Journal of Medical and Veterinary Mycology (1993), 31, 427~-33

Effect of trifluoperazine on growth, morphogenesis and pathogenicity of Candida albicans

A. D. B. BUCHAN 1, V. A. KELLY 2, O. S. KINSMAN 2, G. W. GOODAY 1 AND N. A. R. GOW 1

1Department of Molecular and Cell Biology, Marisehal College, University of Aberdeen and 2Chemotherapy Department, Glaxo Group Research, Greenford, Middlesex, UK

(Accepted 29 April 1993)

The calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ) and R24571 inhibited the yeast-to-mycelial transition of Candida albicans in vitro. This dimorphic switch is thought to potentiate the invasion of host tissues in superficial and systemic candidosis. Growth of six strains of C. albicans and a range of other pathogenic and saprophytic fungi was inhibited by TFP in vitro with an MIC (minimum inhibitory concentration) of <31~g ml-1 (65,uM) indicating that this compound is a general inhibitor of fungal growth. Between 20 and 60/IM TFP inhibited dimorphism markedly without completely inhibiting growth in the yeast form. This suggests that calmodulin is important in the regulation of yeast-hyphal morphogenesis and that TFP antag- onizes this process thereby suppressing the yeast-to-hypha conversion. The potential of TFP as a therapeutic agent was investigated further in superficial and systemic animal models. TFP was used topically against vaginal candidosis in rats, but no significant protective antifungal effect was observed. Also, when administered orally and subcutaneously against systemic C. albicans infections in mice the ED50 was determined to be >25 mg kg- t dose 1, too large to be considered of therapeutic value. This is in contrast to previous reports describing protective effects of TFP against candidiasis. Therefore, although TFP can inhibit growth and dimorphism of C. albicans in vitro, we conclude that this calmodulin antagonist is unsuitable as a chemotherapeutic agent.

Trifluoperazine (TFP) is a phenothiazine which is used widely as a tranquillizer in antipsychotic therapy [1]. The phenothiazines inhibit calmodulin activity [5, 24] and thereby elicit multiple effects on cellular physiology [5-10, 21]. Previous studies indicated that TFP [2, 9] and its analogue chlorpromazine (CPZ) [24] inhibited growth in vitro of several common pathogenic yeasts including Candida albicans and showed that low concentrations of TFP inhibited germ tube formation [20, 22] without inhibiting the growth of the yeast form.

Eilam et al. [9] found that treatment with TFP at 4-7 mg kg- 1 body weight greatly increased the survival rate of mice infected systemically with lethal doses of C. albicans and Cryptococcus neoformans. The ability of phenothiazines to cross the blood-brain barrier [1, 3] makes them particularly interesting candidate drugs for the treatment of fungal meningitis and encephalitis. Daily doses of up to 56 mg kg - 1, much higher than the therapeutic dose, have been reported to have no toxic effect on mice, causing only mild drowsiness [9].

Here we report an investigation of the effects of TFP on dimorphism and growth of C albicans in vitro and on C albicans infections, both systemically in the mouse and vaginally using the rat vaginitis model. We show that TFP inhibits both growth and temperature/pH-mediated dimorphism of C. albicans in vitro, and that dimorphism was

Correspondence address: N. A. R. Gow, Department of Molecular and Cell Biology, Marischal College, University of Aberdeen, AB9 1AS, UK.

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428 B U C H A N ET AL.

inhibited at concentrations of phenothiazines that did not prevent growth in the yeast form. However, the levels of TFP required to exhibit antifungal effects in animal models in vivo were too high to contemplate its use in chemotherapy.

METHODS

Organisms and culture conditions C albicans (Robin) Berkhout strain 3153A was from the Mycological Reference Laboratory, Colindale, London, UK. All other strains of C. albicans and other fungi were from the Glaxo Group Research culture collection, Greenford, Middlesex. C albicans strain 2402E is a clinical isolate with a known virulence in animal models and was used for pathogenicity testing as described below. The organism was stored in aliquots in liquid nitrogen at - 140°C and, when required, subcultured onto Sabouraud maltose agar slopes (Oxoid) and incubated overnight at 37°C. C. albicans strain C316 was stored on Sabouraud maltose agar slopes and subcultured in Sabouraud broth (Oxoid). Inocula were prepared from overnight shaking cultures in Sabouraud broth at 37°C.

For all experiments in vitro, C. albicans was grown on Sabouraud dextrose slopes or in the defined medium of Lee et al. [15], modified according to Buffo et al. [4] and referred to as Soil's medium (SM), at an initial pH of 4-5 and temperature of 25°C. To induce mycelial growth, stationary phase cells were harvested and resuspended at an inoculation density of 5 x 106 yeast cells ml- ~ in fresh pre-warmed SM at pH 6"5 and 37°C. The cells were grown in shaking culture at 37°C for a period of 6 h. Samples were removed hourly, fixed in formalin and examined microscopically. Percentage germ tube and bud formation was calculated from at least 300 cells per sample.

Phenothiazines and dimorphism TFP, CPZ and R24571 were from Sigma (Poole, UK). These compounds were dissolved initially in DMSO (dimethylsulphoxide), diluted subsequently in distilled H20, titrated to neutral pH with NaOH and sterilized by filtration. Final concentrations of TFP were 0 (control), 1, 10, 20, 50 and 100/tM (048/zg ml - 1). The concentration of DMSO in the medium was never >1% (v:v) and did not affect growth or dimorphism (data not shown).

TFP and growth MIC values for TFP were determined on a range of C albicans strains and other fungi over a 24 h growth period using standard agar dilution in Isosensitest agar (Oxoid Ltd., Basingstoke, UK). The MIC was the lowest concentration that prevented visible growth of colonies on this agar. The effect of TFP, CPZ and R24571 on the growth of yeast cells in liquid cultures was also determined spectrophotometrically by absorbance at

600 nm.

Animals For the vaginitis experiments, ovarectomised CD female rats (Charles River Breeding Laboratories Ltd., Margate, UK) of between 150-200 g were used. For the systemic infections, female mice (CRH) of 18-22 g were used. For the experimental vaginitis experiments ovarectomy was performed at least 1 week before infection.

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EFFECT OF T F P O N CANDIDA 429

Rat vaginal candidosis Rats were induced to pseudoeostrous by intraperitoneal injection of 0.2 ml of 5 mg ml-1 oestradiol benzoate in ethyl oleate ('Benztrone', Paines and Byrne, Greenford, Middlesex, UK), 4 days before infection. This hormonal state is essential for predis- position to infection [14]. The oestrous state of the rats was monitored daily throughout the experiment as described previously [14] using the Papanicolaou method of staining vaginal epithelial cells [11, 23]. Rats found to be no longer in the pseudoestrous state were removed from the experiment.

The effects of TFP were compared to treatment with clotrimazole (Bayer, Newbury, UK) as a positive control. Yeast cells of C. albicans 2402E were suspended in 0"8% saline, 10 mg ml- ~ clotrimazole or 20/t~a TFP. Rats were inoculated with 0-1 ml saline containing 3.3 × 107 cells applied directly into the vagina, using the barrel of a 1 ml syringe.

Administration of the drugs was by douching at the time of infection (as above) and by subsequent dosing at 24 h and 48 h in volumes of 0' 1 ml of 10 mg ml- 1 clotrimazole or 1 ml of 20/~M TFP. Control treatment used a 1 ml saline douche. In one experiment the initial TFP dosing was delayed until 4 h after inoculation with C. albicans. Vaginal infection of rats was monitored by washing with 1 ml saline at 24, 48 and 72 h after inoculation and determining viable counts expressed as the log CFU (colony forming units)ml 1 vaginal washing [15]. The results were analysed by Levene's test and analysis of variance (ANOVA).

Mouse systemic candidosis Acute systemic candidosis was achieved following an intraperitoneal inoculation of 2.4 × 107 CFU C. albicans C316 in 1-25% (w:v) 'Yestamin' (baker's yeast), in a total volume of 0.5 ml. Addition of the baker's yeast enhances virulence. TFP and keto- conazole (Janssen Biochimica, Beerse, Belgium) were prepared in 0"5% (w:v) sodium carboxy methyl cellulose in 0-8% saline. Treatment with drugs administered orally or subcutaneously was carried out 1, 3 and 5 h after inoculation in groups of five mice. Control mice received no drugs. A separate group of mice was injected with the 'Yestamin' only and treated with the two higher doses of TFP (25 and 6'2 mg kg- ~) to determine the effects of TFP on uninoculated animals. Survivors were determined after 5 days and EDs0 values calculated according to the method of Reed & Meunch [19].

RESULTS

TFP inhibits growth and dimorphism Yeast cells were induced to form germ tubes using the regime of pH and temperature- regulated dimorphism as described by Buffo et al. [4]. TFP inhibited germ tube formation of C. albicans 3153A in a concentration-dependent manner as illustrated in Fig. 1. After 6 h, germ tube formation was 97%o, 37%, 15% and 0% in 0, 20, 50 and 100p~J TFP, respectively. In cultures where germ tube formation was inhibited, evagination by budding continued. No significant reduction in growth rate, as measured by absorbance, or yield at stationary phase was evident in cultures grown in liquid media containing up to 50 pM TFP. However the yield was inhibited by 63% by 100 pM TFP. In the presence of TFP, budding cells clumped in a fashion characteristic of mycelial development. Ultimately this limited the analysis of percentage bud formation, after exposure to TFP, in these experiments.

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430 BUCHAN ~]" AL.

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FIG. 1. The effect of0#M (A), 10,u~ (11), 20/tM (~) , 50/~M (0 ) and 100/~M (O) TFP on percentage germ tube formation of C. albicans 3153A. The error bars indicate standard errors of the mean for tripIicate measurements (n= 300).

TFP was inhibitory to the growth of a range of yeasts and filamentous fungi when tested by the agar dilution method in Isosensitest agar. MICs for six strains of C. albicans, four other Candida species included Candida glabrata, Candida krusei and Candida parapsilosis and one strain each of Aspergillusflavus and C. neoformans were between 16 and 31/tg ml-a (34-65~tM). A. flavus and Schizosaccharomyces pombe showed significantly less sensitivity to TFP with MICs of 62 and > 125/tg ml - 1.

Other calmodulin inhibitors such as R24571 and CPZ also inhibited dimorphism. As reported previously, R24571 was more potent than TFP [22] while CPZ was less potent than TFP in inhibiting morphogenesis. For example, germ tube formation was 20% in 1/~M and 3% in 5/~4 R24571 after 6 h. In comparison, germ tube formation was 97%, 95% and 60% in 1/tM, 5/~M and 50/zM CPZ after 6h. As with TFP, these concentrations had no effect on the specific growth rate or yield of the yeast form of C. albicans 3153A measured spectrophotometrically (data not shown).

The effects of TFP on growth and dimorphism showed that there was a narrow window of concentrations of TFP (approximately 20 60/~M in our experiments) in which the dimorphic transition was markedly inhibited but growth by budding still occurred.

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FIG. 2. Effects of TFP on establishment and maintenance of rat vaginal infections of C. albicans 2402E. Rats were treated with saline (©), 0.1 ml of 10 mg ml- 1 clotrimazole (0 ) or 1 ml of 20 mM TFP (~) at 0, 4, 24 and 48 h. In one experiment (IlL TFP was administered only at 4, 24 and 48 h to allow germ tubes to form before administration of the drug.

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EFFECT OF TFP ON CANDIDA

TABLE 1. Efficacy of TFP and ketoconazole in mouse systemic candidosis with C albicans C316. Yeast cells of C albicans were inoculated intraperitoneally

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Route of administration Dose Survivors EDs0

Compound of drug (mg kg - l) (5 days) (mg kg - 1 dose - l)

TFP Oral 25 1/5 >25 6.2 0/5 1.5 0/5 0.4 0/5

TFP Subcutaneous 25 0/5 >25 6.2 0/5 1.5 0/5 0.4 0/5

Ketoconazole Oral 100 5/5 7.1 25 4/5 6.2 3/5 1.5 0/5

Ketoconazole Subcutaneous 50 5/5 5.4 12.5 4/5 3"1 2/5 0.8 2/5

Control 0 1/15

Antifungal activity of TFP in the rat vaginitis model

The effect o f T F P on the establishment o f superficial Candida infections was studied in the rat vaginitis model with C. albicans strain 2402E. Trea tment with clotrimazole as a positive control resulted in a significant (P=0.004) decline in the number o f C F U compared to controls using saline alone (Fig. 2). In contrast , T F P had little effect on the C F U recovered when administered with the inoculum or 4 h post-infection (Fig. 2). The post-infection t reatment was used to a t tempt to discriminate between the effects o f T F P before and after germ tube format ion in an experimental animal. However , no difference was seen in the numbers o f C F U recovered when the T F P was administered prior to, or after, inoculat ion with yeast cells. Levene's Test and A N O V A showed no significant differences between saline control groups and animals receiving T F P at all concentrat ions tested (F= 0"07; d.f. = 2,15 P = 0"93). In addition, hyphal growth was detected in vaginal smears f rom treated animals. Thus T F P had no therapeutic effect in the rat vaginitis model.

Mouse systemic candidosis

To examine the potential anti-Candida effect o f T F P in systemic candidosis, mice were inoculated intraperi toneally with 2.4 × 107 C F U as described. The survival o f mice receiving T F P or ketoconazole, either orally or subcutaneously, was assessed. Using either me thod o f T F P administrat ion the EDso was determined as >25 mg k g - 1

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432 BUCHAN ET AL.

dose - 1, too high to be considered of therapeutic value (Table 1). Higher concentrations of the drug were not investigated due to marked tranquilizing effects of the drug. For animals receiving TFP but no inoculum, all mice dosed three times with 25 mg kg- 1 dose - 1 or 6"2 mg kg 1 dose 1 were tranquilized by the TFP after 24 h but appeared normal at 48 h. Positive controls using ketoconazole showed an EDs0 of 5-4 and 7" 1 mg kg- ~ dose- 1, with subcutaneous and oral dosing, respectively.

DISCUSSION

We have found that low concentrations of TFP and other calmodulin antagonists inhibited the induction of germ tube formation using the commonly adopted proto- col of temperature and pH-regulated dimorphism [4]. A window of concentrations of TFP, between 20 and 60/~M, was defined in which morphogenesis was inhibited, but growth could occur by budding. MIC values obtained by plate culture are not directly comparable to inhibition experiments in liquid culture but results indicated that other pathogenic fungi were inhibited by TFP within a similar range of concen- trations. TFP-mediated inhibition of germ tube formation by serum-induced [20], N-acetylglucosamine-induced [21] and pH-induced dimorphism at low temperature [22] has been reported although the effects on the growth of the yeast form were not described clearly in every case. Calmodulin-like protein has been reported in C. albicans [13, 17] and calmodulin has been shown to be present in greater amounts in the hyphal form of C. albicans than in yeast cells [16]. The supply of Ca 2÷ has also been reported to influence growth by bud or germ tube formation in C. albicans [12, 22] and other fungi [10]. The formation of Ca2+-calmodulin complexes may lead to calmodulin- mediated stimulation of protein phosphorylation during germ tube formation [18]. Phenothiazones, such as TFP and CPZ have been shown to antagonize these activities but other effects of these compounds are possible. Our data support and extend these findings that suggest an important role for Ca 2+ as a second messenger in the regulation of yeast-hyphat morphogenesis in C. albicans [10, 22].

Despite the marked effect of TFP on growth and germ tube formation in vitro we find that the concentration of TFP that was required to protect against superficial or systemic Candida infections in vivo was higher than that reported previously [9], and too high to promote further interest in developing these drugs with a view to therapy.

ACKNOWLEDGEMENTS

We thank the Scottish Home and Health Department and Glaxo Group Research for financial assistance throughout this project and E. A. Harrison for help with statistical analyses and Geoff Gadd for useful discussions.

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EFFECT OF TFP ON CANDIDA 433

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