Small bacterial nucleic acids called ‘cyclic dinucleotides’, among which the best known is cyclic-di-GMP, are important regulators of bacterial adaptation strategies such as biofilm formation, persistence, cytotoxicity and development. These molecules and their derivatives play also a crucial role in bacteria-host interactions, as they are able to elicit an innate immune response.
Persistent bacterial infections caused by bacterial biofilms endanger human health: biofilms are often refractory to antibiotics and disinfectants to which planktonic bacteria are susceptible, causing more than 70% of all infections in developed countries. The second messenger 3′, 5′-cyclic diguanylic acid (c-di-GMP) controls many aspects of the switch between planktonic and biofilm lifestyles, including the formation of persisters, dormant or slow-growing cells. C-di-GMP synthesis and breakdown are controlled by specific diguanylate cyclases (DGCs) and phosphodiesterases (PDEs).
Our aim is to study the metabolism of c-di-GMP and to find new targets for effective anti-biofilm drugs in Pseudomonas aeruginosa, a known model system and an opportunistic human pathogen, major cause of infection in tissues and medical devices.
A brief summary of our recent results is given below.
C-di-GMP synthesis/breakdown explored in real-time. An innovative approach to quantify in real-time c-di-GMP by circular dichroism (CD) spectroscopy was devised ( Stelitano et al 2013, Nucleic Acids Res.), based on the property of dimers of c-di-GMP to form stable complexes with the divalent cation Mn2+. This novel approach will allow unveiling the catalytic properties of enzymes involved in c-di-GMP turnover (DGCs and PDEs).
DGCs and PDEs structure and function: we have determined the structure and function of selected DGCS and PDEs from P. aeruginosa.
PDEs of the HD-GYP subclass hydrolyse c-di-GMP into GMP via the intermediate pGpG, producing GMP at a surprisingly low rate in vitro (Stelitano et al. 2013, PLOS ONE). We found that affinity for pGpG is higher than that for c-di-GMP, suggesting that in vivo pGpG could also be a signal molecule.We recently solved the structure of PA4781 and shown that the protein displays unselective metal binding sites, thus reconsidering the evolution of this HD-GYP containing proteins (Rinaldo et al.2015, J. Bacteriol.)
We solved the crystal structure of the catalytic domain of the DGC PA1120 (YfiN). Unlike other DGCs, YfiN does not undergo product feedback inhibition. Coupling structural, kinetic and in silico data we propose a model for the allosteric regulation of YfiN and other bacterial transmembrane receptors (Giardina et al. 2013, PLOS ONE).
Inhibitors targeting c-di-GMP metabolism:
- GTP and c-di-GMP analogues. Over 50 putative inhibitors of DGCs and/or PDEs were identified either by in silico virtual screening (with A.Paiardini, Rome, I) or designed and synthesized (with L. Cappellacci, Camerino, I). Recent publications: Fernicola et al, J.Med. Chem 2015 and Fernicola et al., J Bacteriol. 2015
- Inhibitors targeting nucleotide biosynthesis. In collab. with P.Landini (Milan,I), nucleotide biosynthesis was identified as the intracellular target of a known drug (azathioprine) (Antoniani et al, 2013, Applied Microb Biotechnol): this strategy might be used to decrease c-di-GMP in the cell.
We are now starting a new project aiming at understanding the structural and functional properties of the innate immune receptor STING.