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.

Identification of Anti-Inflammatory and Anti-Hypertensive Drugs as Inhibitors of Bacterial Diguanylate Cyclases

“…These results correlate very well with the inhibitory activity and affinity of the compounds against WspR and YdeH measured in the biochemical assays, suggesting that a reduction in intracellular c-di-GMP levels promoted either by compounds that direct inhibits DGCs or by interference with the bacterial nucleotide pool, might be responsible for the reducing of cell aggregates. Finally, in agreement with previous results reported by Antoniani et al,26,27 the potent anti-biofilm agent sulfathiazole almost completely abolished DGC-induced cell aggregation, validating our assays. Growing evidence has established that high c-di-GMP intracellular concentrations inhibit bacterial motility while stimulates the production of extracellular polysaccharides and other adhesion factors, leading to a transition between planktonic and biofilm lifestyles.…”

“…26,27 In this report, we used an in silico strategy to identify DGC inhibitors. Since the selected compounds are FDA-approved drugs, their scaffolds lie within a privileged chemical-biology space, where their pharmacokinetic properties and toxicological profiles are well established.…”

“…24,25 In vivo screenings showed that sulfathiazole and azathioprine have an impact on biofilm formation by indirectly decreasing c-di-GMP concentrations, probably targeting the biosynthesis of GTP. 26,27 Alternatively, strategies based on c-di-GMP modification and GTP analogs also identified specific DGCs inhibitors targeting both active and the allosteric binding sites. 28,29 In this report, we identified novel inhibitors of bacterial DGCs by virtual screening methods (VS) from the DrugBank database.…”

Infections associées aux biofilms

“…Une autre piste serait d'interférer avec la voie du di-GMPc qui est impliquée dans la régulation du mode de vie biofilm (Figure 2). L'inhibition de la biosynthèse de di-GMPc par le sulfathiazole ou le fluorouracile a été décrite [52]. Bien qu'une étude clinique ait montré que l'utilisation de cathéters imprégnés de 5-fluorouracile permettait de réduire le risque de colonisation bactérienne, la toxicité chez l'homme du sulfathiazole ou du fluorouracile reste à évaluer [53].…”

Current Research Approaches to Target Biofilm Infections.

“…The signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) accumulates under conditions that promote EPS production and biofilm formation, and appears to be universally conserved in Gram-negative bacteria (64). Therefore, researchers have screened for antibiofilm molecules that block the synthesis of c-di-GMP or that reduce the expression of c-di-GMP-controlled promoters.…”