# Anti-Biofilm Agents to Overcome _Pseudomonas aeruginosa Antibiotic Resistance_
## 2 The Biofilm Life Cycle
### Irreversible Adhesion and Biofilm Formation  
The main intracellular signaling pathway governing biofilm formation and maturation mechanisms is quorum sensing (QS) [26]. QS is a bacterial communication system which regulates gene expression in proportion to cell density. When the quorum is reached, small extracellular molecules called autoinducers (AIs) are released and bind to their receptors in a neighboring cell to activate the transcription of virulence genes. In _P. aeruginosa_, three QS circuits named _las_, _rhl_ and _pgs_ are described. The _las_ and _rhl_ systems can be found  
Figure 2: Synthesis and enzymatic degradation of c-di-GMP and its role in cell function. Diguanylate cyclases (DGCs) synthesize c-di-GMP from two molecules of guanosine triphosphate (GTP). Phosphodiesterases (PDEs) hydrolyze c-di-GMP into pGpG, then two molecules of guanosine monophosphate (GMP). c-di-GMP controls bacterial motility, biofilm formation, cell cycle progression and virulence by binding to different receptor/effector proteins. The c-di-GMP molecule appears in purple.  
in several pathogens. They produce and recognize acyl-homoserine lactone-like AI: the \(N\)-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C\({}_{12}\)-HSL) odDHL and the \(N\)-butanoyl-L-homoserine lactone (C\({}_{4}\)-HSL) BHL, that activate their transcription factors LasR and RhIR, respectively. A third specific _pqs_ system produces and detects the 2-heptyl-3-hydroxy-4(1\(H\))-quinolone PQS (_Pseudomonas_ quinolone signal). This AI activates its transcription factor PqsR.  
The extracellular aqueous polymeric matrix is composed of proteins, rhamnpids, extracellular DNA (eDNA) and exopolysaccharide named Pel, Psl and alginate [32]. Psl and Pel maintain structural integrity and are necessary for the cell adhesion to biotic or abiotic surfaces. Their production is stimulated by c-di-GMP. The alginate is essentially secreted by mucoid strains in the lungs of CF patients in response to stressful conditions (nutrients deprivation, antibiotics, slower cell growth, high osmotic pressure). In addition to preserving matrix structural cohesion, the alginate helps evade the immune system by resisting phagocytosis and macrophage-mediated free radical damage, but also diminishes antibiotic diffusion [33; 34; 35]. eDNA, which is excreted following eukaryotic cell lysis induced by pyocyanin, is mainly a cell-cell interconnecting component. It contributes to the mushroom-like structure of the biofilm, can serve as a nutrient source and appears as a proinflammatory molecule by activating neutrophils. It can also reduce antibiotic penetration and promote horizontal gene transfer, and it takes part in the coordination of cell movements during twitching motility, thus contributing to biofilm expansion [33; 36]. In the early stages of biofilm formation, soluble lectins A and B (LecA and LecB), aggregating proteins implemented at the outer membrane surface, strengthen the biofilm stability by binding to matrix and membrane saccharides from other cells [37]. At this stage, rhamnpids stimulate the LPS composition adaptation and the swarming motility. After irreversible adhesion and production of the extracellular matrix, bacteria grow in microcolonies and proliferate in the biofilm.