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The opportunistic pathogen Candida albicans causes infection in immunocompromised hosts. 1) C. albicans is a dimorphic fungus that transforms from yeast to hyphal form depending on environmental conditions. The transition from yeast to hyphae of C. albicans is influenced by temperature, pH and Ca 2ϩ , and these signal transduction cascade is modulated by RAS1 which controls the cAMP pathway and MAP kinase cascade in C. albicans. [2][3][4][5] As adenylate cyclase (CYR1) is a component of cAMP pathway and involved in growth signal, RAS1 activates both cell growth and hyphal transformation of C. albicans.6,7) The kind of antifungal drugs is few compared with that of drugs for bacteria. Thus, the discovery of a new antifungal element is expected.Hinokitiol is a chemical compound of aromatic sevenmembered tropolon and a component of essential oils isolated from Cupressaceae. Although hinokitiol exhibits antibacterial and antifungal activity, 8,9) the antimicrobial mechanism has not been sufficiently clarified. Thus, in this report, we studied the antifungal mechanism of hinokitiol against C. albicans. MATERIALS AND METHODSReagent Hinokitiol (Wako Pure Chemicals Co., Ltd.) was used in this study.Fungus C. albicans TIMM1768 was generously donated by the Institute of Medical Mycology, Teikyo University, Japan. The C. albicans JCM1542 and 2076 strains were obtained from the Japan Collection of Microorganisms, Riken, Japan. These strains and C. albicans NIH A-207 were maintained for 24 h at 27°C in Sabouraud's medium with shaking. After incubation, these cells were washed with sterilizd water, and suspended in RPMI1640 medium (Nissui Pharmacetuical Co., Ltd., Japan). The cells were incubated at 37°C in 5% CO 2 to induce the hyphal form.Antifungal Activity of Hinokitiol C. albicans NIH A-207 (1ϫ10 6 cells/ml in RPMI1640 medium) was mixed with hinokitiol (final 2.5, 5.0 and 10.0 mg/ml), and the growth rates were monitored spectrophotometrically by absorbance at OD 620 nm at every hour until 12 h. The accuracy of this measurement was verified using NucleoCounter TM YC-100 (MS Techono Systems). NucleoCounter TM YC-100 can count the amount of nucleus and converted into the number of cells. When the number of hyphal cells of C. albicans was counted using this system, the resulted number was similar to the number calculated from the absorbance (data not shown). To establish anaerobic conditions, sodium sulfite (final concentration 10 mg/ml) was added to RPMI1640 medium. Na 2 SO 3 absorbs oxygen dissolved in RPMI1640 medium (Na 2 SO 3 ϩO 2 ϭ2Na 2 SO 4 ).Amount of ATP in C. albicans C. albicans NIH A-207 (1ϫ10 6 cells/ml in RPMI1640 medium) was mixed with hinokitiol (final concentration 5 mg/ml), and incubated at 37°C in 5% CO 2 . After the incubation, the amount of ATP in C. albicans was measured using ChekLite 250 plus (Kikkoman Co., Ltd., Japan).Quantification of mRNAs in C. albicans C. albicans (1ϫ10 6 cells/ml in RPMI1640 medium) was incubated with hinokitiol (5 mg/ml) at 37°C in 5% CO 2 for 4 h. After the incubation, the cel...

Hyphal growth of Candida albicans was observed at neutral condition, whereas the yeast growth was increased below pH 5. Eight out of 12 strains of C. albicans grow in hyphal form at pH 7, and hyphal formation was inhibited in all strains at pH 4. The addition of GMP, an alternative oxidase (AOX) activator, induced the hyphal growth at pH 4. Although C. albicans grew in hyphal form in pH 7, SHAM, an AOX inhibitor, enhanced the yeast proliferation at this pH. The relative expression level of RAS1 mRNA was higher at pH 7 than at pH 4, indicating that the hyphal formation signal was defective under acidic conditions. Based on these findings, we concluded that AOX-RAS1 signal transduction is the main pathway of hyphal formation of C. albicans, and that the signal was controlled by pH condition.

Candida albicans are opportunistic fungi which generally exist in the oral cavity, skin, vagina and intestinal organs, 1,2) and are dimorphic fungi which undergo a transition from the yeast form to the hyphal form depending on the growth conditions.3,4) They grow in yeast form at membranes and the surface of the skin, but in hyphae form at the deep-seated mycosis.Temperature, pH and Ca 2ϩ are known as external factors of the transformation.5-7) The hyphae transformation is induced by the activation of the MAP kinase and cAMP pathways, which are controlled by RAS protein. 8,9) Polyamines are synthesized from ornithine by ornithine decarboxylase (ODC) and known as inducers of adenylate cyclase. 10)In this report, we discovered that polyamines participated in the transformation of C. albicans, and the activation of adenylate cyclase was related to the transformation. MATERIALS AND METHODSOrganism and Growth Conditions C. albicans NIH A-207 strain was routinely grown in Sabouraud's medium (peptone 10 g, glucose 20 g and yeast extract 5 g, per liter). To induce hyphae formation, the cells were grown in RPMI1640 medium at 37°C.Reagents 1,4-Diamino-2-butanone (DAB) was pur-The Amount of Polyamines in C. albicans C. albicans cells (1ϫ10 5 cells/ml in RPMI1640) were incubated with DAB (0, 960 mM) in 5% CO 2 at 37°C for 1 and 3 h. Cells were washed twice with sterile water and disrupted with a Mini-Beadbeater (diameter of glass beads, 0.3 mm, Central Scientific Commerce, Inc.). Total proteins in the supernatant were analyzed at 280 nm and polyamines levels were determined using a Labosearch polyamine auto (A and T Corp.).Yeast Induction of C. albicans by Polyamine Synthesis Inhibitor C. albicans cells were adjusted to 1ϫ10 5 cells/ml in RPMI1640. DAB (0, 30, 60, 120, 240, 480, 960 mM) was added and incubated in 5% CO 2 at 37°C for 3 h. The ratio of hyphae cells was counted microscopically.Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Gene arrangement participated in the hyphae formation of C. albicans referenced using the Entrez System of the National Center for Biotechnology Information (U.S.A.). PCR primers were designed using the Search Launcher in the Human Genome Sequencing Center (U.S.A.). C. albicans cells (1ϫ10 5 cells/ml in RPMI1640) were incubated with DAB (0, 960 mM) in 5% CO 2 at 37°C for 3 h. The cells were washed twice with sterile water. The cDNA library of the cells was synthesized with a Cells-tocDNA kit (Ambion Inc., U.S.A.). PCR reaction was conducted with a KOD-plus kit (Toyobo Co., Ltd.). Primers used in this assay are shown in Table 1. PCR products were analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies). The result was shown as a relative expression of mRNA compared with that of ACT1 (actin) as described by McCreach's method. 11)cAMP Concentration in C. albicans C. albicans cells (1ϫ10 5 cells/ml in RPMI1640) were incubated with DAB (0, 960 mM) in 5% CO 2 at 37°C for 3 h. Cells were washed twice with sterile water and disrupted with a Mini-Beadbeater. Total proteins in the s...