TiO2 photocatalysis damages lipids and proteins in Escherichia coli

Abstract

This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO2) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO2 photocatalysis on E. coli survival was monitored by counting on agar plate and by assessing lipid peroxidation and performing proteomic analysis. We observed through malondialdehyde quantification that lipid peroxidation occurred during the photocatalytic process, and the addition of superoxide dismutase, which acts as a scavenger of the superoxide anion radical (O2·(-)), inhibited this effect by half, showing us that O2·(-) radicals participate in the photocatalytic antimicrobial effect. Qualitative analysis using two-dimensional electrophoresis allowed selection of proteins for which spot modifications were observed during the applied treatments. Two-dimensional electrophoresis highlighted that among the selected protein spots, 7 and 19 spots had already disappeared in the dark in the presence of 0.1 g/liter and 0.4 g/liter TiO2, respectively, which is accounted for by the cytotoxic effect of TiO2. Exposure to 30 min of UV-A radiation in the presence of 0.1 g/liter and 0.4 g/liter TiO2 increased the numbers of missing spots to 14 and 22, respectively. The proteins affected by photocatalytic oxidation were strongly heterogeneous in terms of location and functional category. We identified several porins, proteins implicated in stress response, in transport, and in bacterial metabolism. This study reveals the simultaneous effects of O2·(-) on lipid peroxidation and on the proteome during photocatalytic treatment and therefore contributes to a better understanding of molecular mechanisms in antibacterial photocatalytic treatment.

FIG 1

Influence of photocatalytic treatment on E. coli cell viability, determined by counting on agar plate. Bars indicate standard deviations. , in the dark; , under UV-A irradiation. * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and **** (P < 0.0001) indicate the significance of the difference between sample means, obtained using Student's t test.

FIG 2

Impact of UV-A photocatalysis with TiO₂ and influence of the addition of SOD at 1,000 U on E. coli lipid peroxidation. A duration of 60 min and a TiO₂ concentration of 0.4 g/liter were chosen. Membrane lipid peroxidation is measured by assessing production of malondialdehyde (MDA). MDA is a biomarker of lipid peroxidation and is used to measure the lipid peroxidation rate relative to controls (MDA concentration obtained without and with treatment). Bars indicate standard deviations. , in the dark; , under UV-A irradiation. ** (P < 0.01), **** (P < 0.0001), and ns (not significant) indicate the significance of the difference between sample means obtained with Student's t test.

FIG 3

Impact of TiO₂ photocatalysis on the whole-cell proteome of E. coli ATCC 8739. Representative 2-DE gel pictures (pH range, 4 to 7) of whole-cell protein lysates after different treatments for 30 min of UV-A light irradiation are shown. Previously, bacteria were grown during 16 h to 18 h on tryptic soy agar at 37°C. (A) Standard conditions (without TiO₂ in the dark). (B) Under UV-A light irradiation. (C and E) In the dark at a TiO₂ concentration of 0.1 g/liter (C) or 0.4 g/liter (E). (D and F) Under UV-A light irradiation at a TiO₂ concentration of 0.1 g/liter (D) or 0.4 g/liter (F). Spots present (black numbers) under standard conditions (gel A) but not detected (white numbers) after one of the applied treatments (gels B to F) are shown. Proteins corresponding to these spots were identified by chip-LC-QTOF analysis.