Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors
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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.
Keywordsc-di-GMP Diguanylate cyclase Biofilm formation High-throughput screening Antimicrobial drugs Sulfathiazole
We thank Grant Burgess for a critical reading of the manuscript, Pierfausto Seneci for providing us with the Prestwick Chemical Library, and Susanna Marcandalli for technical assistance. This study was supported by the Italian Foundation for Research on Cystic Fibrosis (project FFC#9/2006, adopted by “Gruppo Rocciatori di Belluno”) and by the CHEM-PROFARMA-NET Research Program of the Italian Ministry for University and Research (Project RBPR05NWWC_004).
- Bardonnet N, Blanco C (1992) uidA antibiotic resistance cassettes for insertion mutagenesis, gene fusion and genetic constructions. FEMS Microbiol Lett 93:243–248Google Scholar
- Bennhold H (1922) Eine spezifische Amyloidfärbung mit Kongorot [Specific staining of amyloid with Congo red]. Münch Med Wochenschr 69:1537–1538Google Scholar
- Clinical and Laboratory Standards Institute (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A7, 7th edn. Clinical and Laboratory Standards Institute, Wayne, PAGoogle Scholar
- Haasum Y, Ström K, Wehelie R, Luna V, Roberts MC, Maskell JP, Hall LM, Swedberg G (2001) Amino acid repetitions in the dihydropteroate synthase of Streptococcus pneumoniae lead to sulfonamide resistance with limited effects on substrate K(m). Antimicrob Agents Chemother 45:805–809CrossRefGoogle Scholar
- Kulasakara H, Lee V, Brencic A, Liberati N, Urbach J, Miyata S, Lee DG, Neely AN, Hyodo M, Hayakawa Y, Ausubel FM, Lory S (2006) Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence. Proc Natl Acad Sci U S A 103:2839–2844CrossRefGoogle Scholar
- Miller JH (ed) (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold spring Harbor, NYGoogle Scholar
- Perry JR, Miller GR (1989) Quality control slide for potassium hydroxide and cellufluor fungal preparation. J Clin Microbiol 27:1411–1412Google Scholar
- Ross P, Mayer R, Benziman M (1991) Cellulose biosynthesis and function in bacteria. Microbiol Rev 55:35–58Google Scholar
- Simm R, Morr M, Remminghorst U, Andersson M, Römling U (2009) Quantitative determination of cyclic diguanosine monophosphate concentrations in nucleotide extracts of bacteria by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Anal Biochem 386:53–58CrossRefGoogle Scholar