A Peptide Derived from GAPDH Enhances Resistance to DNA Damage in Saccharomyces cerevisiae Cells

Abstract

Social behaviors do not exist only in higher organisms but are also present in microbes that interact for the common good. Here, we report that budding yeast cells interact with their neighboring cells after exposure to DNA damage. Yeast cells irradiated with DNA-damaging UV light secrete signal peptides that can increase the survival of yeast cells exposed to DNA-damaging stress. The secreted peptide is derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and it induced cell death of a fraction of yeast cells in the group. The data suggest that the GAPDH-derived peptide serves in budding yeast's social interaction in response to DNA-damaging stress. IMPORTANCE Many studies have shown that microorganisms, including bacteria and yeast, display increased tolerance to stress after exposure to the same stressor. However, the mechanism remains unknown. In this study, we report a striking finding that Saccharomyces cerevisiae cells respond to DNA damage by secreting a peptide that facilitates resistance to DNA-damaging stress. Although it has been shown that GAPDH possesses many key functions in cells aside from its well-established role in glycolysis, this study demonstrated that GAPDH is also involved in the social behaviors response to DNA-damaging stress. The study opens the gate to an interesting research field about microbial social activity for adaptation to a harsh environment.

Keywords: DNA damage; GAPDH; Saccharomyces cerevisiae; microbial social behavior; signal peptide.

FIG 1

UV-irradiated cells induce expression of RNR3 in intact cells. (A) Schematic view of the dual-reporter system. Yeast cells of the P_RNR3-mCherry strain were injected into the microtube through a transparent quartz glass tube with a diameter of 100 µm at the speed of 500 µL/min using a pump. During this process, the tube was exposed to UV radiation. The UV-induced DNA-damaged cells were immediately mixed with UV-untreated cells of the P_RNR3-GFP strain at a concentration ratio of 1:1 and cocultured for 5 h in the dark. (B) Representative confocal laser scanning microscopy images of non-treated cells (P_RNR3-mCherry), UV-untreated cells (P_RNR3-GFP strain) mixed with UV-irradiated cells (P_RNR3-mCherry), UV-irradiated SD medium, and high-dose UV-killed cells (P_RNR3-mCherry), respectively. Note that the P_RNR3-mCherry strain cells exposed to high-dose UV exhibit autofluorescence, and the fluorescence is observed in both 488 nm and 561 nm channels (scale bar, 10 μm). (C) Statistical results of (B) after counting cells. Percentage of UV-untreated cells (P_RNR3-GFP strain) expressing GFP after mixture are shown. A total of 900 cells were analyzed per condition in three or more independent experiments. (mean ± SD; n = 3, *P < 0.05).

FIG 2

UV-irradiated yeast cells secrete small molecular peptides that stimulate the expression of the RNR3 gene. (A) Conditioned medium from UV-irradiated cell culture induces P_RNR3-GFP expression. Cells are treated with conditioned medium for 2h. (B) The signal molecule is secreted into medium within 15 min after UV irradiation and induces P_RNR3-GFP expression.CM from non-treated cells was used a normalized control. (C) Proteinase K digestion completely abolishes the inducing activity of the signal molecule. Culture medium of UV-irradiated cells was treated with heat inactivation, proteinase K, inactive proteinase K, RNase A, or DNase before incubation with cells of P_RNR3-GFP strain. (D) Stimulating effects of different medium fractions separated through ultrafiltration on P_RNR3-GFP expression. More than 50,000 cells were detected by flow cytometry per condition in three or more independent experiments. (mean ± SD; n = 3, *P < 0.05; **<0.01; ***<0.001).

FIG 3

Stressed yeast cells secrete a GAPDH1-derived extracellular substance that protected cells from DNA-damaging stress. Cells were treated with UV radiation (50 J/m²) after incubation with different conditioned medium. (A) Conditioned medium from UV-irradiated cells; (B) conditioned medium from high-dose UV-irradiated cells. Living cells are counted by CFU assay, and the survival rates are shown in (A) and (B), respectively. (C and D) Conditioned medium from UV-irradiated cells protects yeast cells from treatment with MMS (0.3%), and 4-NQO (1.5 µg/mL). (E) The ability of conditioned medium to enhance the resistance to 0.3% (wt/vol) MMS is greatly decreased in supernatants from tdh1Δ, yca1Δ, and tdh1Δ yca1Δ mutants. (F) The ability of conditioned medium to induce the expression of P_RNR3-GFP is greatly decreased in tdh1Δ, yca1Δ, and tdh1Δ yca1Δ mutants. (mean ± SD; n = 3, *P < 0.05; **<0.01).

FIG 4

Functional characters of SP1. (A) The synthesized peptide SP1 (6 µM) induces P_RNR3-GFP expression. The representative flow cytometry analysis of P_RNR3-GFP induction experiments are shown. More than 30,000 cells per condition were detected by flow cytometry in three or more independent experiments. (B) The statistical result of (A).(C) The survival rate of yeast cells pre-incubated with SP1 (6 µM) or control peptide (6 µM) under the stress of UV radiation (50 J/m²). (D) The survival of SP1 pre-treated yeast cells at different time points with the stress of MMS killing. Cells that were pretreated with SP1 (6 µM) or with control peptide (6 µM) for 2 h, followed by the exposure to 0.15% (wt/vol) MMS. The MMS killing time points are indicated at the bottom. (E) The induction of P_RNR3-GFP is not correlated with the increased concentration of SP1. (F) The yeast cells expressing P_RNR3-GFP are more prone to decreased cell viability. The CFU of cells isolated from sub-cluster R1 and R2 (GFP+) are shown. (G) SP1 causes the death of a portion of the cells. The yeast cells were treated with 6 μM SP1 for 2 h and spread on plates for counting CFU. (mean ± SD; n = 3, *P < 0.05; **<0.01; ***<0.001).

FIG 5

Cells in various conditions show different sensitivities to SP1 treatment. (A, B) Yeast cells in late stationary phase or pretreated with the DNA damaging agent MMS are more sensitive to SP1 than those in log phase. (A) Cells in stationary phase. (B) Cells pretreated with MMS. The survival rate of the control group is set as 100%. (C) The bna2Δ mutant cells are more sensitive to SP1 than wild type cells in SD medium with limitation of nicotinic acid. (D) The survival rate of wild type and bna2Δ cells showed no significant difference after SP1 treatment in YPD medium without limitation of nicotinic acid. (mean ± SD; n = 3, *P < 0.05; **<0.01; ***<0.001).

FIG 6

SP1 enhanced resistance to DNA damaging stress by killing a fraction of the cells. (A) Representative confocal laser scanning microscopy images of cells pretreated with 6 µM SP1 or control peptide in cell culture cavities (scale bar, 15 μm). Dividing cells are labeled with a red square. (B) Percentage of dividing yeast cells pretreated with SP1or control peptide (the number of dividing cells/the total number of cells in each cavity at the beginning of cell culturing × 100%). (C) The average number of cells per cluster formed by dividing cell pretreated with SP1 or control peptide in each cavity after injecting into cavities for 2 h. (D) same as (A), except the yeast cells were then treated with 0.3% (wt/vol) MMS killing. The cells that have stopped dividing are labeled with a black circle. Dividing cells are labeled with a red square. (E) Percentage of dividing cells in yeast cells pretreated with SP1 or control peptide in response to 0.3% (wt/vol) MMS killing. (F) The average number of cells per cluster formed by a dividing cell pretreated with SP1 or control peptide in response to 0.3% (wt/vol) MMS killing in each cavity after injecting into cavities for 7 h. Each dot or triangle in the plots corresponds to an independent experiment, of which at least 10 cavities were analyzed. The orange or pink bar indicates average of each group. Data were from at 10 independent experiments. (mean ± SD; n = 10, *P < 0.05; **<0.01).