Abstract
Regulated cell death (RCD) encompasses the activation of cellular pathways that initiate and execute a self-dismissal process. RCD occur over a range of stressors doses that overcome pro-survival cellular pathways, while higher doses cause excessive damage leading to passive accidental cell death (ACD). Hydrogen peroxide (HP) has been proposed as a potential tool to control harmful cyanobacterial blooms, given its capacity to remove cyanobacterial cells and oxidize cyanotoxins. HP is a source of hydroxyl radicals and is expected to induce RCD only within a limited range of concentrations. This property makes this compound very useful to better understand stress-driven RCD. In this work, we analyzed cell death in microcystin-producing Microcystis aeruginosa by means of a stochastic dose response model using a wide range of HP concentrations (0, 0.29, 1.76, 3.67, 7.35, 14.70, and 29.5 mM). We used flow cytometry and unsupervised classification to study cell viability and characterize transitional cell death phenotypes after exposing cells to HP for 48 and 72 h. Non-linear regression was used to fit experimental data to a logistic cumulative distribution function (cdf) and calculate the half maximal effective concentration (EC50). The EC50 of M. aeruginosa exposed to HP were 3.77 ± 0.26 mM and 4.26 ± 0.22 mM at 48 and 72 h, respectively. The derivative of cdf (probability density function; pdf) provided theoretical and practical demonstration that EC50 is the minimal dose required to cause RCD in 50% of cells, therefore maximizing the probability of RCD occurrence. 1.76 mM HP lead to an antioxidant stress response characterized by increased reactive oxygen species (ROS) levels and HP decomposition activity. The exposure of 3.67 mM HP induced a dose-related transition in cell death phenotype, and produced several morphological changes (a less dense stroma, distortion of the cell membrane, partial disintegration of thylakoids, extensive cytoplasmic vacuolation and highly condensed chromatin). The EC50 and the stochastic cdf and pdf together with the multidimensional transitional phenotypic analysis of single cells contribute to further characterize cell death pathways in cyanobacteria.
Highlights
Cyanobacteria are a group of organisms with great ecological and economic importance (Dittmann and Wiegand, 2006)
The main consensus is that Accidental Cell Death (ACD), which is unpreventable and is caused by intense damage to which the cell cannot respond, can be distinguished from Regulated Cell Death (RCD), an active process that relies on the intracellular molecular machinery and can be modulated pharmacologically or genetically (Galluzzi et al, 2015)
A similar approach has been recently introduced for studies on cell death in cyanobacteria, including recommendations to characterize of RCD pathways by combining molecular, biochemical, and morphologic aspects following the directions of the (NCCD) (Aguilera et al, 2021)
Summary
Cyanobacteria are a group of organisms with great ecological and economic importance (Dittmann and Wiegand, 2006). The conditions that promote their massive proliferations have been extensively studied, but considerably less is known about mechanisms and molecular machinery of cell death in cyanobacteria. RCD can be activated in the context of physiological processes, such as post-embryonic development and ontogeny of the immune system (Taylor et al, 2008; Fuchs and Steller, 2011), and in that particular case it is designated as programmed cell death. It is important to emphasize that this terminology follows the conceptual framework and recommendations of the nomenclature committee on cell death (NCCD) (Galluzzi et al, 2018). A similar approach has been recently introduced for studies on cell death in cyanobacteria, including recommendations to characterize of RCD pathways by combining molecular, biochemical, and morphologic aspects following the directions of the (NCCD) (Aguilera et al, 2021). We will hereon refer to RCD in all cases where a stressor triggers an active form of cell death regardless the specific cellular pathway involved (i.e., ferroptosis, pyroptosis, apoptosis, necroptosis, etc.) and whether these pathways are characterized or not
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