The Role of Pseudo-Orthocaspase (SyOC) of Synechocystis sp. PCC 6803 in Attenuating the Effect of Oxidative Stress

Abstract

Caspases are proteases, best known for their involvement in the execution of apoptosis-a subtype of programmed cell death, which occurs only in animals. These proteases are composed of two structural building blocks: a proteolytically active p20 domain and a regulatory p10 domain. Although structural homologs appear in representatives of all other organisms, their functional homology, i.e., cell death depending on their proteolytical activity, is still much disputed. Additionally, pseudo-caspases and pseudo-metacaspases, in which the catalytic histidine-cysteine dyad is substituted with non-proteolytic amino acid residues, were shown to be involved in cell death programs. Here, we present the involvement of a pseudo-orthocaspase (SyOC), a prokaryotic caspase-homolog lacking the p10 domain, in oxidative stress in the model cyanobacterium Synechocystis sp. PCC 6803. To study the in vivo impact of this pseudo-protease during oxidative stress its gene expression during exposure to H₂O₂ was monitored by RT-qPCR. Furthermore, a knock-out mutant lacking the pseudo-orthocaspase gene was designed, and its survival and growth rates were compared to wild type cells as well as its proteome. Deletion of SyOC led to cells with a higher tolerance toward oxidative stress, suggesting that this protein may be involved in a pro-death pathway.

Keywords: Synechocystis sp. PCC6803; orthocaspase; programmed cell death; proteomics; pseudo-enzyme.

FIGURE 1

Growth of the Synechocystis 6803 wild type (WT) and ΔOC mutant. (A) Growth curve of Synechocystis 6803 wild type and ΔOC mutant strains monitored during 13 days by optical density (OD) measurements at 730 nm. Values are means ± SEM, n = 3. This experiment was repeated three times with similar results. (B) Quantification of the cell number of Synechocystis 6803 wild type and ΔOC mutant strains during growth for 9 days at control conditions. Values are means ± SEM, n = 3. This experiment was repeated three times with similar results. (C) Photos of representative wild type and ΔOC mutant strains at day 3 of growth at control conditions.

FIGURE 2

Growth of Synechocystis 6803 wild type and ΔOC mutant strains is impaired in the presence of hydrogen peroxide. Synechocystis 6803 wild type and ΔOC mutant strains in their exponential growth phase (OD ∼0.8) were exposed to different concentrations of H₂O₂ (B: 2, C: 3.5, D: 5, or E: 10 mM); non-treated cells were used as control (A). Bacterial growth as OD₇₃₀ (left panel) and quantum yield (Fv/Fm) (middle panel) was measured after 0, 1, 3, 6, 12, and 24 h of treatment. Right panel: Relative expression of SyOC in WT exposed to H₂O₂ (2, 3.5, 5, or 10 mM) determined by real-time PCR. Relative expression to housekeeping genes rnpB and 16s was determined at five different time points (0, 30, 60, 180, and 360 min) and plotted relative to 0 h (control condition). Data points are means (± SEM) calculated from three biological and three technical replicates and represent two independent experiments. Asterisk indicate a significant difference using ANOVA test post-hoc Fisher LSD (α = 0.05).

FIGURE 3

ΔOC survives growth in higher concentrations of hydrogen peroxide compared to the wild type. Photos (A) and evaluation (B) of drop dilutions (ranging from 10^–1 to 10^–4 fold) of WT and ΔOC Synechocystis strains using BG-11 plates in the absence or presence of different concentrations of H₂O₂. This experiment was repeated three times with similar results. Logarithmic values of CFU/ml are means (±SEM) calculated from three biological and three technical replicates. Photos (C) and quantification (D) of the cell death assay after growth in the presence of 2 or 3.5 mM H₂O₂ for 24 h. Dead cells (Sytox green-positive) were counted under a microscope. The experiment is representative of three independent replicates. Asterisks indicate statistical difference by t-test (α = 0.05).

FIGURE 4

Proteomic changes in WT and ΔOC in response to H₂O₂. Volcano plots were constructed using Perseus. Logarithm of p-values plotted against the logarithm of fold change (FC) of the conditions indicated in each graph. (A) WT vs. ΔOC in control conditions; (B) WT vs. ΔOC exposed to H₂O₂ stress; (C) effect of H₂O₂ stress on WT; (D) effect of H₂O₂ stress on ΔOC. Up- or down-regulated proteins are shown in black and gray, respectively; significantly up- or down-regulated proteins in blue and yellow, respectively. (E) Significantly differentially regulated proteins involved in nucleotide excision repair, oxidative, lipopolysaccharide (biosynthesis and export), and stress-related processes (Heat shock and peptidases). Names as annotated in the UniProtKB database. Letters A–D correspond to the volcano plots (A–D).

FIGURE 5

Strain-specific changes in protein interactions. Interaction networks for WT (A) and ΔOC (B,C), protein- and KEGG pathways were annotated using the STRING databases and highlighted with different colors.