Date Published: July 12, 2017
Publisher: Public Library of Science
Author(s): Wafa Mihoubi, Emna Sahli, Ali Gargouri, Caroline Amiel, Carl G. Maki.
p53 over expression in yeast results in cell death with typical markers of apoptosis such as DNA fragmentation and phosphatidylserine externalization. We aimed to substitute/supplement classical fluorescent techniques (TUNEL, Annexin V, ROS detection) usually used to detect biochemical changes occurring during yeast apoptosis mediated by p53 over expression and the effect of anti-apoptotic purified molecules from Nigel (Nigella sativa) extracts on these same yeasts by the label free technique of FTIR spectroscopy. The comparison of the entire IR spectra highlighted clear modifications between apoptotic p53-expressing yeasts and normal ones. More precisely, DNA damage was detected by the decrease of band intensities at 1079 and 1048 cm-1. While phosphatidylserine exposure was followed by the increase of νsCH2 and νasCH2 bands of unsaturated fatty acids that were exhibited at 2855 and 2926 cm-1, and the appearance of the C = O ester functional group band at 1740 cm-1. In a second step, this FTIR approach was used to estimate the effect of a purified fraction of the Nigel extract. The modulation of band intensities specific to DNA and membrane status was in agreement with apoptosis supression in presence of the Nigel extracts. FTIR spectroscopy is thus proven to be a very reliable technique to monitor the apoptotic cell death in yeast and to be used as a means of evaluating the biomolecules effect on yeast survival.
Fourier-transform infrared (FTIR) spectroscopy is a vibrational spectroscopic technique used to solve the chemical composition information of a sample and to provide its “molecular fingerprint” . This technique has been previously explored for different measurement modes in many biological systems [2,3] to become a powerful tool for the cell components analysis, such as nucleic acids  proteins  and membranes . This technique has also been useful for complex biological systems, such as tissues  and microorganisms [3,8,9] because of its ability to analyze both qualitative and quantitative, molecular and structural indications for samples components. FTIR spectroscopy is a non-destructive technique [10,11], that allows a rapid analysis of biochemical and structural changes (without the need for reagents) such as DNA, protein and cell membrane [10,12–14]. The analysis of spectral data provides qualitative and quantitative information of a cell component on the basis of peak’s shifts, bandwidths and band intensities. Under stress, data can simultaneously determine changes in proteins, lipids, carbohydrates and nucleic acids at the level of functional groups [10, 15]. FTIR spectroscopy was used by lot of researchers to study metabolic alterations in stressed microorganisms such as the bacterial response to antibiotics , the effect of starvation [17,18], and of different environmental stresses [19–24], as well as yeast alterations by chemical compounds such as ethanol [25,26]. Bellisola and Sorio  consider that FTIR spectroscopy in medical biology is an interesting emerging opportunity.
The most typical markers of apoptosis are DNA fragmentation and phosphatidylserine externalization. Both markers are well conserved in yeast and are harnessed here to show that p53 overexpression induces apoptosis (; this work). The present FTIR study of yeast apoptosis highlighted several intensity changes of bands that are characteristic of the same markers. These findings prove that the FTIR technique is a good tool to study the biochemical changes in yeast during apoptosis.
This study confirms that FTIR spectroscopy technique is a very sensitive and useful tool for the detection and monitoring of biochemical changes of apoptosis mediated by p53 over-expression in yeast S. cerevisiae, previously analyzed by flow-cytometry. Moreover, highlighted apopotosis marker bands were used to evaluate the protective effect of Nigella sativa extract on p53-mediated apoptosis. This rapid and specific method could undoubtedly be used for a large screening of other natural biomolecules on yeast p53-mediated apoptosis.