Inhibition of Pseudomonas aeruginosa biofilms: new molecular strategies targeting cyclic-di-GMP metabolism

Biofilms formed by bacterial pathogens are responsible of more than 70% of all infections in developed countries and are less sensitive to treatments with antimicrobial agents. The ubiquitous second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP) is used in most bacteria to control the switch to the biofilm lifestyle; c-di-GMP has no counterpart in eukaryotic cells and thus it is an ideal target to develop effective anti-biofilm strategies. The aim of this proposal is to study the metabolism of c-di-GMP in the opportunistic human pathogen Pseudomonas aeruginosa in order to find new targets for effective antibiofilm drugs. We plan to characterize strategic P. aeruginosa proteins involved in c-di-GMP metabolism (diguanylate cyclases-DGCs and phosphodiesterases-PDEs) and to study the connection between the c-diGMP pathway and other metabolic pathways relevant for pathogenesis. We developed the enzymatic and c-di-GMP binding assays using the reference DGC PleD from Caulobacter crescentus. We have measured the DGC activity of purified PleD and confirmed, by titration of PleD with c-di-GMP (employing Isothermal Titration CalorimetryITC), that PleD binds c-di-GMP (2 mol c-di-GMP/mol protein; Kd 0,9 μM) in agreement with literature data. We have tested putative DGC inhibitors, starting from selected compounds, previously identified in microbiological screening of a chemical library by Landini and coworkers. These compounds do not significantly inhibit PleD in vitro (up to 500 μM) suggesting that they do not inhibit c-di-GMP biosynthesis through direct interaction with DGC proteins, but likely affect the availability of nucleotide substrate(s). We have characterized putative P.aeruginosa DGCs or PDEs, focusing our attention on selected targets, i.e. the DGC PA1120 and the PDEs PA4781 and PA4108. The PA1120 protein (TpbB) is a DGC linking Las-induced quorum sensing (QS) to the formation of matrix exopolysaccharide (EPS) and extracellular DNA, necessary for biofilm development and coordinated group response in P. aeruginosa. We have produced the isolated catalytic domain of TpbB and measured its catalytic activity (0,1 nmol/min/mg); PA1120 is not significantly inhibited by c-di-GMP at low GTP concentrations. Identification and characterization of inhibitors are in progress. Few biochemical data are available on PDEs containing the HD-GYP domain, despite their importance in pathogenesis and their role in controlling biofilm formation. While EALtype PDEs hydrolyze c-di-GMP to linear diguanylate (pGpG), HD-GYPs completely hydrolyze c-di-GMP to GMP in a single reaction. In P.aeruginosa, 2 HD-GYP proteins are found, which are able to decrease c-di-GMP levels in vivo; these proteins contain the PDE catalytic