Publications (9)32.2 Total impact
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Dataset: j.1462-5822.2011.01722.x
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Article: The role of tryptophan in protein fibrillogenesis: relevance of Trp7 and Trp14 to the amyloidogenic properties of myoglobin.
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ABSTRACT: In order to understand the role of tryptophan in the mechanisms of fibrils formation, the ability of a series of analogs of the residue 7-18 span of myoglobin to form amyloid-like fibrils was investigated. Alternatively one or both tryptophans were substituted with alanine and leucine, to determine the contribution of hydrophobicity and aromaticity. The scale of aggregation propensity of the peptides determined indicates that tryptophan is crucial for the amyloidogenic process. Since the rare tryptophan residue is generally engaged in structural roles in proteins, or when exposed serves as binding sites, we surmise that its exposure in the amyloidogenic fragments allows for intermolecular clustering with residues from other molecules leading to the formation of amyloid aggregates.Protein Engineering Design and Selection 02/2012; 25(4):199-203. · 2.94 Impact Factor -
Article: Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.
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ABSTRACT: NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA.Cellular Microbiology 11/2011; 14(3):368-85. · 5.46 Impact Factor -
Article: The soluble recombinant Neisseria meningitidis adhesin NadA(Δ351-405) stimulates human monocytes by binding to extracellular Hsp90.
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ABSTRACT: The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadA(Δ351-405,) devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadA(Δ351-405) cellular effects in monocytes. We show that NadA(Δ351-405) (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadA(Δ351-405) cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadA(Δ351-405) /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadA(Δ351-405) and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadA(Δ351-405) determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadA(Δ351-405) alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by anti-TLR2 antibodies. We propose that hsp90-dependent recruitment into an hsp90/hsp70/TLR4 transducing signal complex is necessary for the immune-stimulating activity of NadA(Δ351-405) anti-MenB vaccine candidate.PLoS ONE 01/2011; 6(9):e25089. · 4.09 Impact Factor -
Article: Mapping of the Neisseria meningitidis NadA cell-binding site: relevance of predicted {alpha}-helices in the NH2-terminal and dimeric coiled-coil regions.
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ABSTRACT: NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH(2)-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH(2)-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH(2) globular head domain and the NH(2) dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.Journal of bacteriology 10/2010; 193(1):107-15. · 3.94 Impact Factor -
Article: The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA- OMVs, without further stimulating their proinflammatory activity on circulating monocytes.
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ABSTRACT: Hypervirulent MenB causing fatal human infections frequently display the oligomeric-coiled coil adhesin NadA, a 45-kDa intrinsic outer membrane protein implicated in binding to and invasion of respiratory epithelial cells. A recombinant soluble mutant lacking the 10-kDa COOH terminal membrane domain (NadA(Delta351-405)) also activates human monocytes/macrophages/DCs. As NadA is physiologically released during sepsis as part of OMVs, in this study, we tested the hypothesis that NadA(+) OMVs have an enhanced or modified proinflammatory/proimmune action compared with NadA(-) OMVs. To do this we investigated the activity of purified free NadA(Delta351-405) and of OMVs from MenB and Escherichia coli strains, expressing or not full-length NadA. NadA(Delta351-405) stimulated monocytes and macrophages to secrete cytokines (IL-1beta, TNF-alpha, IL-6, IL-12p40, IL-12p70, IL-10) and chemokines (IL-8, MIP-1alpha, MCP-1, RANTES), and full-length NadA improved MenB OMV activity, preferentially on macrophages, and only increased cytokine release. NadA(Delta351-405) induced the lymphocyte costimulant CD80 in monocytes and macrophages, and NadA(+) OMVs induced a wider set of molecules supporting antigen presentation (CD80, CD86, HLA-DR, and ICAM-1) more efficiently than NadA(-) OMVs only in macrophages. Moreover, membrane NadA effects, unlike NadA(Delta351-405) ones, were much less IFN-gamma-sensitive. The activity of NadA-positive E. coli OMVs was similar to that of control OMVs. NadA in MenB OMVs acted at adhesin concentrations approximately 10(6) times lower than those required to stimulate cells with free NadA(Delta351-405).Journal of leukocyte biology 05/2009; 86(1):143-53. · 4.99 Impact Factor -
Article: Human monocytes/macrophages are a target of Neisseria meningitidis Adhesin A (NadA).
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ABSTRACT: Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial-monocyte association, but a NadA-positive strain induced a significantly higher amount of TNF-alpha and IL-8 compared with the parental NadA-negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadA(Delta351-405), a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL-8, IL-6, MCP-1, and MIP-1alpha and low levels of the main vasoactive mediators TNF-alpha and IL-1. NadA(Delta351-405) also inhibited monocyte apoptosis and determined its differentiation into a macrophage-like phenotype.Journal of Leukocyte Biology 06/2008; 83(5):1100-10. · 4.99 Impact Factor -
Article: IFN-gamma and R-848 dependent activation of human monocyte-derived dendritic cells by Neisseria meningitidis adhesin A.
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ABSTRACT: A soluble recombinant form of Neisseria meningitidis adhesin A (NadADelta351-405), proposed as a constituent of anti-meningococcal B vaccines, is here shown to specifically interact with and immune-modulate human monocyte-derived dendritic cells (mo-DCs). After priming with IFN-gamma and stimulation with NadADelta351-405, mo-DCs strongly up-regulated maturation markers CD83, CD86, CD80, and HLA-DR, secreted moderate quantities of TNF-alpha, IL-6, and IL-8, and produced a slight, although significant, amount of IL-12p70. Costimulation of mo-DCs with NadADelta351-405 and the imidoazoquinoline drug R-848, believed to mimic bacterial RNA, increased CD86 in an additive way, but strongly synergized the secretion of IL-12p70, IL-1, IL-6, TNF-alpha, and MIP-1alpha, especially after IFN-gamma priming. CD86/CD80 overexpression correlated with the occupation of high-(kd approximately 80 nM) and low-(kd approximately 4 muM) affinity binding sites for NadADelta351-405. Alternatively, secretion of IL-12p70 and TNF-alpha, IL-6, and IL-8 corresponded to the occupation of high- or low-affinity receptors, respectively. Mo-DCs matured by IFN-gamma and NadADelta351-405 supported the proliferation of naive CD4+ T lymphocytes, inducing the differentiation of both IFN-gamma and IL-4 producing phenotypes. Our data show that NadA not only is a good immunogen but is as well endowed with a proimmune, self-adjuvating, activity.The Journal of Immunology 10/2007; 179(6):3904-16. · 5.79 Impact Factor -
Article: NadA da Neisseria meningitidis interagisce con Hsp90 sulla superficie dei monociti modulando la loro produzione di citochine
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ABSTRACT: Neisseria meningitidis is an encapsulated, Gram-negative bacterium that colonizes the upper respiratory tract of ~10% of humans. With a frequency of one to three cases per 100.000 of the population, the bacterium enters the bloodstream, where it multiplies to high density and cause sepsis. From the bloodstream the bacterium can cross the blood-brain barrier and cause meningitis. The invasive infection is very dramatic, affecting mostly infants, children, and adolescents who do not have bactericidal antibodies against the infecting strains. Immunity against the disease can be acquired naturally or induced by vaccination and correlates with the presence of antibodies able to kill the bacterium in the presence of complement. There are no effective vaccines currently licensed in the Unit States or Europe for prevention of the disease caused by serogroup B meningococcus. The genome sequencing of Neisseria meningitidis, serogroup B, allowed the identification of unknown surface proteins termed GNA (Genome derived Neisseria Antigens) among which NadA (Neisseria Adhesin A). NadA is a highly conserved protein among disease-associated strains and capable of eliciting bactericidal antibodies in mice. Structure prediction and homology comparison with know virulence-associated factors suggest that NadA belongs to the group of OCA (Oligomeric Coiled-coil Adhesin) nonfimbrial adhesins. Recently, NadA has been characterized as a new meningococcic factor, involved in the colonization and the invasion of host cells. Probably there is a unique receptor expressed in different cellular lines. In this thesis, we have focused on unravelling the identity of this possible receptor for NadA in these cells. To identify the specific receptor for NadA, experiments of co-immunoprecipitation and overlay were performed in Chang total cell lysates. Our data suggest as possible receptor a protein of 90 kDa, that was present in the co-immunoprecipitation samples incubated with NadA and absent in controls. Subsequently, this protein obtained by co-immunoprecipitation of NadA, was enriched, separated by 12% SDS-PAGE, then excised from the gel and subjected to tryptic proteolysis; resulting peptides were analyzed by liquid chromatography/MS and data were analyzed with the Mascot software. In this way, we identified the human Heat shock protein 90\beta, as the recognized peptides provide a coverage of 25% of the total protein sequence . To confirm the MS data, co-immunoprecipitation samples and membrane proteins from Chang cells were incubated with antibody anti-Hsp90, in order to demonstrate a membrane localization of Hsp90, which is generally known as a cytoplasmic protein. However, as described in the literature, Hsp90 can be expressed on the surface of various cell types, such as tumor and apoptotic cells, but also on HeLa cells, monocytes, macrophages and dendritic cells. Our results confirm that Hsp90 is also found in the cell membrane of Chang cells. Moreover, we found that in co-immunoprecipitation experiments addition of polymyxin B, a cationic antibiotic similar to antimicrobic peptides produced by monocytes that binds both to NadA and Hsp90, is able to interfere in the interaction of NadA with Hsp90. In order to investigate the effect of the association between NadA and Hsp90 in cells, we quantified the superficial expression of Hsp90 on Chang cells and on human monocytes, isolates from Buffy coat , which were found to be 2% and 5% of total cell surface protein, respectively Since the mechanism of transport to the plasma membrane remains enigmatic, we quantified the superficial expression of Hsp90 in Chang cells after the incubation with rHsp90, but we didn’t see an increase of protein expression. We also performed experiments on human monocytes, which are the main agonist of the innate immune system, in order to study the receptorial function of this association, and also the involvement of the immune system. Monocytes were incubated with antibodies anti-Hsp90, with or without Polymyxin B, and then they were analyzed by FACS, to quantify the binding of NadA-alexa. These experiments showed that the NadA binding to the cells is not influenced by the presence of the anti-Hsp90 antibody. We also investigated whether the superficial expression of Hsp90 in monocytes changed after pre-incubation with NadA. By FACS analysis, we quantified the fluorescence of a PE conjugated secondary antibody after the incubation with anti-Hsp90 antibodies. The results showed that pre-incubation with NadA interferes with the recognition of the superficial Hsp90 by its specific antibodies, showing a decrease of 40%, which could be explained by the competive association between NadA and Hsp90 on the plasmatic membrane. Moreover, when monocytes were incubated with NadA for 3h at 37°C in presence of Polymyxin B, we did not observe decrease on the signal. Taken together, these data indicate that NadA-Hsp90 association is not a receptorial one, indicating that, under physiological conditions, these proteins bind closely and strongly each others, probably producing clusters on the plasma membrane. Finally, to analyze the effect of these complexes on the activation of the immune system, we analyzed the pattern of cytokines produced by human monocytes stimulated for 24h with NadA, antibodies anti-Hsp90 and antibodies anti-TLR2 and 4, and with or without Polymyxin B. These results show a synergic effect of NadA and antibody anti-Hsp90 on cytokines production, mainly IL-6, TNF? and MCP-1; on the contrary, Polymyxin B and antibody anti-TLR4 inhibited cytokines production. The cytokine pattern secreted demonstrated that NadA and Hsp90 induce a macrophage-like phenotype and that these two agonists promote a Th2 response.
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Institutions
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2007–2013
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University of Padua
- Interdepartmental Research Centre for Innovative Biotechnologies CRIBI
Padova, Veneto, Italy
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2009–2011
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University-Hospital of Padova
Padova, Veneto, Italy
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