Biofilm is a microbial community whose formation is regulated by the dinucleotide cyclic-di-GMP. The GGDEF diguanylate cyclases (DGCs) and EAL or HD-GYP phosphodiesterases (PDEs) control the balance of c-di-GMP. DGCs synthesize the second messenger from two GTP molecules through the conserved GGDEF catalytic domain, while PDEs degrade it by producing pGpG through the EAL domain or GMP through the HD-GYP domain. Moreover there are proteins bearing both the GGDEF and EAL or HD-GYP domains named hybrid proteins. Most of these proteins have N-terminal sensing domains which can perceive environmental signals likely involved in modulating the catalytic moiety. The identification of extracellular signals acting on these sensory domains is crucial to understand the final output and the regulation of hybrid proteins. More in general the environmental stimuli able to regulate c-di-GMP levels (and the corresponding proteins involved) are still largely unknown.
We are currently characterizing the hybrid protein PA0575 from Pseudomonas aeruginosa, a model system for studying biofilm and a well known human pathogen. We have identified L-arginine as the small molecule able to trigger the activation of the downstream catalytic domains which lead to c-di-GMP hydrolysis to finally decrease biofilm formation.
Given that literature data suggest that arginine could be involved in controlling biofilm formation, we plan to deeply characterize the arginine dependent PA0575 signaling pathway. Moreover, since previous data indicated that the diatomic gas NO promote biofilm dispersal, we plan to design arginine analogues able to release NO to test whether a combined action on decreasing c-di-GMP levels could enhance the biological effect on biofilm dispersal.
Our knowledge about c-di-GMP signaling pathways remains limited, given that molecular mechanisms of regulation and signals affecting specific c-di-GMP-dependent circuits as well as targets of c-di-GMP action are often missing. Moreover, it is known that individual components involved in c-di-GMP homeostasis are controlled by specific environmental stimuli widening the complexity of the system. Up to now, few environmental signals controlling c-di-GMP-mediated signaling pathways have been identified and analyzed (Xu et al., 2017).
The innovation of this project is to identify signals controlling an hybrid protein such as PA0575 by integrating protein engineering and biophysical studies. Following a structure based approach, we demonstrated that this protein acts a sensor of arginine, as an environmental stimulus, to trigger the final output i.e. c-di-GMP degradation. P. aeruginosa is known to possess a variety of alternative anabolic and catabolic pathways as compared to other bacteria; it presents the ability to utilize many metabolites as energy and/or carbon sources; arginine is at the crossroad of peculiar metabolic pathways, such as the ADI, ADH, ADC ones, leading to N-metabolites and/or ATP (Lu et al., 2004).
On the other hand our study will contribute to obtain data to design arginine-based NO-donors molecules. Indeed controlling biofilms on surfaces of clinical and industrial importance has emerged as an important goal but up to now the approaches for controlling existing biofilms are significantly more limited. For these reasons the production of a small molecule able to (I) activate a PDE reaction leading to decrease c-di-GMP levels and (II) to release NO in the Pseudomonas aeruginosa periplasmic space at the same time could represent a powerful tool to treat biofilm, magnifying the positive effect of NO.
In conclusion this analysis will enrich our knowledge on c-di-GMP related signaling mechanism
and could contribute to propose novel targets and molecular data to design specific antibiofilm
strategies (Rasamiravaka et al., 2015).
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