Silvia Fernicola
Sapienza University of Rome
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Publication
Featured researches published by Silvia Fernicola.
PLOS ONE | 2013
Giorgio Giardina; Alessandro Paiardini; Silvia Fernicola; Stefano Franceschini; Serena Rinaldo; Valentina Stelitano; Francesca Cutruzzolà
Pseudomonas aeruginosa is responsible for a plethora of biofilm mediated chronic infections among which cystic fibrosis pneumonia is the most frightening. The long-term survival strategy of P. aeruginosa in the patients lungs is based on a fine balance of virulence vs dormant states and on genetic adaptation, in order to select persistent phenotypes as the small colony variants (SCVs), which strongly correlate with antibiotic resistance and poor lung function. Recent studies have coupled SCV with increased levels of the signaling molecule cyclic di-GMP, and demonstrated the central role of the diguanylate cyclase YfiN, part of the tripartite signaling module YifBNR, in c-di-GMP dependent SCV regulation. YfiN, also called TpbB, is a multi-domain membrane enzyme connecting periplasmic stimuli to cytosolic c-di-GMP production by an allosteric inside-out signaling mechanism that, due to the lack of structural data, is still largely hypothetical. We have solved the crystal structure of the catalytic domain (GGDEF), and measured the enzymatic activity of the cytosolic portion in real-time by means of a newly developed method. Based on these results we demonstrate that, unlike other diguanylate cyclase, YfiN does not undergo product feedback inhibition, and that the presence of the HAMP domain is required for dimerization and catalysis. Coupling our structural and kinetic data with an in silico study we are now able to propose a model for the allosteric regulation of YfiN.
Nucleic Acids Research | 2013
Valentina Stelitano; Annegret Brandt; Silvia Fernicola; Stefano Franceschini; Giorgio Giardina; Andrea Pica; Serena Rinaldo; Filomena Sica; Francesca Cutruzzolà
Bacteria react to adverse environmental stimuli by clustering into organized communities called biofilms. A remarkably sophisticated control system based on the dinucleotide 3′–5′ cyclic diguanylic acid (c-di-GMP) is involved in deciding whether to form or abandon biofilms. The ability of c-di-GMP to form self-intercalated dimers is also thought to play a role in this complex regulation. A great advantage in the quest of elucidating the catalytic properties of the enzymes involved in c-di-GMP turnover (diguanylate cyclases and phosphodiesterases) would come from the availability of an experimental approach for in vitro quantification of c-di-GMP in real-time. Here, we show that c-di-GMP can be detected and quantified by circular dichroism (CD) spectroscopy in the low micromolar range. The method is based on the selective ability of manganese ions to induce formation of the intercalated dimer of the c-di-GMP dinucleotide in solution, which displays an intense sigmoidal CD spectrum in the near-ultraviolet region. This characteristic spectrum originates from the stacking interaction of the four mutually intercalated guanines, as it is absent in the other cyclic dinucleotide 3′–5′ cyclic adenilic acid (c-di-AMP). Thus, near-ultraviolet CD can be used to effectively quantify in real-time the activity of diguanylate cyclases and phosphodiesterases in solution.
Journal of Medicinal Chemistry | 2015
Silvia Fernicola; Ilaria Torquati; Alessandro Paiardini; Giorgio Giardina; Giordano Rampioni; Marco Messina; Livia Leoni; Fabio Del Bello; Riccardo Petrelli; Serena Rinaldo; Loredana Cappellacci; Francesca Cutruzzolà
Cyclic di-GMP (c-di-GMP) is a widespread second messenger that plays a key role in bacterial biofilm formation. The compounds ability to assume multiple conformations allows it to interact with a diverse set of target macromolecules. Here, we analyzed the binding mode of c-di-GMP to the allosteric inhibitory site (I-site) of diguanylate cyclases (DGCs) and compared it to the conformation adopted in the catalytic site of the EAL phosphodiesterases (PDEs). An array of novel molecules has been designed and synthesized by simplifying the native c-di-GMP structure and replacing the charged phosphodiester backbone with an isosteric nonhydrolyzable 1,2,3-triazole moiety. We developed the first neutral small molecule able to selectively target DGCs discriminating between the I-site of DGCs and the active site of PDEs; this molecule represents a novel tool for mechanistic studies, particularly on those proteins bearing both DGC and PDE modules, and for future optimization studies to target DGCs in vivo.
Journal of Bacteriology | 2016
Silvia Fernicola; Alessandro Paiardini; Giorgio Giardina; Giordano Rampioni; Livia Leoni; Francesca Cutruzzolà; Serena Rinaldo
UNLABELLED Biofilm formation is responsible for increased antibiotic tolerance in pathogenic bacteria. Cyclic di-GMP (c-di-GMP) is a widely used second-messenger signal that plays a key role in bacterial biofilm formation. c-di-GMP is synthesized by diguanylate cyclases (DGCs), a conserved class of enzymes absent in mammals and hence considered attractive molecular targets for the development of antibiofilm agents. Here, the results of a virtual screening approach aimed at identifying small-molecule inhibitors of the DGC PleD from Caulobacter crescentus are described. A three-dimensional (3D) pharmacophore model, derived from the mode of binding of GTP to the active site of PleD, was exploited to screen the ZINC database of compounds. Seven virtual hits were tested in vitro for their ability to inhibit the activity of purified PleD by using circular dichroism spectroscopy. Two drug-like molecules with a catechol moiety and a sulfonohydrazide scaffold were shown to competitively inhibit PleD at the low-micromolar range (50% inhibitory concentration [IC50] of ∼11 μM). Their predicted binding mode highlighted key structural features presumably responsible for the efficient inhibition of PleD by both hits. These molecules represent the most potent in vitro inhibitors of PleD identified so far and could therefore result in useful leads for the development of novel classes of antimicrobials able to hamper biofilm formation. IMPORTANCE Biofilm-mediated infections are difficult to eradicate, posing a threatening health issue worldwide. The capability of bacteria to form biofilms is almost universally stimulated by the second messenger c-di-GMP. This evidence has boosted research in the last decade for the development of new antibiofilm strategies interfering with c-di-GMP metabolism. Here, two potent inhibitors of c-di-GMP synthesis have been identified in silico and characterized in vitro by using the well-characterized DGC enzyme PleD from C. crescentus as a structural template and molecular target. Given that the protein residues implied as crucial for enzyme inhibition are found to be highly conserved among DGCs, the outcome of this study could pave the way for the future development of broad-spectrum antibiofilm compounds.
Journal of Bacteriology | 2015
Serena Rinaldo; Alessandro Paiardini; Valentina Stelitano; Paolo Brunotti; Laura Cervoni; Silvia Fernicola; Carmela Protano; Matteo Vitali; Francesca Cutruzzolà; Giorgio Giardina
XXIII National Meeting on Medicinal Chemistry and 9th Young Medicinal Chemists Symposium (XXIII NMMC – 9th NPCF) | 2015
Riccardo Petrelli; Ilaria Torquati; Mirko Scortichini; Serena Rinaldo; Silvia Fernicola; Giorgio Giardina; Alessandro Paiardini; Livia Leoni; Giordano Rampioni; Marco Messina; Francesca Cutruzzolà; Loredana Cappellacci
31th meeting of SIMGBM-MICROBIOLOGY 2015 | 2015
Silvia Fernicola; Serena Rinaldo; Giorgio Giardina; Paolo Brunotti; Valentina Stelitano; Alessandro Paiardini; Riccardo Petrelli; Ilaria Torquati; Loredana Cappellacci; Francesca Cutruzzolà
YOUNG RESEARCHER IN LIFE SCIENCES | 2014
Silvia Fernicola; Alessandro Paiardini; Serena Rinaldo; Ilaria Torquati; Riccardo Petrelli; Loredana Cappellacci; Francesca Cutruzzolà
XXII National Meeting on Medicinal Chemistry | 2013
Riccardo Petrelli; Ilaria Torquati; Silvia Fernicola; V. Stelitano S. Rinaldo Stelitano S Rinaldo; Giordano Rampioni; Marco Messina; Livia Leoni; Francesca Cutruzzolà; Loredana Cappellacci
Microbiology | 2013
Silvia Fernicola; Serena Rinaldo; Giorgio Giardina; Ilaria Torquati; Riccardo Petrelli; Loredana Cappellacci; Francesca Cutruzzolà