Intracellular pH changes induced in Propionibacterium acnes by UVA radiation and blue light

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Journal of Photochemistry and Photobiology B: Biology 31 (1995) 125-131

Intracellular pH changes induced in Propionibacterium acnes by UVA radiation and blue light C.M. Futsaether a, B. Kjeldstad b, A. Johnsson b • Departmentof Agricultural Engineering, Agricultural Universityof Norway, N-1432 ~s, Norway b Departmentof Physics, AVH, Universityof Trondheim, N-7055 Dragvoll, Norway Received 3 January 1995; accepled 17 May 1995

~bstract

The intracellular pH changes induced in the Gram-positive skin bacterium Propionibacterium aches by blue light and UVA radiation were ;tudied. Two methods (3 ~p nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy using a pH-sensitive fluoroprobe 12',7'-bis-(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein)) were used to determine the intracellular pH. The pH changes induced by rradiation were found to be a function of cell survival. These changes as a function of cell survival followed the same pattern for blue light and UVA radiation. A reduction of the pH gradient across the cell membrane (inside alkaline) was found for lethal doses (less than 15% :;urvival). This reduction corresponded to a decrease in intracellular pH and may indicate a proton influx. An increase in the pH gradient, :¢hich corresponded to an increase in the intracellular pH, was observed for sublethal doses. This increase appears to be reversible. Thus two :;eparatemechanisms, which appear to be the same in UVA and blue light regions, may be responsible for the irradiation-induced pH alterations. (eywords: Propionibacterium aches; UVA radiation; Visible radiation; Intracellular pH; 3~p NMR spectroscopy; 2',7'-bis-(2-carboxyethyl)-5-(and -6-),:arboxyfluorescein (BCECF)

1. Introduction

The cell membrane has been suggested (see, for example, ~,ef. [1]) as a target of UVA radiation (320-400 rim). i~ncreased sensitivity to inorganic salts, membrane leakage and lipid peroxidation have been observed after UV irradia'ion of E s c h e r i c h i a coli [1-3], indicating that membrane ,Jamage occurs, but its relative importance to cell death is still ,mclear [4,5 ]. The Gram-positive skin bacterium P r o p i o n i b a c t e r i u m ,zcnes, implicated in the skin disease acne vulgaris [6], pro,iuces endogenous porphyrins, mainly protoporphyrin and ,:oproporphyrin [ 7 ]. According to an action spectrum for the :nactivation ofP. a c n e s by UVA radiation and blue light [ 8 ], ~he sensitivity of P. a c h e s is highest for shorter wavelengths and decreases with increasing wavelength. However, a local :;ensitivity maximum, which is dependent on the endogenous !~orphyrin concentration, is found in the blue region (maxi:hum, 415 nm) [8]. Further results obtained from photonactivation studies ofP. a c n e s indicate that blue and red light ~nactivation involves porphyrin sensitization induced via sin:~let oxygen, whereas inactivation by UVA radiation (360 or 320 nm) appears to involve oxygen-dependent mechanisms ,)ther than singlet oxygen processes [ 8,9]. An increase in 011-1344/95/$09.50 © 1995 Elsevier Science S.A. All rights reserved

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polyphosphates and a dissipation of the pH gradient across the cell membrane have been observed in 3tp nuclear magnetic resonance (NMR) studies of P. a c h e s irradiated with broad-band UVA, resulting in approximately 10% survival [ 10]. These results indicate that proton pumping across the cell membrane may be affected by UVA irradiation of P. aches. A decrease in the electrochemical proton gradient has also been found in E. coli after UVA irradiation, resulting in more than 70% inhibition of the respiration rate [ 11 ]. In this study, we investigate the intracellular pH changes induced in P. a c n e s by irradiation. Due to the involvement of different inactivation mechanisms in the UVA and visible light regions, the effects of broad-band blue light, monochromatic blue light (415 rim) and UVA radiation (360 nm) were examined. Two methods (31p NMR spectroscopy and fluorescence spectroscopy based on a pH-sensitive fluorescent probe (2',7'-bis-(2-carboxyethyl)-5-(and -6-)- carboxyfluorescein (BCECF))) have previously been used to determine the intracellular pH of P. a c h e s [ 10,12]. The NMR technique requires a high cell density and has a limited time resolution, but enables the monitoring of a sample for several hours [ 13]. The use of pH-sensitive fluorescent probes does not require such a high cell density and provides a higher time resolution, but can be hampered by probe leakage which

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limits the length of time a sample can be monitored [ 12]. We therefore chose to use both methods to examine the pH changes induced by irradiation.

2. Materials and methods 2.1. Growth conditions P. acnes (Serotype I, CN 6278) was grown anaerobically on bovi~ae blood-agar plates under dark conditions at 37 °C [7 ]. Four days prior to the experiments, bacteria were transferred to a complex yeast extract medium (20 g bacto-agar (Difco Laboratories, Detroit, MI), 10 g tryptone (Difco), 5 g yeast extract (Difco), 10 g NaCI (Merck, Darmstadt, Germany) in 1 I distilled water) or to Eagle's synthetic medium (basal medium Eagle 10× concentrated with Earle's balanced salt solution without sodium bicarbonate and L-glutamine (Whittaker Bioproducts, Walkersville, MD) modified with agar noble (Difco) and buffered to pH 6.7 with 30 mM Na2HPO4.2H20 and 20 mM KH2PO 4 (Merck)) [ I0]. The bacteria were then incubated until harvesting in the late exponential phase. 2.2. 31p NMR spectroscopy studies 2.2.1. Cell suspensions P. aches ( (1.5-2.5) × 10 II cells ml- 1) was suspended in

buffer containing an equal volume of 10 mM Na2HPO4, 10 mM KH2PO4, 85 mM NaC1, 60 mM 2-(N-morpholino)ethanesulphonic acid (MES; Sigma Chemical Co., St. Louis, MO), 100 mM piperazine-N,N'-bis(2-ethanesulphonic acid) (PIPES; Sigma) and 10% D20 (Hydro, Rjukan, Norway) and was adjusted to pH 6.8 using 1 M NaOH. The bacteria constituted approximately 30%--40% of the total sample volume. 2.2.2. 3lp NMR spectroscopy

NMR tubes (10 mm) containing 1.5 ml cell suspension were used to examine the sample. The NMR spectra were recorded at 22 °C using a Bruker WM-400 spectrometer at 161.81 MHz. Accumulation was conducted using a 71 ° pulse (30/~s), a 1.0 s repetition rate and 200-720 transients. The experiments were conducted in a D20 locking mode. In order to avoid radial concentration gradients in the sample, it was not spun in the sample holder. The chemical shift values were referred to the shift of external 85% phosphoric acid (Merck). Two peaks corresponding to intracellular and extracellular inorganic phosphate can be identified in the 31p NMR spectra of P. acnes suspensions [ 10]. The chemical shifts of these peaks are pH dependent and can therefore be used to determine the intracellular and external pH [ 13 ]. A titration curve of inorganic phosphate [ 13 ] was used as a pH calibration curve.

2.2.3. Irradiation conditions

Samples to be irradiated were removed from the NMR tubes and diluted in 100 ml buffer [10]. The diluted cell suspension was distributed in Petri dishes. Four blue light tubes (Philips TL 20W/06), covered with a 5 mm glass plate, were used to irradiate the samples. Maximum emission was at 425 nm. The irradiance was 17 W m - 2 and was measured using a calibrated thermopile detector (type S15, Sensors Inc.). The Petri dishes containing the cell suspension were placed on the glass plate [ 10]. During irradiation, the suspension was stirred manually at regular intervals. The temperature was 22 °C. A survival curve for P. acnes irradiated by these light tubes was obtained and was used to estimate the surviving fraction after irradiation; 50% survival was obtained after approximately 5 min of irradiation. After irradiation, the cell suspensions were centrifuged (3500 X g, 15 min) and resuspended to their original cell density [ 10]. They were then used in further NMR measurements. 2.3. Fluorescence spectroscopy studies 2.3.1. Cell suspensions and determination of intraceUular pH Cell suspensions of P. aches (5 x 108 cells m1-1) were

prepared using 100 mM MES, titrated to pH 6.5 using 3 M NaOH. The fluorescent probe BCECF was introduced into the cells using the membrane-permeable acetoxymethyl ester of BCECF (BCECF-AM). BCECF-AM (5 /zM) (stock solution, 1 mM in dimethyl suiphoxide (DMSO); Molecular Probes, Inc., Eugene, OR) was added to the suspension. The cell suspensions were then incubated at 6 °C for 2 h such that intracellular esterases could hydrolyse BCECF-AM to form BCECF which is then retained intracellularly [ 12]. The suspension was then centrifuged at 12 9 0 0 × g and 3 °C for 5 min. The pellet was resuspended and washed once before final resuspension. Corrected fluorescence excitation spectra of BCECF-incubated P. acnes were recorded using a Perkin Elmer LS 50B spectrofluorometer. Excitation was performed at 440-515 nm and the emission wavelength was set at 535 nm. The excitation and emission monochromator bandwidths were 5 nm and 10 nm respectively. The ratio of the fluorescence intensities of BCECF at 505 nm and 450 nm was used to determine the intracellular pH [ 14]. To relate the fluorescence ratio 505/ 450 to the pH for a given cell suspension, the procedure described in Ref. [ 12] was used. Briefly, the ionophore nigericin (final concentration, 5 ~M, Sigma) and KCi (final concentration, 100 mM) were added to the suspension to eliminate pH gradients across the cell membrane. By titrating the cell suspension with small amounts of 0.5 M NaOH or 0.5 M HC1, the fluorescence ratio at different pH values was obtained. Using the method of least squares, a calibration curve of the fluorescence ratio as a function of pH was constructed from which the intracellular pH of the sample was determined (in all cases, the correlation coefficient was greater than 0.99). This procedure was repeated for each

C.M. Futsaether et al. / Journal of Photochemistry and Phowbiology B: Biology 31 (1995) 125-131

sample such that a calibration curve corresponding to that particular sample was obtained. 2.3.2. Radiation source and irradiation conditions

The samples were irradiated at two wavelengths, 360 and ~15 rim. As the cell density required for fluorescence measurements was several orders of magnitude smaller than that required for NMR spectroscopy, a different irradiation source was used. A 1000 W xenon lamp coupled to a monochromator 1Applied Photophysics, London, UK), combined with a Coming UV filter (0-32 (360 rim) or 0-52 (415 nm)), was ~sed to irradiate the sample. At the sample position, the fluence rate was 15 W m -2 (360 nm) or 17 W m -2 (415 nm). In both cases, the half-power bandwidth was 20 nm. Lamp fluence rates were measured using an Optronic OL 752 spectroradiometer (Optronic Laboratories, Orlando, FL). To test for the possible phototoxicity of BCECF at the ~rradiation wavelengths of 360 and 415 nm, the surviving tractions after irradiation of a control sample (not incubated with BCECF-AM) and a BCECF-AM-incubated sample were compared. After final resuspension after incubation, the eli suspension was diluted (2 × 104 cells m l - ~). Aliquots of ml of diluted cell suspension were irradiated at the desired wavelength. During irradiation, 20/.tl of cell suspension was removed every 3-5 min and seeded on bacto-agar plates. The urviving fraction was determined by examining the colonyiorming ability as described in Ref. [8]. For the irradiation wavelengths 360 nm and 415 nm, the irradiation times required to produce 50% survival were approximately I 0 min ~nd 7 min respectively. The intracellular pH after irradiation was determined as follows. A cell suspension was prepared and incubated as ttescribed above. After final resuspension after incubation, the suspension was divided into two samples. An aliquot of I ml of one sample (5 × 108 cells ml-~) was irradiated at ~;60 or 415 nm with a fluence resulting in a chosen surviving traction. In each case, the surviving fraction after irradiation was determined. The other (control) sample was kept in the c~trk. After irradiation, both samples were washed once to remove leaked BCECF [12] and resuspended in 2 ml of buffer. The intracellular pH values of the cells in each suspension were then determined using the method described ~.bove. Due to washing and resuspension, the intracellular pH was not determined until 5 min after irradiation. During irradiation, a homogeneous solution was mainl ained by continuously bubbling air through the suspension. All irradiation experiments were conducted at room temper~ture (22 °C) and in randomized order to avoid systematic rrors.

2,. Results ~. 1.31p NMR spectra of P. acnes irradiated by broad-band Hue light

NMR spectra of P. acnes were recorded before and after iT-radiation by broad-band blue light. The samples were mon-

127

itored for up to 3 h after irradiation. Due to the dilution of the samples prior to irradiation and the subsequent resuspension to the original cell density after irradiation, the NMR spectra of irradiated cells could not be recorded until approximately 30 min after irradiation. The cellular response immediately after irradiation could therefore not be determined. The dilution process was found to have no effect on the NMR spectra (data not shown). The results from a representative experiment in which a P. acnes sample was irradiated for 30 s by broad-band blue light are shown in Figs. 1 and 2. Fig. 1 shows the 3~p NMR spectra 15 rain before and 40 min after 30 s of irradiation. The surviving fraction after irradiation was approximately 90%. It can be seen that the amount of polyphosphate (PP) increased after irradiation. The amount of internal inorganic phosphate (Pi i") decreased simultaneously. At approximately 1.5 h after irradiation, the amount of polyphosphate had decreased to pre-irradiation levels. The increase in polyphosphate levels is similar to that observed after near-UV irradiation of P. aches and is discussed in detail in Refs. [ 10] and [ 15]. As can also be seen in Fig. 1, the chemical shift of the internal inorganic phosphate peak was displaced from 2.07 to 2.55 ppm after irradiation, indicating an increase in intracellular pH from 6.6 to 7.0. The external pH decreased by 0.1 pH units as estimated by the chemical shift of the external inorganic phosphate peak. Thus the pH gradient across the cell membrane increased after blue light irradiation. Fig. 2 shows the intracellular pH, the external pH and the pH gradient (ApH) across the cell membrane as a function of time after irradiation. Both the intracellular pH and pH gradient increased after irradiation. After approximately 3 h after irradiation, the intracellular pH decreased to its preirradiation value. The pH gradient, however, remained at a level higher than its pre-irradiation value due to a decrease in the external pH. It appears that the pH changes induced after irradiation are at least partly reversible. The NMR spectra of P. acnes irradiated by lethal doses (less than 40% survival) of blue light were also recorded. In this case, the internal and external inorganic phosphate peaks coincided after irradiation. This seems to indicate that lethal doses of blue light lead to a dissipation of the pH gradient across the cell membrane. In addition, an increase in the amount of polyphosphate was observed after irradiation by lethal doses of blue light. Similar results have been observed after irradiation of P. aches with lethal doses (less than 10% survival) of broad-band UVA [ 10] and after photodynamic treatment of haematoporphyrin derivative (HpD)-sensitized P. acnes[ 16]. 3.2. Irradiation-induced intracellular pH changes detected using BCECF 3.2.1. Phototoxicity of BCECF Incubation ofP. acnes under dark conditions with BCECF-

AM or BCECF for up to 4 h did not affect the cell viability [ 12]. Thus, under dark conditions, BCECF does not have a

128

C.M. Futsaether et al. / Journal of Photochemistry and Photobiology B: Biology 31 (1995) 125-131

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Fig. 1.3tp NMR spectra 15 rain before (A) and 40 rain after (B) 30 s irradiation of P. acnes with broad-band blue light. P. aches was grown on bacto-agar medium. The surviving fraction after irradiation was 90%. The peaks are as follows: SP, sugar phosphate; Plj", internal inorganic phosphate; pjo, external inorganic phosphate; PP, polyphosphate [ 10]. The spectra show a representative experiment.

toxic effect on P . a c n e s . To determine whether BCECF is phototoxic during UVA or blue light irradiation, the cell survival after irradiation was examined for cells incubated with ( BCECF sample) or without (control sample) BCECFAM. The survival curves are shown in Fig. 3. In most cases, BCECF enhanced the inactivation of P. a c h e s after irradiation

at 360 nm or 415 nm. The degree of BCECF phototoxicity was dependent on the irradiation wavelength and the growth medium. BCECF phototoxicity was greatest for cells grown on Eagle's minimum essential medium. Significantly less phototoxicity was observed for P. a c h e s grown on bacto-agar medium, as exemplified by the low phototoxicity observed after 360 nm irradiation of bacto-agar-grown P . a c n e s . Cel-

C.M. Futsaether et al. / Journal of Photochemist~ and Photobiology B: Biology 31 (1995) 125-131 1.2

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i ular BCECF uptake was, however, independent of the growth medium used (data not shown). The phototoxicity nduced by 415 nm irradiation was greater than that induced 9y 360 nm irradiation. Although the BCECF absorbance at 360 nm and 415 nm is relatively small, absorption may con:ribute to the observed phototoxicity. Some fluorescein derivatives have been shown to have a photosensitizing effect in the visible light region [ 17,18]. At pH 7.0, the approximate intracellular pH of control cells, maximum BCECF absorbance occurs at approximately 500 nm. The absorbance at 360 nm and 415 nm is approximately one order of magnitude smaller than that at 500 nm. The slightly larger absorbance at 415 nm than at 360 nm may partly explain the greater phototoxicity observed at 415 nm.

Intracellular pH changes induced in P. acnes by 360 nm or 415 nm irradiation were examined. Due to the low BCECF phototoxicity (Fig. 3), bacto-agar-grown P. aches was used to investigate the effects of 360 nm irradiation. P. acnes grown on either Eagle's or bacto-agar medium was used to investigate the effect of 415 nm irradiation. Little BCECF phototoxicity was observed after 415 nm irradiation (for survival of greater than 30%) of bacto-agar-grown P. aches The average intracellular pH in irradiated P. acnes was determined approximately 5 min after irradiation. This pH was compared with that of control cells which had not been irradiated. In all cases, the external pH (pHo) was 6.5 and was constant throughout the experiment. At pHo 6.5, the intracellular pH in the control cells was in the range pH 6.87.0 (mean, pH 6.9). In Fig. 4, the results are presented as the difference between the intracellular pH in irradiated and control cells ( p H i . - pHco.) as a function of the surviving fraction after irradiation. Thus, when p h i , - pH¢on > 0, the intraeellular pH in irradiated cells is larger than that in control cells. As can be seen from Fig. 4, the same pattern of irradiationinduced pH changes was obtained regardless of the irradiation wavelength, growth medium and degree of BCECF phototoxicity. The same pattern, but with greater spread in the data, was observed if pHi,~- pH¢o, was plotted as a function of the fluence. Cells irradiated with fluences resulting in more than 10% survival experienced an increase in intracellular pH relative to the control cells. The maximum increase, 0.3 pH units, appears to occur at approximately 60%-80% survival. For fluences resulting in less than 10% survival, a decrease

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C M. Futsaether et al. / Journal of Photochemistry and Photobiology B: Biology 31 (1995) 125-131

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Fig. 4. Intracellular pH changes (pH,,~-pH,~,) 5 min after irradiation at 360 or 415 nm as a function of the surviving fraction, pH~ is the intracellular pH in irradiated ceils and p H ~ is the intracellular pH in control calls. The pH was determined 5 min after irradiation. P. aches was grown on two different media. The data points represent P. aches grown on and irradiated at: × , bacto-agar medium, 360 nm; <>, bacto-agar medium, 415 rim; 4~, Eagle's medium, 415 nm. For 4', the mean ± SE ( n = 3 ) is shown. For × and <>, the points are single measurements and the error bars show the estimated error in pH,~ - primo, ( ± 0.02 pH units) obtained experimentally by comparing the intracellular pH in two samples treated identically but not irradiated.

in intracellular pH relative to the control cells was observed. For less than 1% survival, the intracellular pH approached the external pH such that pHir~- pHco, approached - 0.4 pH units. This corresponds to the results found in the NMR experiments where the intracellular and external inorganic phosphate peaks coincided after lethal doses of radiation.

4. Discussion

In this study, we have shown that intracellular pH changes are induced in the skin bacterium P. acnes after irradiation with UVA or blue light. The intracellular pH changes are dependent on the cell survival after irradiation. Sublethal doses of irradiation result in an increase in the intracellular pH by up to 0.4 pH units. This increase appears to be reversible. After lethal doses of irradiation, the intracellular pH decreases and approaches the external pH. This pattern is obtained in both the UVA and blue light regions despite evidence indicating that different inactivation mechanisms are involved in these two regions. In the case of 415 nm light, Kjeldstad and Johnsson [8] proposed that photoinactivation involves porphyrin sensitization induced via singlet oxygen. However, inactivation at 360 nm appears to involve oxygendependent mechanisms other than solely singlet oxygen processes [9]. We used two separate methods (31p NMR spectroscopy and fluorescence spectroscopy) to determine whether intracellular pH changes were induced in P. acnes. Both methods predict the same type of pH changes. It is therefore unlikely that the phototoxicity observed in some cases when using the fluorescent probe BCECF is the cause of these pH changes,

Rather, the pH changes appear to be a cellular response to irradiation. The irradiation-induced pH changes may be a result of membrane damage. As sublethal fluences result in an increase and lethal fluences in a decrease in the intracellular pH, it is possible that two mechanisms may be responsible for the pH changes. Increased pumping activity, inducing an increase in the pH gradient across the membrane (inside alkaline), may be involved in the pH alterations observed at sublethal fluences. Changes in pumping activity, perhaps involving proton extrusion, may indicate modifications of an ion channel. Galio et al. [19] reported changes in membrane potential induced by low doses of broad-band UV and suggested that these changes may reflect specific damage to an ion channel. Peak et al. [20] observed leakage ofS6Rb + (K + analogue) from &aminolaevulinic acid-sensitized E. coil after monochromatic UVA irradiation and suggested that damage to a proton K + pump may be involved. The pH homeostasis in bacteria is believed to be controlled by the concerted action of a number of electrogenic and electroneutral alkali cation transport systems [21,22]. It is possible that UVA- or blue light-induced modification of one or more of these transport systems may lead to a disruption of pH homeostasis, characterized by an increase in intracellular pH, perhaps as a result of proton extrusion. Such a mechanism could be responsible for the increase in intracellular pH obtained in P. aches at sublethal fluences. Lethal fluences result in a decrease in the pH gradient across the cell membrane (inside alkaline). The decrease in intracellular pH may be a result of proton influx due to increased membrane permeability. Changes in membrane permeability after UVA irradiation have been observed in other studies. Kelland et al. [3] detected leakage of S6Rb+, [ methyl-3H ] thymidine and cell components absorbing at 260 nm after UVA irradiation of E. coli. Lipid peroxidation, which has been linked to increased ion permeability, and membrane lysis have been detected after UVA or blue light irradiation ofE. coli [ 1,23-25 ]. The decrease in electrochemical proton gradient observed in E. coli after exposure to high doses of UVA radiation was thought to be coupled to damage to a proton-amino acid cotransport system [11]. Thus increased passive membrane permeability caused by ion channel damage, lipid peroxidation or membrane lysis may be responsible for the decrease in pH gradient observed after lethal fluences.

5. Conclusions

Irradiation of P. acnes with UVA or blue light induces intracellular pH changes which are a function of the cell survival. The induced pH alterations follow the same pattern regardless of the irradiation wavelength. It is therefore likely that the mechanisms leading to pH alterations are the same in the UVA and blue light regions.

C.M. Futsaether et al. / Journal of Photochemistr), and Photobiology B: Biology 31 (1995) 125-131

Acknowledgments We wish to thank Dr. Jostein Krane and his colleagues at the NMR Laboratory in Trondheim for their assistance with the NMR experiments. This investigation was supported by ihe Research Council of Norway.

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