TY - JOUR
AU - Phoenix, David A.
AB - Abstract “Comet assay” showed light activated (3.15 J cm−2 over 30 min) phenothiazinium based photosensitisers (PhBPs) to induce photo-damage of Staphylococcus aureus DNA, as indicated by DNA “tails” between 80 and 120 µm. In general, PhBPs exhibited significant singlet oxygen yields (ΦΔPhBP > 0.7), suggesting the use of type II mechanisms of photo-oxidation. However, the photodynamic action of PhBPs on DNA showed generally insignificant production of 7,8-dihydro-8-oxo-2′-deoxyguanosine, normally a major product of type II DNA photo-oxidation. These combined results show DNA to be a major site of action of PhBPs and suggest that this action may involve type II attack on a nucleoside(s) other than guanosine. Phenothiazinium based photosensitiser, Staphylococcus aureus, DNA, 7,8-Dihydro-8-oxo-2′-deoxyguanosine 1 Introduction Pathogens with multiple resistance to conventional antibiotics are responsible for a global pandemic of infectious diseases, precipitating an urgent need for new agents with novel mechanisms of antibacterial action [1,2]. In response, there have been extensive investigations into the antimicrobial properties of photosensitisers [3–5], which are a class of molecules with well-established therapeutic use in the field of photodynamic therapy [6–8]. The underlying strategy in the therapeutic use of photosensitisers is the directed induction of photo-toxicity. Uptake of a photosensitiser by target cells is followed by irradiation with light at a suitable wavelength, generally within the therapeutic window of 600–750 nm [4]. Light absorption by the photosensitiser can then lead to an electronically excited state and the possibility that this excitational energy can be passed onto other molecules by two major mechanisms [9,10]. Type I mechanisms involve hydrogen abstraction or electron transfer between the excited photosensitiser and nearby biomolecules, yielding oxygenated free radicals, whilst type II mechanisms involve energy transfer between the excited photosensitiser and molecular oxygen, yielding singlet oxygen, 1O2[11,12]. The products of both mechanisms are highly reactive species and are able to initiate further interactions with outcomes that are generally toxic to the cell including: damage to membranes [13,14], the inactivation of essential enzymes [15,16] and mutagenetic effects due to DNA modification [17]. An important example of such DNA modification is the oxidation of guanosine to produce 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). This compound is a principle product of both type I and type II mechanisms of photosensitisation and is often used as an in vivo biomarker of DNA photo-damage [18]. A variety of photosensitisers have been shown to possess antibacterial properties with much recent attention focusing on phenothiazinium dyes and their derivatives [19]. These phenothiazinium based photosensitisers (PhPBs) are generally cationic and possess a core structure, which is a planar tricyclic heteroaromatic ring system [20]. PhBPs are highly effective against a variety of bacterial species but of particular interest is the ability of these dyes to inactivate Gram-positive pathogens. Dimethyl methylene blue (DMMB) and methyl methylene blue (MMB) each show high photo-toxicity to vancomycin-resistant Enterococcus faecalis and Enterococcus faecium[21]. These Gram-positive organisms are leading nosocomial pathogens with intrinsic resistance to many conventional antibiotics, and moreover, possess the ability to acquire resistance to novel antibiotics in pace with their introduction into therapeutic practice [22]. Other studies have shown DMMB, NMB, toluidine blue O (TBO) and methylene blue (MB) to be similarly photo-toxic to epidemic strains of methicillin-resistant Staphylococcus aureus (MRSA) with DMMB and NMB demonstrating a higher efficacy than vancomycin [23]. Most recently a number of novel PhBPs have been shown to be highly photo-toxic to S. aureus[24] and previous studies have suggested that the planar and cationic nature of PhBPs may promote intercalation with the nucleosides of DNA [3–6]. In the present study, we have investigated the ability of these PhBPs to inflict photo-damage on the DNA of S. aureus using a modified form of the “Comet” assay [25], which quantifies strand breaks in cellular DNA. In addition, we have investigated the possibility that DNA damage induced by PhBPs may involve 8-oxodG production and type II mechanisms of photo-oxidation. 2 Methods and materials 2.1 Reagents Alkaline phosphatase and nuclease P1 were purchased from ICN (UK) and each stored at −80 °C. Nutrient broth No 2 and all agars were purchased from Lab ‘M’ (UK). All other reagents were purchased from Sigma (UK) unless otherwise stated. Acetonitrile and acetic acid were of spectroscopic grade. Proteinase K, lysozyme and phage λ DNA were stored at −80 °C. New methylene blue (NMB) and dimethyl methylene blue (DMMB), azure A (AA), azure C (AC), azure B (AB), toluidine blue O (TBO), brilliant crystal blue (BCB), pyronin Y (PYY) and neutral red (NR) were stored in solid form at 4 °C. 2.2 Light adsorption characteristics of PhBPs The absorbance of the PhBPs described above was monitored across the wavelength range 500 nm to 750 nm using a scanning spectrophotometer (Pye Unicam SP8-400 UV/VIS). The resulting spectra were used to determine the wavelength within this range that gave maximum absorbance (λmax) for each of the PhBPs analysed (Table 1) and these λmax values were then taken to be those which would be optimal for the photo-activation of their parent dyes. To provide light, which covered the range of these λmax wavelengths, samples were incubated under a bank of fluorescent lights, which provided low energy incoherent light with a maximum emission of 700 nm and a fluence rate of 1.7 mW cm−2, as measured with a Skye SKP 200 meter (Skye Instruments, UK). Under these conditions, 30 min of illumination gave a total light dose of 3.15 J cm−1 with no significant changes in temperature observed. Table 1 Photodynamic properties of PhBPs PhBPs  Relative singlet oxygen yield  Maximal absorption in the range 500–750 nm  Comet assay  ΦΔPhBP  (λmax)  Concentration of PhBPs (µM)  Length of DNA tail produced by action of PhBPs (µm)  NMB  1.35  648  1.25  95  DMMB  1.22  630  0.8  120  TBO  0.86  625  12.5  90  AA  0.77  625  25.0  85  AB  0.77  546  12.5  80  AC  0.77  647  18.8  85  BCB  0.03  605  40  5  NR  0.18  623  12.5  120  PYY  0.05  536  3.1  90  PhBPs  Relative singlet oxygen yield  Maximal absorption in the range 500–750 nm  Comet assay  ΦΔPhBP  (λmax)  Concentration of PhBPs (µM)  Length of DNA tail produced by action of PhBPs (µm)  NMB  1.35  648  1.25  95  DMMB  1.22  630  0.8  120  TBO  0.86  625  12.5  90  AA  0.77  625  25.0  85  AB  0.77  546  12.5  80  AC  0.77  647  18.8  85  BCB  0.03  605  40  5  NR  0.18  623  12.5  120  PYY  0.05  536  3.1  90  The concentrations of PhBPs used in Comet assay correspond to the MLCs of PhBPs under light conditions when directed against S. aureus, as described by [24]. View Large Table 1 Photodynamic properties of PhBPs PhBPs  Relative singlet oxygen yield  Maximal absorption in the range 500–750 nm  Comet assay  ΦΔPhBP  (λmax)  Concentration of PhBPs (µM)  Length of DNA tail produced by action of PhBPs (µm)  NMB  1.35  648  1.25  95  DMMB  1.22  630  0.8  120  TBO  0.86  625  12.5  90  AA  0.77  625  25.0  85  AB  0.77  546  12.5  80  AC  0.77  647  18.8  85  BCB  0.03  605  40  5  NR  0.18  623  12.5  120  PYY  0.05  536  3.1  90  PhBPs  Relative singlet oxygen yield  Maximal absorption in the range 500–750 nm  Comet assay  ΦΔPhBP  (λmax)  Concentration of PhBPs (µM)  Length of DNA tail produced by action of PhBPs (µm)  NMB  1.35  648  1.25  95  DMMB  1.22  630  0.8  120  TBO  0.86  625  12.5  90  AA  0.77  625  25.0  85  AB  0.77  546  12.5  80  AC  0.77  647  18.8  85  BCB  0.03  605  40  5  NR  0.18  623  12.5  120  PYY  0.05  536  3.1  90  The concentrations of PhBPs used in Comet assay correspond to the MLCs of PhBPs under light conditions when directed against S. aureus, as described by [24]. View Large 2.3 Cell cultures and incubation with PhBPs Staphylococcus aureus (NCIMB 6751) was grown aerobically in Nutrient broth (25 g l−1) using a Gallenkamp orbital incubator at 37 °C. The strain was grown for approximately 18 h and these cultures used to inoculate fresh Nutrient broth (200:1 dilution) at 37 °C, which had previously been aerated. These cultures were then grown until in late logarithmic phase (A410= 0.7), which corresponded to a final cell concentration of 1013 cfu ml−1. The cells from these cultures were harvested, centrifuged (1500g, 15 min 5 °C) and resuspended in phosphate buffer (10 mM, pH 7). This procedure was then repeated except that the cells were resuspended in fresh Nutrient broth to give a final cell concentration 105 cells ml−1. This cell suspension was then used to individually solubilise NMB, DMMB, TBO, AA, AB, AC, BCB, NR and PYY with final dye concentrations as described in Table 1. As a control, this procedure was repeated with samples containing no dye. Aliquots (200 µl) of these cell/dye mixtures were then individually added to the wells of flat bottomed 96 well micro-titre plates and either kept in the dark or placed in a light box for 30 min and given a total light dose of 3.15 J cm−2, all as described above. These samples were then immediately used in microgel preparation. 2.4 The preparation of microgels for Comet assay Microgel assembly was adapted from the methodologies of [25] and [26]: Aliquots (100 µl) of 5 % (w/v) agarose in phosphate buffered saline (PBS: NaCl, 80 g l−1; KCl, 2.0 g l−1; Na2HPO4, 14 g l−1; KH2PO4, 2.0 g l−1; pH 7) were evenly spread on microscope slides to form a secure base layer for the microgel assembly. To ensure a flat upper surface, the molten agarose layer was overlain with a cover slip, air-dried at room temperature, and the cover slip removed. For each of the cell/dye samples prepared above, an aliquot (10 µl) was immediately mixed with 50 µl of 0.5% (w/v) low melting agarose in PBS, which had been previously prepared and maintained at 45 °C. These mixtures were then evenly spread over the agarose base layer, overlain with a cover slip and kept at 4 °C until the gel/cell layer had solidified. The cover slip was then removed, 175 µl of 0.5% (w/v) agarose in PBS evenly spread onto the solidified layers and the whole kept at 4 °C until the final agarose layer had solidified. These microgels were then immersed in lysing solution (Triton X-100, 1 % (v/v); NaCl, l2.5 M; EDTA, 100 mM; Tris, 10 mM; pH 10), which was supplemented with 1% (w/v) sodium N-lauroyl sarcosine and lysozyme (0.5 mg ml−1), for bacterial cell wall digestion. After 1 h, microgels were removed from the supplemented lysis solution and immersed in a solution for bacterial enzyme digestion (NaCl, 2.5 M; EDTA, 10 mM; Tris, 10mM; pH 7.4) containing proteinase K (1.0 mg ml−1) for 1 h at 37 °C. After completion of these procedures, treated microgels were placed on the horizontal slab of an electrophoresis unit. This unit was then flooded with electrophoresis buffer (NaOH, 300 mM; EDTA, 1 mM; pH 13) and the microgel allowed to equilibrate with the buffer for 20 min, permitting damaged DNA to unwind under the alkaline conditions. The microgels were then electrophoresed at 25V for 25 min, removed from the electrophoresis unit and immersed in neutralising buffer (0.4 M Tris, pH 7.5) for 30 min. After neutralisation, microgels were stained by placing in a tank with freshly made ethidium bromide (final concentration 100 µg ml−1) for 10 min to permit the visualisation of DNA. The microgel slides were then mounted onto the platform of a confocal fluorescence microscope (Leitz Diaplan) and viewed using an FITC filter combination (excitation 490 nm, dichroic 500 nm and emission 510 nm). Single S. aureus cells were identified and examined for DNA damage, as indicated by the “Comet effect”. Images were recorded using a camera (Spot Insight, USA, model No. 3) attached to the confocal microscope and stored as psp files on a PC (Viglen, P3800), using software supplied by Leitz Diaplan. Analysis of these stored images for “Comet tail” length was performed using the VisComet programme, kindly supplied by Professor N.P. Singh (http://www.impuls-imaging.com/viscomet_intro.html; accessed 15.07.04) and the results of these analyses are shown in Table 1. 2.5 Singlet oxygen production by PhBPs Singlet oxygen production by PhBPs was measured according to the methodology of [27], which monitors the decolourisation of 1,3-diphenylisobenzofuran (DPIBF) in methanol with time at 410 nm. By assuming that the decrease in absorption of DPIBF at 410 nm is directly proportional to its reaction with singlet oxygen, the time taken for a fifty per cent decrease in absorption of DPIBF at 410 nm is directly proportional to its reaction with singlet oxygen. Thus, the time taken by each of the PhBPs in Table 1 to cause a fifty per cent decrease in DPIBF absorption under identical conditions (t1/2PS), provides a measure of its relative photosensitising efficiency. The time for the DPIBF adsorption to decrease by fifty percent due to photosensitization by methylene (t1/2MB) was taken as 1. The singlet oxygen yield for methylene blue (ΦΔMB) is given by [27] as 0.443 and thus the singlet oxygen yields of the PhBPs of the present study (Table 1) were calculated according to   2.6 The incubation of phage λ DNA with PhBPs Aliquots (100 µl) of phage λ DNA (500 µg ml−1) in phosphate buffer (10 mM, pH 7.5), which had been maintained on ice, were placed in the wells of flat bottomed 96 well micro-titre plates. Aliquots (300 µl) of phosphate buffer (10 mM, pH 7.5) containing either: NMB, DMMB, TBO, AA, AB, AC, BCB, NR or PYY were then added to these DNA solutions with final dye concentration as described in Table 1. These micro-titre plates were then either maintained in the dark or placed in a light box for 30 min and given a total light dose of 3.15 J cm−2, all as described above. As a control, this procedure was repeated with samples containing no dye. After incubation, the DNA of all samples was enzymatically digested to its component nucleosides using an adaptation of the method of [28]. Samples were mixed with 50 µl of acetate buffer (1 M, pH 4.75), and enzymatically digested by the addition of 15 units of nuclease P1 at 100 °C for 30 min and cooled to 10 °C. These mixtures were then incubated with 1.2 units of alkaline phosphatase at 50°C for 1 h and the resulting samples immediately analysed used for the presence of 8-oxodG. 2.7 HPLC analysis of phage λ DNA samples for 8-oxodG production These analyses were performed using a JASCO (UK) HPLC system equipped with a UV detector (Waters 486). Samples (20 µl) of phage λ DNA that had been treated with PhBPs as described above were loaded onto an RP-C-18 column (3 µm particle size; Beckman, USA) with a mobile phase of 4% (v/v) acetonitrile and 0.1% (v/v) acetic acid at a flow rate of 1 ml min−1. Eluant levels of 8-oxodG were monitored by observing absorbance peaks at 297 nm and quantified by converting the areas under these peaks to weights of 8-oxodG using standard curves. Preliminary calibration experiments established that a linear relationship existed between the weight of 8-oxodG loaded onto the RP-C-18 column used and the area under the resulting chromatogram peak, within range the 0–10.0 µg. The weight of 8-oxodG resulting from a given phage λ DNA/PhBP incubation was then expressed as a % (w/w) of the total guanosine in the starting amount of phage λ DNA for that incubation, based on the guanosine content of the phage λ genome as 25% (Genbank, 2002; access code JO2459). 3 Results and discussion It has previously been shown that the PhBPs of the present study are photo-toxic to S. aureus and here, we have considered the possibility that the DNA of the organisms may be a target of these dyes. Cells of S. aureus were incubated with PhBPs at levels corresponding to their illuminated MLCs for this organism and these cells analysed for DNA photo-oxidation using the “Comet assay”. DNA from cells of S. aureus that had been treated with PhBPs under dark conditions formed compact structures (Fig. 1(a)) with diameters of the order of 25 µm, which is consistent with the presence of supercoiled, double stranded DNA [29]. These results indicate that under our experimental conditions, the PhBPs tested possess no significant inherent ability to damage the DNA of S. aureus. In contrast to these control data, “Comet” analyses of S. aureus cells that had been incubated with PhBPs under light conditions (Figs. 1 (b)–(k)) showed the majority these cells to display an elongated “tail”. This effect results from the electrophoretic migration of DNA that has suffered strand breakage [19] and clearly suggests that the PhBPs studied are able to inflict such damage on S. aureus DNA. Figure 1 View largeDownload slide Comet assay of S. aureus cells after treatment with PhBPs. S. aureus cells were incubated with PhBPs under light conditions, as described above. Figures 1(b)–1(k) show that the DNA of these cells exhibited high levels of damage, indicated by the elongated DNA tails produced under the conditions of the Comet assay [25]. In contrast, “Comet” assay of S. aureus cells that had been incubated with PhBPs under dark conditions showed the DNA of these cells to possess small compact structures, indicating supercoiled, double stranded DNA and the absence of significant damage. Figure 1(a) shows a typical result, which is for DMMB. Comparable results were recorded for S. aureus cells that had been similarly analysed in the absence of PhBPs. Figure 1 View largeDownload slide Comet assay of S. aureus cells after treatment with PhBPs. S. aureus cells were incubated with PhBPs under light conditions, as described above. Figures 1(b)–1(k) show that the DNA of these cells exhibited high levels of damage, indicated by the elongated DNA tails produced under the conditions of the Comet assay [25]. In contrast, “Comet” assay of S. aureus cells that had been incubated with PhBPs under dark conditions showed the DNA of these cells to possess small compact structures, indicating supercoiled, double stranded DNA and the absence of significant damage. Figure 1(a) shows a typical result, which is for DMMB. Comparable results were recorded for S. aureus cells that had been similarly analysed in the absence of PhBPs. The “tails” shown in Fig. 1 were generally between 80 and 120 µm (Table 1), which is consistent with the presence of high levels of DNA photo-damage [25] and suggests that DNA is a major photo-target for the action of PhBPs on S. aureus. Moreover, the limited range of these DNA “tail” lengths suggests that under our experimental conditions, the PhBPs studied are able to inflict comparable levels of photo-damage on S. aureus DNA although BCB produced no detectable “tail” (Fig. 1(b)) and would appear to have no significant photodynamic ability to damage the DNA of the organism. Broadly correlating with these results, in vitro assay showed the majority of the PhBPs studies to exhibit high singlet oxygen yields (ΦΔPhBP generally >0.7; Table 1), except in the case of BCB (ΦΔBCB= 0.03; Table 1). Taken in combination, these results suggest that type II mechanisms of DNA photo-oxidation [4] may generally feature in the photo-damage induced by these agents, except for BCB, which might not possess a significant photodynamic ability. Interestingly, PYY also exhibited no significant singlet oxygen yield (ΦΔPYY= 0.05; Table 1) but in contrast to BCB showed a strong photodynamic ability to attack S. aureus DNA, which could indicate the involvement of a type I mechanism of DNA photo-oxidation [4]. A principal product arising from both type I and type II mechanisms of DNA photo-oxidation is 8-oxodG. At levels corresponding to those of our Comet assay (Table 1), the PhBPs of the present study were tested for their ability to generate this compound from phage λ DNA. Control experiments showed that in the absence of PhBPs, there was no significant basal production of 8-oxodG in our experimental system. Moreover, the inclusion of PhPBs within the system led to no significant increases in production of the compound except in the cases of PYY and NMB. For these PhBPs, light activation led to increases in the production of 8-oxodG that represented conversion of 10% and 18% (w/w), respectively, of guanosine in the phage λ genome. In the case of PYY, these levels of 8-oxodG production are consistent with those expected by our predicted use of a type I mechanism of photosensitisation by the dye [18]. However, the type II mechanisms of DNA photo-oxidation predicted for NMB are known to lead to levels of DNA guanosine conversion approaching 100% [17], clearly suggesting that 8-oxodG may not be a major product arising from the DNA photo-oxidative mechanism(s) used by the dye. Indeed, secondary oxidation of 8-oxodG is known to occur since the oxidised purine has been shown to react more efficiently with 1O2 than its parent nucleoside [30] and DNA damage arising from the type II photo-oxidation of other DNA bases has been reported [18,31,32]. In conclusion, our results clearly suggest that DNA is a major site of action for the majority of the PhBPs studied when directed against S. aureus. Nonetheless, PhBPs are known to utilise multiple sites of action within some bacterial cells [2–5] and the participation of other cellular targets in the photo-toxicity of these dyes to S. aureus is a possibility, which we are currently investigating. For the PhBPs studied, our results suggest that PYY may use a type I mechanism of photo-oxidation to attack S. aureus DNA whilst the remaining dyes studied may utilise a type II mechanism. Although guanosine is generally the major nucleoside targeted in type II DNA photo-oxidation, it would appear that the PhBPs of the present study are able to target another nucleoside(s). 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TI - A study on the bacterial photo-toxicity of phenothiazinium based photosensitisers
JF - Journal of the Endocrine Society
DO - 10.1016/j.femsim.2004.09.002
DA - 2005-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-study-on-the-bacterial-photo-toxicity-of-phenothiazinium-based-AH7KZAwonH
SP - 367
EP - 372
VL - 43
IS - 3
DP - DeepDyve
ER -