Nutrients such amino acids play key roles in shaping the metabolism of microorganisms in natural environments and in host¿pathogen interactions. Among nutrients, Arginine can be perceived by the opportunistic human pathogen Pseudomonas aeruginosa to control the second messenger 3¿-5¿cyclic diguanylic acid (c-di-GMP). This dinucleotide is involved in biofilm formation, a multicellular lifestyle which confers to the bacterium protection against antibiotics and unfavorable conditions.
The intracellular levels of c-di-GMP are controlled by the rate of its synthesis and degradation, regulated by diguanylate cyclases (GGDEF signature) and phosphodiesterase (EAL and HD-GYP signatures) respectively.
Our group recently found that RmcA (Redox regulator of c-di-GMP) in the Pseudomonas aeruginosa is able to respond to Arginine to control c-di-GMP levels (Paiardini et al., 2018).
RmcA is a multidomain membrane protein harbouring a Venus Fly Trap (VFT) domain devoted to periplasmic Arginine binding, a transmembrane helix and a cytoplasmic part displays 3 PAS and 1 LOV domains linked to GGDEF-EAL catalytic tandem, where the hydrolysis of c-di-GMP occurs (Mantoni et al., 2018).
The aim is to gain details on the mechanism of control of Arginine on c-di-GMP hydrolysis. Understanding this mechanism will be useful to promote biofilm dispersion, in which cells are more sensitive to antimicrobic compounds, to treat chronic infection with combined strategies.
We starting to investigate about the activity of the same truncated constructs on RmcA from P. aeruginosa and putida, in order to identify possible common structural determinants responsible for the transduction mechanism. Furthermore, the architecture of transducers such RmcA is too complex to be suitable for full-length studies, so we identified a prototype protein able to change c-di-GMP levels in response to Arginine, showing a minimal architecture i.e., VFT, transmembrane helix, catalytic domain(s) (Fig.1).
In 2000, the Center for Disease Control and Prevention (CDC) announced that biofilm-mediated infections are among the seven major health problems facing the 21st century.
Levels of cyclic dinucleotides in prokaryores are linked to crucial cellular mechanisms as they control the remodeling of basic metabolism in response to external stimuli, as in the case of bacterial biofilm. The relevance of these second messengers is known but greater understanding of the molecular details involved in their regulation in response to nutrients is needed to develop effective cell fate control strategies and thus target chronic biofilm infections.
The potential therapeutic impact of biofilm inhibition via c-di-GMP in human disease is enormous. Despite promising in vitro results, so far the direct targeting of enzymes involved in c-di-GMP turnover has been found to be ineffective and poorly selective on cells (Schirmer T et al., 2009; Krasteva PV et al., 2012; Sondermann H et al., 2012). Biofilms cause infections with high mortality (Khatoon et al., 2018), especially chronic and hospital-based ones. They are also dangerous because they colonize implantable devices which are more sensible to contamination than native tissues. Biofilms also create other problems such as deterioration of dental surfaces, surface contamination in the food industry, water pipes and deterioration of air quality in ventilation and air treatment systems (Bryers JD, 2008) . In the sessile state, bacteria are sensitive to antimicrobials and therefore being able to regulate c-di-GMP levels from the environment is important in order to use a combined approach of nutrients and antibiotics to treat infections.
Being the LOV domain found in other GGDEF-EAL proteins, we propose that these results may be relevant to understand the mechanism of c-di-GMP control under different environmental stimuli; the redox switch we have observed and that we plan to deeply characterize may represent the molecular strategy linking oxygen limitation and biofilm homeostasis, an adaptation relevant to chronic infections sites particularly in P. aeruginosa.