Atmospheric Nonthermal Plasma-Treated PBS Inactivates Escherichia coli by Oxidative DNA Damage

Abstract

We recently reported that phosphate-buffered saline (PBS) treated with nonthermal dielectric-barrier discharge plasma (plasma) acquires strong antimicrobial properties, but the mechanisms underlying bacterial inactivation were not known. The goal of this study is to understand the cellular responses of Escherichia coli and to investigate the properties of plasma-activated PBS. The plasma-activated PBS induces severe oxidative stress in E. coli cells and reactive-oxygen species scavengers, α-tocopherol and catalase, protect E. coli from cell death. Here we show that the response of E. coli to plasma-activated PBS is regulated by OxyR and SoxyRS regulons, and mediated predominantly through the expression of katG that deactivates plasma-generated oxidants. During compensation of E. coli in the absence of both katG and katE, sodA and sodB are significantly overexpressed in samples exposed to plasma-treated PBS. Microarray analysis found that up-regulation of genes involved in DNA repair, and E. coli expressing recA::lux fusion was extremely sensitive to the SOS response upon exposure to plasma-treated PBS. The cellular changes include rapid loss of E. coli membrane potential and membrane integrity, lipid peroxidation, accumulation of 8-hydroxy-deoxyguinosine (8OHdG), and severe oxidative DNA damage; reveal ultimate DNA disintegration, and cell death. Together, these data suggest that plasma-treated PBS contains hydrogen peroxide and superoxide like reactive species or/and their products which lead to oxidative changes to cell components, and are eventually responsible for cell death.

Fig 1. ROS scavengers protect cells under stress conditions.

Colony assay demonstrating E. coli cell viability and the effect of ROS scavengers upon exposure to plasma-treated PBS. Plasma-treated PBS (75 seconds; predetermined) has exhibited a significant, and cell contact (holding) time-dependent bacterial inactivation. A significant protection is provided by preincubation with ROS scavengers (vitamin E, 200mM; calatase, 200 units; thiourea, 100 mM). Bar, SEM; *, p <0.05 against plasma untreated (0 min) condition; **, p <0.05 against corresponding conditions with ‘No Scavenger’. (n = 3).

Fig 2. Membrane-associated changes in E. coli cells (wildtype) following plasma-treated PBS exposure.

(A) Malondialdehyde, the marker of lipid peroxidation is detected by TBARS assay demonstrating the peroxidation change in lipid. The findings are normalized against untreated samples. Bar, SEM; *, p <0.05 against 0.3% H₂O₂. (B) Loss of membrane potential detected by DiOC₃ assay. Bar, SEM; *, p >0.05 against corresponding untreated (control; 0 min) condition. H₂O₂ is used as positive control in both assays. (n = 3). The findings together suggest a loss of membrane potential and subsequent membrane lipis peroxidation.

Fig 3. Survival responses of the wildtype and deletion mutants.

Colony assays demonstrating viability of E. coli wildtype and gene deletion mutants of superoxide dismutase (A, C) and catalase (B, D) exposed to dose-dependent plasma-treated PBS without (A, B) or with (C, D) external catalase protection (10 min preincubation with catalase). These findings suggest that external catalase provide a significant protection to wildtype and sod and kat deletion mutants from oxidative stress. Bar, SEM; *, p <0.05 against corresponding condition of wildtype strain; **, p <0.05 against corresponding conditions without catalase. (n = 3).

Fig 4. Plasma-treated PBS inducing oxidative DNA damage.

(A) A representative agarose gel electrophoresis showing DNA integrity. (B) A graph showing corresponding band intensities of DNA (from gel of Fig 4A). The graph suggests that external catalase substantially protects DNA from damage. (C) The accumulation of 8-hydroxy-2’ -deoxyguanosine (8-OHdG; a marker for oxidative DNA damage) in wildtype and representative deletion mutants after exposure to plasma-treated PBS for 1 minute. Catalase prevented the formation of 8-OHdG during this exposure. Bar, SEM; *, p <0.05 against wildtype E. coli (without catalase). (D) Response of E. coli harboring recA::lux fusion construct to plasma-activated PBS. A maximum induction of recA is seen between 3 h to 5 h post plasma-activated PBS exposure. Bar, SEM. (n = 3).

Fig 5. The Graphical presentation showing the change in the levels of gene expression in wildtype E. coli after plasma-activated PBS treatment and the effect of ROS scavenger, catalase.

A differential expression of oxidative stress-responsive oxyRS and soxRS regulons (A), and the regulated genes of superoxide dismutase and catalase (B) are shown. The regulons oxyR, oxyS, soxR and soxS, and the regulated genes sodB and katG exhibited the early response to exposure to plasma-treated PBS showing activation, and preincubation with external catalase (wildtype w/ catalase) substantially inhibited their activation against longer exposures such as 5 min and 10 min. The data are normalized against corresponding untreated (wildtype without external catalase; no exposure to plasma-treated solution) cells. Bar, SEM. (n = 3).

Fig 6. The graphs showing the change in gene expression levels during oxidative stress in gene deficient mutants of E. coli after plasma treated PBS exposure.

The expression is given as fold change against wildtype. The specific RT-PCR probes were used to demonstrate the transcriptional activation response of oxy and sox regulons (A), and the transcription of superoxide dismutase (B) and catalase (C). In all mutants, oxyS, sodC in sodA, sodB and their double mutant, and katG in sodA, sodB, sodAsodB and katE mutants, and katE in all sod mutants, and katG mutant, were activated substantially around 5 min of exposure with plasma-treated PBS. Bar, SEM. (n = 3).