7 minute read
Serena Rinaldo
Sensing Arginine through the Venus Fly Trap domain to control c-di-GMP levels in Pseudomonas aeruginosa: molecular mechanism and metabolic effects of signal transduction.
Serena Rinaldo
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RESEARCH AREA: Genetics and biology of microorganisms
Department of Biochemical Sciences “A. Rossi Fanelli serena.rinaldo@uniroma1.it
RmcA from the opportunistic human pathogen Pseudomonas aeruginosa is a multisensory transducer able to control c-di-GMP (3',5'-cyclic diguanylic acid) levels in response to different environmental stimuli. C-di-GMP is a master regulator of biofilm formation, a multicellular lifestyle which confers to the bacterium protection against antibiotics and unfavourable conditions. RmcA may control c-di-GMP hydrolysis (thus yielding the corresponding linear derivative, i.e. the nanoRNA pGpG) in response to L-arginine (1, 2), a pleiotropic nutrient able to sustain both N/C needs and ATP production; in light of this, this nutrient is associated to chronic infections, biofilm/virulence and antibiotic resistance (3). Interestingly, previous data on RmcA indicated that the catalytic activity is sensitive to electrons availability, and the corresponding mutant phenotype superposes to those related to the phenazine depletion (4). Phenazine represents a class of molecules able to shuttle electrons among the biofilm community and this extracellular redox cycling is crucial to enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors (5,6). We have unveiled the molecular mechanism linking electron perceiving and nutrient sensing to finally control c-di-GMP consumption activity (manuscript in preparation). The relevance of such results prompted us to widen the profiling of RmcA in its cellular background, by analysing the corresponding protein in P. putida, a bacterium relevant for its biotechnological potential, in collaboration with Espinosa Urgel (Spain). This parallel activity was crucial to identify the proper conditions to analyse in a real time fashion the effect of nutrients on the respiratory activity.
RmcA is characterized by a complex architecture encompassing the periplasmic space to the intracellular compartment through a transmembrane (helix) portion; as summarized in Figure 1, the Arginine sensing portion involves a Venus Fly Trap domain, while the cytoplasm part displays 3 PAS and 1 LOV domains linked to the diguanylate cyclases (GGDEF) and the phsphodiesterases (EAL) catalytic tandem, where the hydrolysis of c-diGMP occurs (2).
Figure 1. Domain organization of RmcA (FL); boxed the cytoplasmic portion used in this study (cRmcA)
While it is clear that L-arginine is recognized by the periplasmic domain, little is known about the electron sensing, which is a broad-spectrum activity being associated to the anaerobic metabolism taking place in P. aeruginosa biofilm. We found that the LOV domain is the key portion of the protein able to perceive and directly control the catalytic moiety GGDEF-EAL.
In light of this, we performed the biochemical characterization of this protein by different approaches: 1. Use of truncated versions of RmcA to dissect the catalytic mechanism of c-di-GMP consumption, its allosteric control in a redox dependent way (cRmcA). 2. Use of the full-length protein as such or embedded within nanodisc, by taking advantage of the prototype bearing the minimal architecture required to recognize Arginine to control c-di-GMP turnover, characterized in the first part of the research. 3. Metabolic re-programming occurring upon RmcA depletion in a model system such as P. putida. This set up was found to be ideal to detect the effect of nutrients supplementation. This report includes the relevant outcomes obtained in the period 18-30 months of the project. In summary:
1. cRmcA
R
R
ArginineArginine
R
We found that the phosphodiesterase activity (PDE) of the EAL domain is dramatically controlled upon nucleotide binding (Figure 2). Thanks to a TransNational Access into the EU Facility Figure 2. Schematic model of cRmcA activation; nucleotides exert their MOSBRI (grant agreement N° allosteric role by promoting dimerization of the protein and of the 101004806 MOSBRI, Pasteurcatalytic do GGDEF-EALVenus FlyTrap Venus FlyTrap main. modul E e lectrons negatively tun is shown for simplicity. e the PDE activity. Only LOVPFBMI access provider), we found that FAD, GTP and even cdi-GMP itself promote protein i n i n dimerization, thus confirming the huge P r o t e P r o t e Pas re-arrangement which takes place upon
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R m c R m c GGDEF Lov RmcA activation. Moreover, oxygen GGDEF depletion and FAD reduction finely tune EAL Domain EAL Msp1D1Msp1D1 NanodiscNanodisc PDE activity, thus confirming the tight link between c-di-GMP control and
Figure 3. RmcA and nanodisc. Model of RmcA into a nanodisc.
anaerobic metabolism in biofilm. This evidence has a more general relevance, being P. aeruginosa anaerobic metabolism a hallmark of biofilm-mediated infections; moreover, the PAS-PAS-PAS-LOV architecture has been found in many other transducers.
2. Full-length
By taking advantage of the results presented in the previous report, we purified the fulllength protein both in detergent and embedded within the nanodisc (Figure 3). The proper nanodisc scaffold has been selected and the right lipids:nanodisc ratio found to promote RmcA loading. The protein was found to be active as PDE, both in detergent and in nanodisc, and therefore this result paved the way for studying the arginine dependent RmcA activation. The nanodisc isolation gave us the unique opportunity to study the protein in a more native environment.
3. Nutrient-mediated respiration control
In order to better understand the role of RmcA in response to L-arginine, we used O2 different model systems to define the best protocol. These data were obtained by means of Seahorse measurements in collaboration with Urgel Espinosa, thanks to his TransNational Access into the EU Facility MOSBRI
Figu the re 4. Schemtic figure). Oxyge model n level o s f respi were ratory prof measured ilin in g u a pon real nutri -time ent fas addi hion tion ( and the the apple i oxyge n n (grant agreement N° consumption rate measured to gain a quantitative evaluation of the respiratory 101004806 MOSBRI, DSBactivity. UROM access provider). We obtained a detailed characterization of bacterial respiration which is a unicum with this kind of approach. The effect of arginine supplementation was investigated and found to affect the oxygen levels in P. putida. We plan to take advantage of this study to characterize the P. aeruginosa RmcA mutant, also under hypoxic or anaerobic conditions.
ACKNOWLEDGEMENTS
The European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101004806 MOSBRI is gratefully acknowledged.
REFERENCES
1) Paiardini A, Mantoni F, Giardina G, Paone A, Janson G, Leoni L, Rampioni G,
Cutruzzolà F, Rinaldo S. (2018) A novel bacterial l-Arginine sensor controlling cdi-GMP levels in Pseudomonas aeruginosa. Proteins. 86(10):1088-1096.
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L, Petrelli R, Ricciutelli M, Leoni L, Rampioni G, Arcovito A, Rinaldo S*,
Cutruzzolà F*, Giardina G. (2018) Insights into the GTP-dependent allosteric control of c-di-GMP hydrolysis from the crystal structure of PA0575 protein from
Sciences. 2022, 23 (8), 4386.
4) Okegbe C, Fields BL, Cole SJ, Beierschmitt C, Morgan CJ, Price-Whelan A,
Stewart RC, Lee VT, Dietrich LEP. Electron-shuttling antibiotics structure bacterial communities by modulating cellular levels of c-di-GMP. Proc Natl Acad
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JK, Tender LM, Newman DK. Extracellular DNA Promotes Efficient Extracellular
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Phenazine production promotes antibiotic tolerance and metabolic heterogeneity in Pseudomonas aeruginosa biofilms. Nat Commun. 2019 Feb 15;10(1):762.
Publications
Rossi, C.S., Barrientos-Moreno, L., Paone, A., Cutruzzolà, F., Paiardini, A., EspinosaUrgel, M., Rinaldo, S. Nutrient Sensing and Biofilm Modulation: The Example of Larginine in Pseudomonas. International Journal of Molecular Sciences. 2022, 23 (8), 4386. IF 5,924.
Cutruzzolà, F., Paiardini, A., Scribani Rossi, C., Spizzichino, S., Paone, A., Giardina, G., Rinaldo, S. A conserved scaffold with heterogeneous metal ion binding site: the multifaceted example of HD-GYP proteins. Coordination Chemistry Reviews. 2022 450, 214228. IF 22,315.
Nardella, C., Malagrinò, F., Pagano, L., Rinaldo, S., Gianni, S., Toto, A. Determining folding and binding properties of the C-terminal SH2 domain of SHP2 Protein Science. 2021, 30 (12), 2385-2395. IF 6,725.
Fiorillo, A., Battistoni, A., Ammendola, S., Secli, V., Rinaldo, S., Cutruzzola, F., Demitri, N., Ilari, A. Structure and metal-binding properties of PA4063, a novel player in periplasmic zinc trafficking by Pseudomonas aeruginosa Acta Crystallographica Section D: Structural Biology, 2021, 77, 1401-1410. IF7,652.
Zuliani, I., Lanzillotta, C., Tramutola, A., Barone, E., Perluigi, M., Rinaldo, S., Paone, A., Cutruzzolà, F., Bellanti, F., Spinelli, M., Natale, F., Fusco, S., Grassi, C., Di Domenico, F.
High-fat diet leads to reduced protein o-glcnacylation and mitochondrial defects
promoting the development of alzheimer’s disease signatures. International Journal of Molecular Sciences, 2021, 22 (7), 3746. IF 5,924.
Research Group
Serena Rinaldo Associate Prof. (PI) Alessandro Paiardini Associate Prof. Adele Di Matteo Researcher (IBPMCNR) Alessio Paone Researcher Chiara Scribani Rossi PhD student in Biochemistry Elisabetta Scarchilli Research Fellow
Collaborations
Lars Dietrich, Columbia University (US) Manuel Espinosa Urgel, Estacion Experimental del Zaidin. CSIC (Spain).
