REGULATION OF BACTERIAL ADHESION FACTORS BY THE SIGNAL MOLECULE C-DI-GMP: SPECIFIC EFFECTS AT GENE EXPRESSION LEVELS AND SEARCH FOR NOVEL INHIBITORS

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. 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. In first part of my thesis I developed a screening system for specific inhibitors of DCGs based on 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. I tested over 1,000 chemical compounds in my screening system: I found that azathioprine and sulfathiazole two antimetabolites able to inhibit nucleotide biosynthesis impair c-di-GMP production. My results confirm previous literature data showing that perturbation in intracellular nucleotide pools negatively affect biofilm formation in Gram negative bacteria. In second part of this thesis I discussed the role of yddV-dos operon which encodes a DGC and a PDE acting as a protein complex. Both YddV and Dos proteins affect the production of the main adhesion factors of Escherichia coli: curli and the EPS poly-N-acetylglucosamine (PNAG). In particular, the YddV-Dos complex regulates transcription of the csgBAC operon, which encodes curli structural subunits while not affecting the expression of the regulatory operon csgDEFG. In addition we showed that YddV stimulating the transcription of PNAG biosynthetic operon pgaABCD affects PNAG-mediated biofilm formation. Thus, the yddV-dos operon constitutes a main regulatory element in adhesion factors production. Finally, I was able to show that PNAG production is controlled by polynucleotide phosphorylase (PNPase) at post transcriptional level. My results demonstrate the integration of signal molecules and regulatory protein in adhesion factor production, underling the complexity of biofilm regulation in E. coli.