Different effects of azole-antifungal agents on the regulation of intracellular calcium concentration of Trichophyton rubrum

Different effects of azole-antifungal agents on the regulation of intracellular calcium concentration of Trichophyton rubrum

JOURNALOF Dermatological Science Journal of Dermatological Science 12 (1996) 156- 162 Different effects of azole-antifungal agents on the regulation...

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JOURNALOF

Dermatological Science Journal of Dermatological Science 12 (1996) 156- 162

Different effects of azole-antifungal agents on the regulation of intracellular calcium concentration of Trichophyton rubrum Ineko Inoue, Mariko Seishima”, Kazuko Osada, Yasuo Kitajima Department of Dermatology,

G~jii University School of Medicine, Tsukasamachi 44 Gifu 500, Japan

Received 28 April 1995; revision received 11 August 1995; accepted 21 August 1995

Abstract Prior studies have indicated that intracellular calcium concentration ([Ca’+]i) is involved in fungal cell growth. However, it has not been known whether antifungal drugs affect signal transduction via calcium in fungal cells. In this context, we examined the effects of antifungal drugs, itraconazole, bifonazole and ketoconazole, on [Ca2+]i in Trichophyton rubrum. Itraconazole (l-5 rig/ml) induced a rapid and transient [Ca2‘Ii increase, peaking at 15-20 s in hyphal cells of T. rubrum, but not in spores. The slow descending phase of the [Ca2+ ]i increase induced by itraconazole was depleted by chelating extracellular calcium with ethylene glycol bis(a-aminoethyl ether)-N, N,N’,N’tetraacetic acid (EGTA), suggesting that the increase in [Ca’+]i is biphasic: Ca2+ mobilization from the internal pool and influx from the outside of the cell. At 10 rig/ml and 100 rig/ml, however, itraconazole induced an explosive and sustained calcium increase in both spores and hyphae. At less than 1 rig/ml, no [Ca’+]i increase was caused in both hyphae and spores. On the other hand, although some hyphal cells showed a transient [Ca2+]i increase, most of the cells did not show any changes of [Ca2+]i after the addition of ketoconazole at 10 rig/ml. Both spores and hyphal cells incubated with 100 rig/ml of bifonazole or ketoconazole showed a gradual increase of intracellular calcium concentration until 5 min, when the measurement was ceased. These findings suggest that signal transduction via calcium might be involved in some biological effects of itraconazole on T. rubrum, and that bifonazole and ketoconazole could differently affect [Ca’+]i in T. rubrum from itraconazole. In addition, the determination of [Ca2+]i changes induced by antifungal agents may contribute to clarification of the biological effects on fungal membranes. Keywords: Trichophyton rubrum; Intracellular calcium concentration; Antifungal agent; Signal transduction

1. Introduction

Abbreviations: DMSO, dimethyl sulfoxide; EGTA, ethylene glycol bis(/?-aminoethyl ether)-N, N, N’,N’-tetraacetic acid * Corresponding author.

Intracellular calcium plays an essential role in the regulation of cellular functions by acting as a second messenger in response to extracellular stimuli [l]. These stimuli induce a rapid and tran-

0923-181l/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0923-1811(95)00475-8

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sient rise in intracellular calcium concentration ([Ca’+]i) in target cells either by opening Ca2+ channels in the plasma membrane to allow extracellular Ca2+ to enter the cytoplasm or by activating cell surface receptors that trigger hydrolysis of membrane phosphoinositide lipids and thereby produce cytosolic inositol 1,4,5-trisphosphate (IP,) that releasesfree Ca2+ from internal stores [Z]. There are a variety of factors which regulate the intracellular calcium concentration, such as Ca2+ -adenosine triphosphate (ATP)ase, mitochondria, endoplasmic reticulum, and vacuoles in fungi [3,4]. The distribution of calcium concentration in yeasts has been already rather well studied by Jackson and Health [3]. The Ca2+ gradient was highest and steepestin the apex (within about 3 pm of the tip) and declined rapidly to a lower level 10 to 20 pm behind the tip in SaproZegnia fir-ax, suggesting that a tip-high gradient of cytoplasmic Ca2+ may play a regulatory role in hyphal tip growth in fungi [3]. Recently, antifungal agents for topical application, such as itraconazole, bifonazole and ketoconazole, were developed for fungal infection [5]. These agents have been reported to inhibit lipid synthesis or affect the membrane integrity in fungal cell walls [5]. The inhibition of ergosterol synthesis affects the membrane constituents, molecular structure and function, which should lead to dysfunction of transmembrane signaling. From these viewpoints, it might be of great interest to know how these antifungal agents affect the transmembrane signaling via intracellular calcium to disturb the fungal cell functions. 2. Materials and methods

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tone and 1% yeast extract with different concentrations of antifungal drugs or vehicle. Cultures were shaken at 80 cycles/min at 3°C. After different intervals, cultures were centrifuged at 3000 rev./min for 1 h at 4°C. Dry weights of lyophilyzed pellets were determined. Statistical analysis of the data was carried out by Student’s t-test. 2.2. Measurement of intracellular calcium concentration in T. rubrum T. rubrum was inoculated on the glass cover-

slips (5 mm x 5 mm)overlaying Sabouraud agar. The glass coverslips were set to adhere to the bottom side of flexiperm (Heraeus Biotechnology, Hanau, Germany). After the cells were cultured for 3-4 days at 30°C the agar was removed. The cells were loaded with calcium indicator fura-2/ AM (10 PM) (Molecular Prob., Eugene, OR, USA) by incubation for 2 h at room temperature in 200 ~1 of Hank’s solution. Subsequently, the cells were washed twice with Hank’s solution to be free of extracellular dye and exposed to antifungal drugs at the different concentrations at 3°C. Fluorescence images were obtained at alternating excitation wavelengths of 340 and 360 nm through an SIT vidicon camera, and were processedby an ARGUS-100 image analyzer (Hamamatsu Photonics Corp., Hamamatsu, Japan). The linear 360 nm interpolation was used and the corrected fluorescent emission intensity ratio, by using 340 and 360 nm excitation with background subtraction was monitored continuously in single cell. Conversion from ratio into absolute [Ca’+] was done using the equation previously described

PI. 3. Results

2.1. Culture of Trychophyton rubrum Five strains of T. rubrum were isolated from scales on 5 different patients with tinea pedis. T. rubrum was grown on Sabouraud glucose (2%)

3.1. Effects of antifungal agents on growth rate of T. rubrum The representative growth profile of T. rubrum

agar at 30°C for 3 weeks. Subsequently, the conidial suspension was prepared by filtering through sterilized gauze to remove hyphal fragments and agar. The cells were adjusted to 4 x lo6 spores/ml by counting in a haemocytometer, and then inoculated in 100 ml of the Sabouraud glucose broth containing 4% glucose, 1% polypep-

in Sabouraud glucose broth is illustrated in Fig. 1. Dry weight of T. rubrum was increased until 72 h after inoculation, and then reached plateau. There is no significant difference in growth curve among 5 strains of‘ T. rubrum. The growth rate of T. rubrum in the growing phase was clearly reduced by itraconazole at concentrations of 5 and 100

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rig/ml until 24 h after inoculation, compared to those incubated with dimethyl sulfoxide (DMSO) culture medium alone and even with ketoconazole (5 and 100 rig/ml) and bifonazole (5 and 100 rig/ml) (Fig. 2). However, by 48 h after inoculation, the growth rate of fungus incubated with 5 rig/ml of itraconazole was recovered to the control level, while that with 100 rig/ml of itraconazole kept the much lower level than those with control. Incubation for 24 h with ketoconazole, bifonazole or DMSO did not affect dry weights of T. rubrum (Fig. 2). After the incubation with 1 pg/ml of bifonazole for 24 h and 48 h, dry weights were 0.31 ) 0.11 g and 0.51 + 0.08 g, respectively. Dry weights after the incubation with 1 pg/ml ketoconazole were 0.49 f 0.09 g (24 h) and 0. 61 f 0. 06 g (48 h). The growth rate was significantly reduced by 1 pg/ml of bifonazole (P < 0.005, at both 24 h and 48 h) or ketoconazole (P < 0.005 at 24 h, P < 0.01 at 48 h) compared to DMSO alone (0.98 f 0.01 at 24 h, 1.23 f 0.20 at 48 h).

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DMSO

(a) ITRA

1

1

I

1

1

48 72 96 120 144

INCUBATION TIME (hr) Fig. 1. The growth profile of Trichophyton rubrum until 144 h after inoculation. Dry weight of T. rubrum was increased until 72 h, and then reached plateau. The values are the means of triplicate determination.

Cc) BIFO

(4

INCUBATION TIME (hr)

Fig. 2. The effects of itraconazole (b), ketoconazole (c) and bifonazole (d) on growth of T. rubrum. T. rubrum was incubated with 100 rig/ml (V) or 5 rig/ml or without (V) itraconazole (b), ketoconazole (c) or bifonazole (d). The growth profiles of T. rubrum incubated without (0) or with 0.01% (A) or 0.0001% (A) of DMSO, which was contained in these agent solutions, are shown in panel a. The growth rate of T. rubrum was statistically reduced by itraconazole at 5 rig/ml (P =C 0.01) and 100 rig/ml (P < 0.01) until 24 h compared to that with DMSO. On the other hand, at 48 h, 100 rig/ml of itraconazole, but not 5 rig/ml, reduced the growth rate compared to DMSO (P < 0.01).

3.2. Changes of [Ca’+]i antifungal drugs

0

(b) KETO

in T. rubrum induced by

In the present experiments, approximately 70% of hyphal segments were loaded with 20 PM of fura-2/AM, while 40%-50% of spores were loaded. The resting [Ca2+]i estimated from the fluorescence ratio (F340/F360) was 90.5 f 30.0 nM (n = 40). There is no significant difference in resting level of intracellular calcium concentration between spores (82.3 f 31.7 nM, n = 20) and hyphal cells (98.5 + 26.5 nM, n = 20). In hyphal cells, but not in spores, 1 and 5 rig/ml of itraconazole induced a rapid and transient increase in [Ca’+]i (Fig. 3a). When 1 PM of ethylene glycol bis(j?-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA) was added 5 min before the itraconazole (5 rig/ml) addition in order to chelate extracellular calcium ions, the slow descending phase of [Ca’+]i increase was cancelled (Fig. 3b). At concentrations of 10 and 100 rig/ml, itraconazole caused an explosive and sustained calcium increase in both spores and hyphae (Fig. 3~). However, no [Ca2+]i increase was observed in both types of cells when 0.1 and 0.5 rig/ml of itraconazole were added (Fig. 3d).

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TIME (min) Fig. 3. Intracellular calcium concentration ([Ca2+]i) response to different concentrations of itraconazole in hyphal cells of T. rubrum. Panels a and b show [Ca* +]i responsesafter addition of 5 rig/ml of itraconazole in hyphal cells pretreated with vehicle (a) or 1 ,uM of EGTA for 5 min (b). Time courses of [Ca’+]i responses induced by 100 rig/ml or 0.1 rig/ml of itraconazole or 0.001% of DMSO are shown in panels c, d, and e, respectively.

TIME (mm) Fig. 4. The effects of different concentrations of ketoconazole on intracellular calcium concentration in hyphal cells of T. rubrum. Ketoconazole at a concentration of 10 rig/ml caused a transient [Ca’+]i increase in less than 10% of hyphal cells (a), but no [Ca’ + ]i responsein most of the hyphal cells (b). Panels c and d show [Ca2+]i responses after the addition of 100 rig/ml of ketoconazole in hyphal cells pretreated with vehicle (c) or I /JM of EGTA for 5 min (d). No [Ca’+]i response was caused by 0.1 rig/ml of ketoconazole in hyphal cells (e).

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No effects of DMSO (0.001% and 0.0005%) were observed on [Ca’+]i as examined as a vehicle control (Fig. 3e). In contrast, although less than 10% of hyphal cells showed a rapid and transient increase in [Ca’+]i peaking at 15-20 s (Fig. 4a), ketoconazole at 10 rig/ml did not induce a calcium increase in most of hyphal cells and almost all spores (Fig. 4b). Bifonazole under 10 rig/ml caused no changes of [Ca’ +]i in spores and hyphal cells (not shown). Both spores and hyphal cells incubated with 100 rig/ml of bifonazole or ketoconazole showed a gradual [Ca2+]i increase until 5 min when measurement was stopped (Fig. 4~). Preincubation with EGTA for 5 min to chelate extracellular calcium did not affect this gradual increase (Fig. 4d). No [Ca*+]i increase was caused by 0.1 or 1 rig/ml of ketoconazole and bifonazole (Fig. 4e). 4. Discussion It is well known that azole-antifungal agents affect the lipid metabolism and disturb the membrane lipid composition of fungal cells. Therefore, it may naturally be suspected that azole-antifungal agents may affect any systems of transmembrane signaling. Calcium-signaling is involved in the regulation of numerous processes in fungal cells, including polarity, secretion, gas exchange, growth [7- 113, and mating [12,13]. From this viewpoint, the present study was carried out to clarify whether or not [Ca*+]i change was involved in the effects of azole-antifungal drugs. Itraconazole at a concentration of 5 rig/ml inhibited the growth of T. rubrum until 24 h after

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inoculation. However, at 48 h, no difference was detected in the growth as examined by measuring the dry weights with or without 5 rig/ml of itraconazole. Therefore, at this concentration of itraconazole, the growth of T. rubrum may be enhanced during the second period from 24 h through 48 h after inoculation. As the minimum inhibitory concentration (MIC) of itraconazole in T. rubrum was 6 rig/ml [14], itraconazole might have some effects on T. rubrum other than growth inhibition via a transient intracellular calcium increase at the concentrations of 5 rig/ml and 1 rig/ml, which are less than MIC. This transient increase of [Ca*+]i may have some beneficial effects on the cell growth of the survived hyphal cells from damage by itraconazole treatment at the concentration of 5 rig/ml, which is almost MIC, since an increase of [Ca’ ‘Ii has been suggested to be related with stimulation of fungal growth at the tip [3]. This may be a possible explanation for the increased growth rate during the period from 24 h through 48 h, and the recovery of the dry weight of cells at 48 h after the addition of itraconazole. Chelating of extracellular calcium by EGTA deleted the descending phase of the transient calcium increase caused by 5 rig/ml of itraconazole. This result suggests that both calcium mobilization from the intracellular calcium pool and calcium influx from the extracellular space are involved in this transient [Ca*+]i increase. This [Ca*+]i change was observed in hyphal cells only. The difference in lipid compositions between spores and hyphal cells [15] might be related to the calcium responsiveness.

Table 1 The change of [Ca’+ ]i in T. rubrum induced by antifungal agents Concentration

1 a/ml 100 rig/ml 10 rig/ml 5 rig/ml 1 rig/ml 0.1 rig/ml Vehicle

Itraconazole

Ketoconazole

Bifonazole

NDa Explosive Explosive Transient increase Transient increase NC NC

ND Gradual increase NCb or transient increase NC NC NC NC

Gradual increase Gradual increase NC ND NC NC NC

NDB, not done; NCb, no change

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A high concentration of itraconazole (10 and 100 rig/ml) induced an explosive and sustained increase of the intracellular calcium, suggesting that itraconazole may have caused a rapid and destructive attack on the cell membrane. This severedamage of the plasma membrane due to 10 rig/ml and 100 rig/ml, enough to cause an explosive increase of [Ca’ + Ii, appears to be well associated with the growth inhibition and the drug concentration over the MIC. On the other hand, bifonazole and ketoconazole caused gradual increasesof intracellular calcium at a higher concentration than MIC (MICs are 45.5 rig/ml and 5.1 rig/ml, respectively) [14,16] and no effect basically at a concentration lower than MIC. This suggests that these antifungal agents may have different effects on cell membrane of T. rubrum from itraconazole. It is of interest to note that 100 rig/ml of ketoconazole and bifonazole, which did not affect the dry weight of T. rubrum during growing phase, caused gradual increases of [Ca’+]i. These gradual increases of [Ca2+]i may be concerned with some dysfunctions due to these agents other than growth inhibition by direct disruption of plasma membrane as seen in an explosive [Ca2‘Ii increase by itraconazole. MICs, which are usually determined by agar dilution or liquid microdilusion methods [14], indicate the minimum concentration at which the antifungal agents inhibit the fungal growth. Since the present study did not reveal the inhibitory effects of these agents by measuring dry weights of fungus until 48 h after addition of the drugs, MIC measured by our methods may be higher, or alternatively, 48 h may be too short to detect the inhibitory effects of these two drugs. However, the latter may be more plausible because of the following reasons. Since azoles inhibit the synthsis of ergosterols, which should be supplied to cell membranes as one of the major constituents, leading to a gradual decrease of the content of pooled ergosterols in membranes, the damage of cell membranes appears gradually rather than explosively, if ketoconazole and bifonazole have no direct disruptive effects on membranes. This gradual damage of membrane may be reflected on the gradual [Ca’ ‘Ii increase shown in our present experiments and no appearance of inhibitory effects on cell growth as early as 48 h

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after addition of these azoles. If these agents have any direct effects on membranes enough to disrupt, an explosive [Ca2‘Ii increase may be caused by these agents, as seenin case of itraconazole. In this regard, it appears to be worthwhile to note that itraconazole molecules interact directly with phospholipid molecules in membranes and disturb the membrane structure at a higher concentration, although it does not induce significant changes in lipid membrane parameters measured by differential scanning calorimetry and infrared spectroscopy with lower concentrations [ 171.Although a small number of hyphal cells of one strain, though less than lo%, showed a transient [Ca*+]i increase, other strains did not respond to 10 rig/ml of ketoconazole. There might be some differences in responsiveness to antifungal agents among different strains. The reason why only 10% of cells responded to 10 rig/ml ketoconazole to show a transient [Ca* + Ji increase is unknown. Although numerous antimycotic agents are available for the treatment of fungal infection [5], little is known about the effects of these agents on the fungal membrane in view of signal transduction. Therefore, in order to examine the effects of the agents on the fungal membrane from this point of view, it may be a useful technique to determine the intracellular calcium change by the addition of these agents.

Acknowledgements

We thank Bayer Yakuhin Ltd. for providing bifonazole, and Janssen-Kyowa Co. Ltd. for itraconazole and ketoconazole.

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