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Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors

  • Applied Genetics and Molecular Biotechnology
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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.

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Acknowledgments

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).

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  1. Department of Biomolecular Sciences and Biotechnology, Università degli Studi di Milano, Via Celoria 26, 20133, Milan, Italy

    Davide Antoniani, Anna Maciąg & Paolo Landini

  2. Department of Molecular Pathology and Innovative Therapies, Section of Biochemistry, Università Politecnica delle Marche, Via Ranieri, 60131, Ancona, Italy

    Paola Bocci & Nadia Raffaelli

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  1. Davide Antoniani
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  2. Paola Bocci
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Correspondence to Paolo Landini.

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Antoniani, D., Bocci, P., Maciąg, A. et al. Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors. Appl Microbiol Biotechnol 85, 1095–1104 (2010). https://doi.org/10.1007/s00253-009-2199-x

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