Supplementary Information

Understanding the antimicrobial mechanism of TiO2-based nanocomposite films in a pathogenic bacterium

Anna Kubacka1, María Suárez Diez2, David Rojo3, Rafael Bargiela1, Sergio Ciordia4, Inés Zapico4, Juan P. Albar4, Coral Barbas3, Vitor A.P. Martins dos Santos2,5,Marcos Fernández-García1,*Manuel Ferrer1,*

1Institute of Catalysis, CSIC, 28049 Madrid, Spain, 2Chair of Systems and Synthetic Biology, Wageningen University, 6703 HB Wageningen, The Netherlands, 3Center for Metabolomics and Bioanalysis, University CEU San Pablo, Boadilla del Monte, 28668 Madrid, Spain, 4Proteomic Facility, CNB-National Centre for Biotechnology, CSIC, 28049 Madrid, Spain, 5LifeGlimmer GmbH, 12163 Berlin, Germany.

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Supplementary Methods

Gene chip experiments.The microarray analysis was performed with the Affymetrix P. aeruginosa GeneChip. Each condition was performed in triplicate, and each biological replicate was used for one GeneChip. In total, nine microarrays were used, which allowed changes in gene expression to be determined with a confidence value of P < 0.001. The data analyses were performed with applications belonging to the appropriate software packages from the Bioconductor microarray analysis suite59. The quality of all of the chips was assessed by fitting a linear model to the probe level data using the function fitPLM from the affyPLM package. The expression values were computed using the Robust Multichip Average algorithm60. The computations were performed using function “gc rma” from the package “affy”. The Rank Products algorithm was used to identify differentially expressed genes61. A percentage of false positives (pfp) value of 0.05 was accepted.

Protein mass spectrometric analysis.The samples were centrifuged for 1 min at 16,000 x gand4°C. The supernatant was discarded, and the bacterial cell pellet was lysed in 1.2 ml of BugBuster® Protein Extraction Reagent (Novagen, Darmstadt, Germany) for 30 min at 4°C with further disruption by sonication (using a pin Sonicator® 3000; Misonix) for 2.5 min (10 watts) on ice (5 cycles x 0.5 min). The extracts were then centrifuged for 5 min at 12,000 x gto separate the cell debris and intact cells. Finally, the supernatants were carefully aspirated (to avoid disturbing the pellet) and transferred to a new tube.

Protein digestion and tagging with iTRAQ-4-plex® reagent:The total protein concentration was determined using RC/DC protein assay (Bio-Rad, Madrid, Spain). For digestion, 20 μg of protein from each condition (UV+EVOH and UV-TiO2-coated EVOH) was precipitated by the methanol/chloroform method. Protein pellets were resuspended and denatured in 20 µl of 6 M guanidine hydrochloride/100 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid (HEPES), pH 7.5, (SERVA Electrophoresis GmbH), reduced with 1 µl of 50 mM Tris(2-carboxyethyl) phosphine (TCEP, AB SCIEX, Foster City, CA, USA), pH 8.0, at 60°C for 30 min and then treated with 2 µl of 200 mM cysteine-blocking reagent (methyl methanethiosulphonate (MMTS; Pierce, IL, USA) for 10 min at room temperature. Samples were diluted up to 120 µl with 50 mM TEAB to reduce guanidine concentration. Digestions were initiated by adding 1.5 µl (1 µg/µl) sequence grade-modified trypsin (Sigma Chemical Co.; St. Louis, MO, USA) to each sample in a ratio 1/10 (w/w), which were then incubated at 37°C overnight on a shaker. The digested samples were evaporated to dryness. Each peptide solution was labelled at room temperature for 2 hours with a half unit of iTRAQ Reagent Multi-plex kit (AB SCIEX, Foster City, CA, USA) that had been previously reconstituted with 80 μl of 70% ethanol/50 mM TEAB. In iTRAQ labelling, tags 114 and 115 were used to label UV+EVOH and UV-TiO2-coated EVOH condition, respectively. Afterwards, both labelled samples were combined and the labelling reaction was stopped by the addition of 100 µl of 50% acetonitrile (ACN) and evaporation in a Speed Vac. The digested, labelled and pooled peptide mixture was desalted using a Sep-PAK C18 Cartridge (Waters, MA, USA), the following manufacturer’s instructions. Lastly, the cleaned tryptic peptides were evaporated to dryness and stored at -20°C for further analysis.

Liquid chromatography and mass spectrometer analysis: A 2.5-µg aliquot of the resulting mixture was subjected to 2D-nano LC ESI-MSMS analysis using a nano liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus, AB SCIEX, Foster City, CA) coupled to high speed Triple TOF 5600 mass spectrometer (AB SCIEX, Foster City, CA) with a duo spray ionisation source. The analytical column used was a silica-based reversed phase column C18 ChromXP (75 µm × 15 cm) with a 3-µm particle size and a 120-Å pore size (Eksigent Technologies, AB SCIEX, Foster City, CA). The trap column was a C18 ChromXP (Eksigent Technologies, AB SCIEX, Foster City, CA) with a 3-µm particle diameter and a 120-Å pore size, and it was switched on-line with the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 2 µl/min. The nano-pump provided a flow-rate of 300 nl/min and was operated under gradient elution conditions, using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in ACN as mobile phase B. Gradient elution was performed according the following scheme: isocratic conditions of 98% A: 2% B for 1 minutes, a linear increase to 30% B in 210 minutes, a linear increase to 40% B in 10 minutes, a linear increase to 90% B in 5 minutes, isocratic conditions of 90% B for 5 minutes and a return to the initial conditions in 2 min. The injection volume was 5 µl. Data acquisition was performed with a TripleTOF 5600 System (AB SCIEX, Concord, ON). Data were acquired using an ion spray voltage floating (ISVF) 2800 V, curtain gas (CUR) 20, interface heater temperature (IHT) 150, ion source gas 1 (GS1) 20, declustering potential (DP) 85 V. All data were acquired using information-dependent acquisition (IDA) mode with Analyst TF 1.5 software (AB SCIEX, Foster City, CA, USA). For the IDA parameters, 0.25 s MS survey scan in the mass range of 350–1250 Da were followed by 15 MS/MS scans of 250 ms in the mass range of 100–1800 (total cycle time: 4.04 s). The switching criteria were set to ions with mass to charge ratio (m/z) greater than 350 and smaller than 1250 with a charge state of 2–5 and an abundance threshold of more than 90 counts (cps). Former target ions were excluded for 20 s. IDA rolling collision energy (CE) parameters script was used for automatically controlling the CE.

Data analysis:MS and MS/MS data obtained for pooled samples were processed using Analyst® TF 1.5.1 Software (AB SCIEX, Foster City, CA, USA). Raw data file conversion tools generated mgf files, which were also searched against a customised UniProtKB/SwissProt database from P. aeruginosa (taxonid:208964), containing 5,572 protein coding genes and their corresponding reversed entries using the Mascot Server v. 2.3.02 (Matrix Science, London, UK). Search parameters were set as follows: enzyme, trypsin; allowed missed cleavages, 1; fixed modifications, iTRAQ4plex (N-term and K) and beta-methylthiolation of cysteine; variable modifications, oxidation of methionine. The peptide mass tolerance was set to ± 20 ppm for precursors and 0.05 Da for fragment masses. The confidence interval for protein identification was set to ≥ 95% (p<0.05), and only peptides with an individual ion score above the 1% false discovery rate (FDR) threshold were considered correctly identified. Only proteins having at least two quantified peptides and p-values of ≤ 0.1 were considered in the quantitation62.

Metabolomic analysis by CE-TOF-MS.The samples were centrifuged for 1 min at 16,000 x gand4°C, and the cell pelletsimmediately mixed with1.2 ml of cold (-80ºC) HPLC-grade methanol(Sigma Chemical Co.; St. Louis, MO, USA). The samples were then stored at -80ºC for 60 min. Then, the samples were vortex-mixed (for 10 seg) and sonicated for 30 s (10 watts) on ice (5 cycles x 0.5 min). After sonication, the pellet was removed by centrifugation at 16,000 g for 20 min at 4ºC and the methanol solution was stored at -80ºC until use. Immediately, 1.2 ml milli-Q H2O was added to the cell pellet, and, after re-suspension, the samples were sonicated for 30 s (10 watts) on ice (5 cycles x 0.5 min). After sonication, the pellet was removed by centrifugation at 16,000 xg for 20 min at 4ºC and the water solution was separated. Finally, equal volumes (1 ml) of each of the methanol and H2O solutions were mixed and stored at -80ºC until analysis. The analysis was performed as follows: Samples for CE-MS analysis were prepared from a volume of 100µl by evaporating to dryness using a Speedvac Concentrator (Thermo Fisher Scientific Inc., Waltham, MA). Each sample was then reconstituted in 100µl of MilliQ® water with the internal standard (0.4mM L-methionine sulphone) and 0.1M formic acid. A capillary electrophoresis system (7100 Agilent) coupled with a TOF Mass Spectrometer (6224 Agilent) was employed for the CE-MS analysis of the samples. The CE mode was controlled by ChemStation software (B.04.03, Agilent) and MS mode by Mass Hunter Workstation Data Analysis (B.02.01, Agilent). The separation occurred in a fused-silica capillary (Agilent) (total length, 100 cm; i.d., 50 μm). All separations were carried out in normal polarity with a background electrolyte containing a 0.8 M formic acid solution in 10% methanol (v/v) at 20ºC. New capillaries were pre-conditioned with a flush of 1.0 M NaOH for 30 min followed by MilliQ® water for 30 min and background electrolyte for 30 min. Before each analysis, the capillary was conditioned with a flush of background electrolyte for 5 min. The sheath liquid (6 µl/min) was MeOH:H2O (1:1) containing 1.0 mM formic acid with two reference masses: m/z121.0509 ([C5H4N4+H]+) and m/z 922.0098 ([C18H18O6N3P3F24+H]+), which allowed correction and higher mass accuracy in the MS. The samples were hydrodynamically injected at 50 mBar for 50 s. The stacking was carried out by applying background electrolytes at 100 mBar for 20 s. The separation voltage was 30 kV, the internal pressure was 25 mBar and the analyses were carried out for 35 min. The MS parameters were as follows: fragmentor, 100 V; skimmer, 65 V; octopole, 750 V; drying gas temperature,200ºC; flow rate, 10l/min; and capillary voltage, 3500 V. The data were acquired in positive mode with a full scan from m/z 85 to 1000 at a rate of 1.41 scan/s. The resulting data files were cleaned of background noise and unrelated ions by Mass Hunter Qualitative Analysis software (B.05.00, Agilent). This tool was used to look for the target list of the compounds (±20 ppm) that were interesting for the project in the corresponding electropherograms. The identity of each compound was confirmed in a second analysis by adding the corresponding standard (10 ppm) to the samples. Then, each sample was checked for an increase in the peak; the result was positive in all cases. The following reagents have been used for the metabolomic analysis: formic acid (MS grade - Sigma-Aldrich, Steinheim, Germany), L-methionine sulphone (Sigma-Aldrich, Taufkirchen, Germany), sodium hydroxide (Panreac-Montcada I Reixac, Spain), and thereference masses were purine and hexakis (1H,1H,3H-tetrafluoropropoxy)phosphazine (HP)) from Agilent (API-TOF reference mass solution kit). All solutions were prepared using MilliQ® water (Millipore, Billerica, MA, USA).

Supplementray references

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Supplementary Fig. S1 Upper panel: EPR spectra obtained in presence of the nanocomposite film and DMPO spin trapping molecule under light excitation in water or ethanol. Signals from the solvent were subtracted. Lower panel: Evolution of the DMPO-OH• signal intensity obtained in water as a function of the irradiation time.

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Supplementary Fig. S2 Expression level of genes encoding enzymes involved in the catabolic pathway of glycine to the one carbon pool and related reactions under TiO2 photocatalysis. The genes encoding enzymes implicated in each particular reaction and their expression levels are specifically indicated. Gene code as follows: gbcA, glycine betaine demethylase subunit A (Rieske subunit); gbcB, glycine betaine catabolism protein; dgcB, Dimethylglycine catabolism protein; soxABC, sarcosine oxidase subunits; katA and katB, catalases; glyA2, serine hydroxymethyltransferase/glycine hydroxymethyltransferase; gcvP2, glycine dehydrogenase (decarboxylating); gcvT2, aminomethyltransferase (glycine cleavage system aminomethyltransferase); gcvH2, glycine cleavage system H protein; sdaB, L-serine dehydratase. Colour code as follows: green, genes expressed (and thus reaction active) at higher level; red, genes expressed (and thus reaction active) at lower level. The numbers in parentheses are expression fold changes.

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Supplementary Fig. S3Cartoon representing the major effects in cells of P. aeruginosa when confronted to TiO2 photocatalysis.Colour code as follows: green, genes expressed (and thus reaction active) at higher level; red, genes expressed (and thus reaction active) at lower level. Abbreviations used: LPS, lipopolysaccharide; HSL, homo-serinelactone; THF, tetrahydrofolate; 5,10-MTHF, 5,10-methylenetetrahydrofolate.

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Supplementary Table S1Cell inactivation rates and time for 50% reduction in experiment concerning the elimination of several microorganisms with initial concentration 8 x 108 cell ml-1.

Microorganism / Rate / 10-7 CFU ml-1 s-1 a / T1/2 / min a
Pseudomonas aeruginosa / 63.1 / 5.5
Escherichia coli / 1.9 / 10.1
Staphylococcus aureus / 3.3 / 7.4
Enterococcus faecalis / 1.7 / 19.1
Pichia jadinii / 0.5 / 19.4

a)Standard error; ± 8; 0.5 min.

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Supplementary Table S2 List of P. aeruginosa PAO1 genes most strongly induced in response to OH radicals. Datawere statistically scored, using the Student´s t-test. p-values were then adjusted for multiple testing, using the False Discovery Rate procedure of Benjamini and Hochberg63. Statistical analyses were performed in R using the mulltest package. The data represent the mean of three independent experiments, with each chip experiment performed in triplicate.

Gene name / Gene name / EC number / Gene product / Biological category / FC:(class1/class2) / P value
PA1540 / - / Putative uncharacterized protein / Unknown / 224.2408 / 0
PA0140 / ahpF / EC 1.6.4.- / Alkyl hydroperoxide reductase subunit F / Detoxification / 145.2743 / 0
PA2850 / ohr / Organic hydroperoxide reductase (Ohr subfamily peroxiredoxin) / Detoxification / 74.0643 / 0
PA1982 / exaA / EC 1.1.2.8 / Quinoprotein ethanol dehydrogenase/ Glucose dehydrogenase / Central metabolic reaction (carbohydrate metabolism) / 73.7251 / 0
PA1541 / - / Drug efflux transporter / Transport (ABC/Efflux pump transport) / 73.374 / 0
PA3183 / zwf / EC 1.1.1.49 / Glucose-6-phosphate 1-dehydrogenase / Central metabolic reaction (carbohydrate metabolism) / 67.3921 / 0
PA3584 / glpD / EC 1.1.1.8 / Glycerol-3-phosphate dehydrogenase / Central metabolic reaction (carbohydrate metabolism) / 64.1499 / 0
PA4613 / katB / Catalase / Detoxification / 59.7164 / 0
PA2825 / ospR / Transcriptional regulator / Regulation (transcription regulation) / 58.0861 / 0
PA4623 / - / Putative uncharacterized protein / Unknown / 56.1644 / 0
PA2444 / glyA2 / EC 2.1.2.1 / Serine hydroxymethyltransferase 2 / glycine hydroxymethyltransferase / Central metabolic reaction (amino acid metabolism) / 50.5342 / 0
PA3283 / - / Putative uncharacterized protein / Unknown / 48.9415 / 0
PA2826 / - / EC 1.11.1.9 / Glutathione peroxidase / Detoxification / 46.5293 / 0
PA1332 / - / Putative uncharacterized protein / Unknown / 44.4957 / 0
PA3182 / pgl / EC 3.1.1.31 / 6-Phosphogluconolactonase / Central metabolic reaction (carbohydrate metabolism) / 42.3234 / 0
PA2445 / gcvP2 / EC 1.4.4.2 / Glycine dehydrogenase (decarboxylating] 1 / Central metabolic reaction (amino acid metabolism) / 39.1214 / 0
PA4714 / - / Putative uncharacterized protein / Unknown / 35.7571 / 0
PA0567_i / - / Putative uncharacterized protein / Unknown / 33.6694 / 0
PA0848 / ahpB / EC 1.6.4.- / Alkyl hydroperoxide reductase / Detoxification / 33.1195 / 0
PA3195 / gapA / EC 1.2.1.12 / Glyceraldehyde-3-phosphate dehydrogenase / Central metabolic reaction (carbohydrate metabolism) / 32.107 / 0
PA0069 / - / Putative uncharacterized protein / Unknown / 31.8945 / 0
PA1983 / exaB / EC 1.9.3.1 / Cytochrome c550 / central metabolic reactions (energy production and conversion) / 31.8417 / 0
PA0849 / trxB2 / EC 1.8.1.9 / Thioredoxin reductase / Posttranslational modification, protein turnover, chaperones / 31.7961 / 0
PA4763 / recN / EC 3.1.-.- / DNA repair protein RecN / DNA replication, recombination, modification and repair / 30.4606 / 0
PA3282 / - / Putative uncharacterized protein / Unknown / 29.8014 / 0
PA3181 / - / EC 4.1.2.14 / 2-Dehydro-3-deoxy-phosphogluconate aldolase / Central metabolic reaction (carbohydrate metabolism) / 27.5867 / 0
PA3126 / ibpA / Heat-shock protein IbpA / Posttranslational modification, protein turnover, chaperones / 24.9903 / 0
PA1981 / - / Putative uncharacterized protein / Unknown / 24.9542 / 0
PA4356 / xenB / EC 1.6.99.1 / NADH:flavin oxidoreductases (Old Yellow enzyme family) / central metabolic reactions (energy production and conversion) / 24.0298 / 0
PA2000 / dhcB / EC 2.8.3.8 / 3-Oxoacid CoA-transferase B subunit (Acyl CoA:acetate/3-ketoacid CoA transferase, beta subunit ) / Cell membrane structure (lipid metabolism) / 22.9917 / 0
PA3281 / - / Putative uncharacterized protein / Unknown / 22.1633 / 0
PA0506 / - / EC 1.3.99.3 / Acyl-CoA dehydrogenase / Cell membrane structure (lipid metabolism) / 21.3395 / 0
PA2288 / - / Putative uncharacterized protein / Unknown / 20.6712 / 0
PA0671 / - / Putative uncharacterized protein / Unknown / 20.2575 / 0
PA0922 / - / Putative uncharacterized protein / Unknown / 20.0785 / 0
PA2001 / atoB / EC 2.3.1.9 / Acetyl-CoA acetyltransferase / Cell membrane structure (lipid metabolism) / 19.5771 / 0
PA0613 / - / Putative uncharacterized protein / Unknown / 19.2665 / 0
PA3194 / edd / EC 4.2.1.12 / Phosphogluconate dehydratase / Central metabolic reaction (carbohydrate metabolism) / 19.1734 / 0
PA4994 / - / EC 1.3.99.3 / Acyl-CoA dehydrogenase / Cell membrane structure (lipid metabolism) / 19.0445 / 0
PA4354 / - / Putative uncharacterized protein / Unknown / 18.7651 / 0
PA4881 / - / Putative uncharacterized protein / Unknown / 18.284 / 0
PA1999 / dhcA / EC 2.8.3.8 / 3-Oxoacid CoA-transferase, A subunit (Acyl CoA:acetate/3-ketoacid CoA transferase, alpha subunit ) / Cell membrane structure (lipid metabolism) / 17.1444 / 0
PA3007 / lexA / Peptidase S24 LexA-like repressor / Regulation (transcription regulation) / 16.9571 / 0
PA0670 / - / Putative uncharacterized protein / Unknown / 16.8393 / 0
PA3284 / - / Putative uncharacterized protein / Unknown / 16.7417 / 0
PA0565 / - / Putative uncharacterized protein / Unknown / 16.4815 / 0
PA1736 / - / EC 2.3.1.9 / Acetyl-CoA acetyltransferase or acyl-CoA thiolase / Cell membrane structure (lipid metabolism) / 16.4216 / 0
PA5053 / hslV / ATP-dependent protease hslV / Posttranslational modification, protein turnover, chaperones / 16.1266 / 0
PA3930 / cioA / EC 1.10.3.- / Cytochrome Ubiquinol Oxidase / central metabolic reactions (energy production and conversion) / 15.9921 / 0
PA1737 / - / EC 1.1.1.35 / 3-Hydroxyacyl-CoA dehydrogenase, NAD binding domain (Crotonase/Enoyl-Coenzyme A (CoA) hydratase superfamily) / Cell membrane structure (lipid metabolism) / 15.7455 / 0
PA4542 / clpB / Chaperone protein clpB / Posttranslational modification, protein turnover, chaperones / 14.8436 / 0
PA2116 / - / Putative uncharacterized protein / Unknown / 14.7732 / 1,00E-004
PA4612 / ankB / Ankyrin-like protein AnkB / Detoxification / 14.6468 / 1,00E-004
PA3616 / recX / Recombination regulator RecX / DNA replication, recombination, modification and repair / 14.3783 / 1,00E-004
PA4202 / - / Putative uncharacterized protein / Unknown / 14.019 / 1,00E-004
PA5023 / - / Putative uncharacterized protein / Unknown / 14.0036 / 1,00E-004
PA2759 / - / Putative uncharacterized protein / Unknown / 13.8818 / 1,00E-004
PA2830 / htpX / Protease HtpX (heat shock protein) / Posttranslational modification, protein turnover, chaperones / 13.666 / 1,00E-004
PA5523 / hemL / EC 2.6.1.- / Pyridoxal phosphate dependent aminotransferase (acetyl ornithine aminotransferase / glutamate-1-semialdehyde aminotransferase / 4-aminobutyrate aminotransferase) / Central metabolic reaction (unclear: amino acid metabolism / cofactor metabolism) / 13.3908 / 1,00E-004
PA1420 / - / Putative uncharacterized protein / Unknown / 13.3016 / 1,00E-004
PA3008 / - / Putative uncharacterized protein / Unknown / 13.2319 / 1,00E-004
PA0779 / - / ATP-dependent Lon protease (S16) / Posttranslational modification, protein turnover, chaperones / 13.0406 / 1,00E-004
PA0132 / bauA / Beta-alanine-pyruvate transaminase / Central metabolic reaction (amino acid metabolism) / 13.013 / 1,00E-004
PA2597 / - / Putative uncharacterized protein / Unknown / 12.954 / 1,00E-004
PA5106 / - / EC 3.5.3.13 / Metallo-dependent hydrolase or N-formimino-L-glutamate deiminase / Central metabolic reaction (amino acid metabolism) / 12.8775 / 1,00E-004
PA4762 / grpE / Heat shock protein GrpE / Posttranslational modification, protein turnover, chaperones / 12.8368 / 1,00E-004
PA1847 / - / Putative uncharacterized protein / Unknown / 12.7353 / 1,00E-004
PA3014 / faoA / EC 1.1.1.- / Fatty acid oxidation complex subunit alpha (crotonase/Enoyl-Coenzyme A (CoA) hydratase superfamily or 3-hydroxyacyl-CoA dehydrogenase) / Cell membrane structure (lipid metabolism) / 12.7 / 1,00E-004