title: Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors.
abstract: In Gram-negative bacteria, production of bis-(3',5')-cyclic diguanylic acid (c-di-GMP) by diguanylate cyclases (DGCs) is the main trigger for production of extracellular polysaccharides and for biofilm formation. Mutants affected in c-di-GMP biosynthesis are impaired in biofilm formation, thus making DGCs interesting targets for new antimicrobial agents with anti-biofilm activity. In this report, we describe a strategy for the screening for DGC inhibitors consisting of a combination of three microbiological assays. The primary assay utilizes an Escherichia coli strain overexpressing the adrA gene, encoding the DGC protein AdrA, and relies on detection of AdrA-dependent cellulose production as red colony phenotype on solid medium supplemented with the dye Congo red (CR). Presence of DGC inhibitors blocking AdrA activity would result in a white phenotype on CR medium. The CR assay can be performed in 96-well microtiter plates, making it suitable for high-throughput screenings. To confirm specific inhibition of c-di-GMP biosynthesis, chemical compounds positive in the CR assay are tested for their ability to inhibit biofilm formation and in a reporter gene assay which monitors expression of curli-encoding genes as a function of DGC activity. Screening of a chemical library using the described approach allowed us to identify sulfathiazole, an antimetabolite drug, as an inhibitor of c-di-GMP biosynthesis. Sulfathiazole probably affects c-di-GMP biosynthesis in an indirect fashion rather than by binding to DGCs; however, sulfathiazole represents the first example of drug able to affect biofilm formation by interfering with c-di-GMP metabolism.
authors.affiliation: Department of Biomolecular Sciences and Biotechnology, Università degli Studi di Milano, Milan, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Antoniani
authors.forename: Paola
authors.initials: P
authors.lastname: Bocci
authors.forename: Anna
authors.initials: A
authors.lastname: Maciag
authors.forename: Nadia
authors.initials: N
authors.lastname: Raffaelli
authors.forename: Paolo
authors.initials: P
authors.lastname: Landini
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000187:drug effects*; D003224:Congo Red; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D008826:Microbial Sensitivity Tests / Q000379:methods*; D019761:Phosphorus-Oxygen Lyases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D003224:Congo Red; C062025:bis(3',5')-cyclic diguanylic acid; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP