Cell Death Mechanisms Induced by Photo-Oxidation Studied at the Cell Scale in the Yeast Saccharomyces cerevisiae

Abstract

Blue light (400–430 nm) is known to induce lethal effects in some species of fungi by photo-oxidation caused by the excitation of porphyrins but the mechanisms involved remain poorly understood. In this work, we exposed the yeast Saccharomyces cerevisiae to a high density light flux with two-photon excitation (830 nm equivalent to a one-photon excitation around 415 nm) and used quasi real-time visualization with confocal microscopy to study the initiation and dynamics of photo-oxidation in subcellular structures. Our results show that the oxidation generated by light treatments led to the permeabilization of the plasma membrane accompanied by the sudden expulsion of the cellular content, corresponding to cell death by necrosis. Moreover, excitation in the plasma membrane led to very fast oxidation and membrane permeabilization (<60 s) while excitation at the center of the cell did not induce permeabilization even after a period exceeding 600 s. Finally, our study shows that the relationship between the laser power used for two-photon excitation and the time required to permeabilize the plasma membrane was not linear. Thus, the higher the power used, the lower the energy required to permeabilize the plasma membrane. We conclude that fungal destruction can be generated very quickly using a high density light flux. Better knowledge of the intracellular processes and the conditions necessary to induce necrosis should make it possible in the future to improve the efficiency of antimicrobial strategies photo-oxidation-based.

Microbial Photoinactivation by Visible Light Results in Limited Loss of Membrane Integrity

“…These results substantiate our assumption that direct membrane destruction might not be a substantial cause of microbial death. Furthermore, no permeabilization of the membrane was observed for S. cerevisiae [ 23 ] and S. enterica [ 21 ]. A decrease of membrane integrity as a function of photosensitizer concentration was noticed in antimicrobial photodynamic inactivation (aPDI) applying external photosensitizers [ 55 ], but high concentrations of photosensitizers are necessary to destroy the membrane.…”

“…Those damages could be of a kind that are not ROS induced, but due to disintegration of dead cells, which were inactivated by other causes than membrane damage. Dynamically investigated S. cerevisiae under 415 nm irradiation [ 23 ] demonstrated a sudden expulsion of intracellular components, while the membrane itself was intact until this event. Increased absorbance at 260 nm in the supernatant [ 15 ] could also be explained out of disintegrated cell envelopes, occurring just after cell death instead of being a part of the cause of death by photoinactivation.…”

“…Studying underlying conditions and optimal parameters, the mode of action was addressed, determining different inactivation mechanisms in multiple investigations. Among the cellular targets, the cell membrane was often named as an important site [ 15 , 16 , 17 , 18 , 19 ], but also other molecular targets have been recognized [ 20 , 21 , 22 , 23 , 24 ]. Oxidation of nucleic acids was often observed [ 21 , 22 , 24 , 25 ].…”

Optogenetic control of beta-carotene bioproduction in yeast across multiple lab-scales

“…The effective amount of light reaching the culture volume can be difficult to evaluate due to the geometry of the devices, the type of material and the thicknesses of the materials or media the light has to pass through. However, it is necessary to determine the minimal amount of light needed for maximal optogenetic activation as high doses of blue light can result in phototoxic effects 31,32 .…”

Use of red, far-red, and near-infrared light in imaging of yeasts and filamentous fungi

“…Laser power-dependence of photo-oxidation was studied in S. cerevisiae by two-photon microscopy ( λ = 830 nm) as well (Grangeteau et al 2018 ). Fungal destruction (plasma membrane permeabilization, cell necrosis) was initiated easily using high-density light flux, which may facilitate the development of future photo-oxidation-based antifungal therapies (Grangeteau et al 2018 ).…”