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Antimicrobial Agents and Chemotherapy, November 2001, p. 3037-3045, Vol. 45, No. 11
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.11.3037-3045.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

In Vitro and In Vivo Effects of 14alpha -Demethylase (ERG11) Depletion in Candida glabrata

Hironobu Nakayama,1,* Noboru Nakayama,2,dagger Mikio Arisawa,1,Dagger and Yuko Aoki1,Dagger

Department of Mycology1 and Department of Spectroscopic Analysis,2 Nippon Roche K. K. Research Center, Kanagawa 247-8530, Japan

Received 2 October 2000/Returned for modification 13 November 2000/Accepted 14 August 2001

Sterol 14alpha -demethylase (ERG11) is the target enzyme of azole antifungals that are widely used for the treatment of fungal infections. Candida glabrata is known to be less susceptible to fluconazole than most Candida albicans strains, and the incidence of C. glabrata infection has been increasing mostly in conjunction with the use of azole antifungals. Recently, it has been reported that C. glabrata can rescue the defect of ergosterol biosynthesis by incorporating cholesterol from serum. To explore the effect of inactivating Erg11p in C. glabrata, we generated mutant strains in which the ERG11 gene was placed under the control of tetracycline-regulatable promoters. In these mutants, expression of the ERG11 gene can be repressed by doxycycline (DOX). All mutants showed a growth defect in the presence of DOX. The numbers of CFU of the mutants were lowered by only 1/10 with DOX treatment. In these mutants, accumulation of 4,14-dimethylzymosterol, which differs from an accumulated abnormal sterol detected in C. albicans and Saccharomyces cerevisiae treated with fluconazole, was observed by DOX treatment. Although such phenotypes were also observed in serum-containing media by DOX treatment, they were alleviated. Furthermore, the mutant could grow in DOX-treated mice without a severe reduction in the number of cells. Thus, depleting the expression of the ERG11 gene lowered the number of CFU by only 1/10 due to the accumulation of 4,14-demethylzymosterol in vitro, and it did not result in the defective growth of fungal cells in mice. These results suggested that Erg11p is not an ideal target molecule of antifungals for C. glabrata.

* Corresponding author. Present address: Department of Oncology, Nippon Roche K. K. Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan. Phone: 81-467-47-2218. Fax: 81-467-45-6782. E-mail: hironobu.nakayama{at}roche.com.

dagger Present address: Department of Chemistry, Nippon Roche K. K. Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan.

Dagger Present address: Department of Oncology, Nippon Roche K. K. Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan.

Antimicrobial Agents and Chemotherapy, November 2001, p. 3037-3045, Vol. 45, No. 11
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.11.3037-3045.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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