## 5 Coating Materials with Embedded Antibiotics and Other Antibofilm Agents
### Biofilm Control by Inhibitors or Modulators of Bacterial Signaling Processes
#### 5.5.2 Antibiofilm Coatings Are Interfering with c-di-GMP Signaling  
Nitric Oxide-Releasing Coatings and Other Small Molecule Modulators of c-di-GMP-Dependent Signal Transduction Pathways  
The central role played by the second messenger cyclic-diguanosine monophosphate (c-di-GMP, Scheme 11) in biofilm formation and dispersion was well established, although the precise molecular mechanisms of the signal transduction remain mostly unelucidated [55]. However, as we previously pointed out in Part I of this review, it is known that high cellular levels of c-di-GMP promote sessility and biofilm formation by binding to the transcription factor FleQ protein, c-di-GMP down-regulates expression of flagella biosynthesis genes and up-regulates transcription of genes in the pel operon encoding exopolysaccharides (EPS), which are main constituents of the extracellular sheltering matrix required for biofilm formation in _P. aeruginosa_[56; 57]. Conversely, low c-di-GMP levels down-regulates the production of biofilm matrix components and promote biofilm dispersal and returning to the planktonic mode of life. Targeting c-di-GMP signaling systems appears as a promising way to control biofilms with two major advantages over other therapeutic strategies. First, c-di-GMP-mediated signaling was not observed in higher eukaryotic organisms, and therefore, inhibition of this signal by some small molecules is expected to be harmless or to exhibit low toxicity to the infected host, while strongly affecting bacterial biofilms (selective toxicity). Second, although c-di-GMP regulates the lifestyle of bacteria, it is not necessary for bacterial growth [58] and, therefore, would not select for resistant bacteria as other classical bactericidal and bacteriostatic therapeutic agents usually do. The intracellular level of this central biofilm life cycle regulator depends on its turn on the rate of c-di-GMP production and degradation. C-di-GMP is synthesized from two GMP molecules by the enzymes called diguanylate cyclases (DGCs) and is degraded under the hydrolytic activity of two types of specific phosphodiesteraseer enzymes (PDEs), one that linearizes the molecules converting it into 5'-phosphouguanylyl-(3'\(\rightarrow\)5')-guanosine (pGpG) and another one that split the molecule into two GMP molecules. Both DGCs and PDEs have conserved catalytic domains as follows Gly-Gly-Asp-Glu-Phe for DGCs and Glu-Ala-Leu/His-Asp--Gly-Tyr-Pro for the PDEs producing pGpG and GMP, respectively [59; 60]. Figure 10: An example is illustrating the click-chemistry strategy used to attach quorum sensing inhibitors dihydropyrrolones (DHPs) to a glass substrate [52]. Adapted with permission from Quorum sensing inhibitory activities of surface-immobilized antibacterial dihydropyrrolones via click chemistry. Copyright (2014) Elsevier. Nitric oxide (NO) is an endogenous antimicrobial agent produced by macrophages in response to bacterial infection. Nitric oxide is a strong oxidizing agent that damages biological membranes, thereby diffusing inside the cell and altering DNA and proteins. In 2006, the research group led by [PERSON] reported that the ubiquitous biologically active signal molecule NO is able to induce biofilm dispersal at low, non-toxic concentrations in _P. aeruginosa_. Based on their discovery, the authors proposed a novel treatment to eradicate biofilm infections with this pathogen, the combined administration of a NO-donor, and an antimicrobial agent [61]. Later, the same researchers showed that non-cytotoxic NO concentrations stimulate PDEs activity leading to an overall decrease in intracellular c-di-GMP and biofilm dispersal [62]. The precise molecular basis for PDEs activation has been elucidated in _Shewanella woodyi_. Over 250 known bacterial species, including _S. woodyi_, encode heme nitric oxide/oxygen binding (HNOXs) proteins, which act as high affinity NO sensors [63]. When a heme-NO complex forms, the HNOX protein of _S. woodyi_ binds and activates a PDE enzyme [64; 65].