Discovery of novel inhibitors of bacterial biofilm formation targeting enzymes involved in the metabolism of the second messenger cyclic-di-GMP

INTRODUCTION: Bacteria are able to switch between a single cell (planktonic) lifestyle and a biofilm (community) lifestyle. In pathogenic bacteria, growth as biofilm protects bacterial cells against the host immune system and increases tolerance to antibiotic treatment, thus resulting in chronic infections. The bacterial second messenger cyclic-di-GMP (c-di-GMP) plays a pivotal role in biofilm formation, by promoting production of adhesion factors such as extracellular polysaccharides (EPS). Two classes of enzymes are involved in c-di-GMP metabolism: diguanylate cyclases (DGCs), which synthesize c-di-GMP, and phosphodiesterases (PDEs) that hydrolyze the signal molecule. Usually, a high intracellular c-di-GMP concentration correlates with EPS production and biofilm formation. However, exposure of Pseudomonas aeruginosa to subinhibitory concentration of the aminoglycoside antibiotic tobramycin stimulates biofilm formation through the PDE activity of the Arr protein. This result suggests that biofilm formation can also respond to low intracellular c-di-GMP levels. The enzymes involved in c-di-GMP metabolism are widely conserved in Bacteria, but they are not present in upper eukaryotes. Thus, the proteins involved in c-di-GMP metabolism are a very interesting target for antimicrobial compounds with anti-biofilm activity. METHODS: To screen for specific inhibitors of enzymes involved in c-di-GMP metabolism, we have developed a set of microbiological assays that rely on detection of c-di-GMP-dependent EPS production using specific dyes such as Congo Red. Intracellular c-di-GMP levels can then be measured directly by HPLC determination. RESULTS: We have tested over 1,000 chemical compounds in our screening system. Interestingly, we found that sulfathiazole, but not other chemically-related sulfonamide antibiotics, can inhibit both DGC and PDE activity, possibly suggesting that sulfathiazole can bind to the c-di-GMP binding domain of these proteins. DISCUSSION: Sulfathiazole represents the first example of a drug able to affect biofilm formation by interfering with c-di-GMP metabolism. The precise mechanism of biofilm inhibition by sulfathiazole is currently under investigation.