μM, whereas for B. cereus we found only a 65 % uptake at 6 μM. Successively, we determined the impact of this lysis on total protein concentration in the medium and an increase was estimated for both bacteria (84 % after 1 h for E. coli and 90 % for B. cereus, respectively). Moreover, we evaluated the changes in the proteolytic activity of the supernatant, that is an important aspect of bacterial resistance, showing that there was a significant reduction for both bacteria, although at higher level in the case of E. coli. The membranolytic activity was evidenced also morphologically with TEM analysis and a different alteration was evidenced for the two bacteria. Moreover, NMR metabolomics analysis showed that peptide induces changes in E. coli and B. cereus extracellular metabolites especially at the higher tested concentrations: this metabolic variation could be used as a fingerprinting of the peptide action on bacteria physiology due to its interaction with cell wall. Finally, we determined the KHS-Cnd cytotoxicity on human primary cell lines to verify its selectivity toward bacterial cell membranes and we found low toxicity until a concentration of 5 μM. Considering that the peptide exerts both membranolytic and anti-virulence activity on E. coli at 1.5 μM, we confirmed the interesting potential of this AMP as a new drug to counteract AMR., Competing Interests: Declaration of Competing Interest The authors have nothing to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2024
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107. T-cells and CD45-cells discovery in the central nervous system of healthy and nodavirus-infected teleost fish Dicentrarchus labrax .

Author

Pianese V, Alvarez-Torres D, Gemez-Mata J, Garcia-Rosado E, Moreno P, Fausto AM, Taddei AR, Picchietti S, and Scapigliati G

Subjects
Animals, Fish Proteins genetics, Fish Proteins immunology, Central Nervous System immunology, Central Nervous System virology, Brain virology, Brain immunology, Nodaviridae physiology, Fish Diseases immunology, Fish Diseases virology, Bass immunology, RNA Virus Infections veterinary, RNA Virus Infections immunology, RNA Virus Infections virology, Leukocyte Common Antigens metabolism, Leukocyte Common Antigens genetics, Leukocyte Common Antigens immunology, T-Lymphocytes immunology
Abstract

To achieve insights in antiviral immune defense of the central nervous system (CNS), we investigated T cells and CD45 cells in the marine fish model Dicentrarchus labrax infected with the CNS-tropic virus betanodavirus. By employing markers for pan-T cells (mAb DLT15) and CD45-cells (mAb DLT22) in immunofluorescence (IIF) of leukocytes from brain, we obtained 3,7 ± 2.3 % of T cells and 7.3 ± 3.2 % of CD45 + cells. Both IIF and immunoelectron microscopy confirmed a leukocyte/glial morphology for the immunoreactive cells. Quantitative immunohistochemistry (qIHC) of brain/eye sections showed 1.9 ± 0.8 % of T + cells and 2 ± 0.9 % of CD45 + cells in the brain, and 3.6 ± 1.9 % and 4.1 ± 2.2 % in the eye, respectively. After in vivo RGNNV infection the number of T cells/CD45 + leukocytes in the brain increased to 8.3 ± 2.1 % and 11.6 ± 4.4 % (by IIF), and 26.1 ± 3.4 % and 45.6 ± 5.9 % (by qIHC), respectively. In the eye we counted after infection 8.5 ± 4.4 % of T cells and 10.2 ± 5.8 % of CD45 cells. Gene transcription analysis of brain mRNA revealed a strong increase of gene transcripts coding for: antiviral proteins Mx and ISG-12; T-cell related CD3ε/δ, TcRβ, CD4, CD8α, CD45; and for immuno-modulatory cytokines TNFα, IL-2, IL-10. A RAG-1 gene product was also present and upregulated, suggesting somatic recombination in the fish brain. Similar transcription data were obtained in the eye, albeit with differences. Our findings provide first evidence for a recruitment and involvement of T cells and CD45 + leukocytes in the fish eye-brain axis during antiviral responses and suggest similarities in the CNS immune defense across evolutionary distant vertebrates., Competing Interests: Declaration of competing interest Authors declare there are no competing financial interests in relation to the work described., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Published
2024
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108. Proteomics characterization of outer membrane vesicles from the extraintestinal pathogenic Escherichia coli DeltatolR IHE3034 mutant.

Author

Berlanda Scorza F, Doro F, Rodríguez-Ortega MJ, Stella M, Liberatori S, Taddei AR, Serino L, Gomes Moriel D, Nesta B, Fontana MR, Spagnuolo A, Pizza M, Norais N, and Grandi G

Subjects
Bacterial Outer Membrane Proteins chemistry, Bacterial Outer Membrane Proteins metabolism, Bacterial Toxins chemistry, Bacterial Toxins metabolism, Cell Membrane ultrastructure, Escherichia coli chemistry, Escherichia coli ultrastructure, Genome, Bacterial, Peptides, Periplasmic Proteins chemistry, Periplasmic Proteins metabolism, Protein Binding, Protein Subunits, Software, Cell Membrane chemistry, Escherichia coli cytology, Escherichia coli genetics, Escherichia coli Proteins genetics, Membrane Proteins genetics, Mutation genetics, Proteomics methods
Abstract

Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a DeltatolR mutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant. The vesicles were analyzed by two-dimensional electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which are localized to the outer membrane and periplasmic compartments. Interestingly based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that outer membrane vesicles represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis.

Published
2008
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109. Outer membrane vesicles from group B Neisseria meningitidis delta gna33 mutant: proteomic and immunological comparison with detergent-derived outer membrane vesicles.

Author

Ferrari G, Garaguso I, Adu-Bobie J, Doro F, Taddei AR, Biolchi A, Brunelli B, Giuliani MM, Pizza M, Norais N, and Grandi G

Subjects
Bacterial Outer Membrane Proteins genetics, Bacterial Outer Membrane Proteins isolation & purification, Bacterial Outer Membrane Proteins ultrastructure, Chromatography, Gel, Computational Biology, Electrophoresis, Gel, Two-Dimensional, Flow Cytometry, Mass Spectrometry, Neisseria meningitidis, Serogroup B classification, Neisseria meningitidis, Serogroup B genetics, Serotyping, Bacterial Outer Membrane Proteins immunology, Detergents pharmacology, Gene Deletion, Neisseria meningitidis, Serogroup B enzymology, Proteomics methods
Abstract

We compared the proteome of detergent-derived group B Neisseria meningitidis (MenB) outer membrane vesicles (DOMVs) with the proteome of outer membrane vesicles (m-OMVs) spontaneously released into culture supernatant by MenB delta gna33, a mutant in which the gene coding for a lytic transglycosylase homologous to the E. coli MltA was deleted. In total, 138 proteins were identified in DOMVs by 1- and 2-DE coupled with MS; 64% of these proteins belonged to the inner membrane and cytoplasmic compartments. By contrast, most of the 60 proteins of m-OMVs were classified by PSORT as outer membrane proteins. When tested for their capacity to elicit bactericidal antibodies, m-OMVs elicited a broad protective activity against a large panel of MenB strains. Therefore, the identification of mutations capable of conferring an OMV-releasing phenotype in bacteria may represent an attractive approach to study bacterial membrane composition and organization, and to design new efficacious vaccine formulations.

Published
2006
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