In vivo NMR metabolic profiling of fabrea salina reveals sequential defense mechanisms against ultraviolet radiation
Authors: Marangoni R., Paris D., Melck D., Fulgentini L., Colombetti G., Motta A.
Autors Affiliation: Dipartimento di Informatica, Università di Pisa, Pisa, Italy; Istituto di Biofisica del CNR, Pisa, Italy; Istituto di Chimica Biomolecolare del CNR, Pozzuoli, Italy
Abstract: Fabrea salina is a hypersaline ciliate that is known to be among the strongest ultraviolet (UV)-resistant microorganisms; however, the molecular mechanisms of this resistance are almost unknown. By means of in vivo NMR spectroscopy, we determined the metabolic profile of living F. salina cells exposed to visible light and to polychromatic UV-B + UV-A + Vis radiation for several different exposure times. We used unsupervised pattern-recognition analysis to compare these profiles and discovered some metabolites whose concentration changed specifically upon UV exposure and in a dose-dependent manner. This variation was interpreted in terms of a two-phase cell reaction involving at least two different pathways: an early response consisting of degradation processes, followed by a late response activating osmoprotection mechanisms. The first step alters the concentration of formate, acetate, and saturated fatty-acid metabolites, whereas the osmoprotection modifies the activity of betaine moieties and other functionally related metabolites. In the latter pathway, alanine, proline, and sugars suggest a possible incipient protein synthesis as defense and/or degeneration mechanisms. We conclude that NMR spectroscopy on in vivo cells is an optimal approach for investigating the effect of UV-induced stress on the whole metabolome of F. salina because it minimizes the invasiveness of the measurement.
Journal/Review: BIOPHYSICAL JOURNAL
Volume: 100 (1) Pages from: 215 to: 224
More Information: Spectrum Pharmaceuticals. Pancreatic Cancer Action, PCA. National Council for Scientific Research, NCSR. – We have demonstrated that in vivo cell NMR spectroscopy is an efficient tool for investigating UV-induced metabolic perturbations of F. salina . The unsupervised pattern-recognition analysis PCA visualized the pattern of metabolic differences between Vis-irradiated CS and TS, irradiated with the entire spectrum (UV-B + UV-A + Vis). We proved that UV radiation induces clearly metabolic perturbations in the hypersaline ciliate, and we were able to distinguish two different trends of UV-induced responses. Acetate, formate, and saturated fatty acids are prevalently discriminant for immediate reaction to UV irradiation, whereas ectoine, glycine betaine, proline, and alanine are more characteristic of a late reaction. Such metabolic alterations suggest several effects caused by UV solicitations, including permeability (saturated fatty acids) and osmoregulatory problems (ectoine and glycine betaine), oxidation (formate) and impairment of protein synthesis (proline and alanine), and degradation (acetate). We thank Alberto Pietrangeli for his contribution in cell culturing. This work was partly supported by the National Research Council of Italy and personal funds of R.M. and G.C.KeyWords: UV-B IRRADIATION; TRANSPORT; FORMATE; PROTEIN; STRESS; DAMAGE; ACID