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

doi:10.1128/AAC.45.11.3037-3045.2001

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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 14 $alpha$ -Demethylase (ERG11) Depletion in Candida glabrata

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

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 14 $alpha$ -demethylase (ERG11) is the target enzyme of azole antifungals that are widely used for the treatment of fungalinfections. Candida glabrata is known to be less susceptible tofluconazole than most Candida albicans strains, and the incidenceof C. glabrata infection has been increasing mostly in conjunctionwith the use of azole antifungals. Recently, it has been reportedthat C. glabrata can rescue the defect of ergosterol biosynthesisby incorporating cholesterol from serum. To explore the effectof inactivating Erg11p in C. glabrata, we generated mutant strainsin which the ERG11 gene was placed under the control of tetracycline-regulatablepromoters. In these mutants, expression of the ERG11 gene canbe repressed by doxycycline (DOX). All mutants showed a growthdefect in the presence of DOX. The numbers of CFU of the mutantswere lowered by only 1/10 with DOX treatment. In these mutants,accumulation of 4,14-dimethylzymosterol, which differs from anaccumulated abnormal sterol detected in C. albicans and Saccharomycescerevisiae treated with fluconazole, was observed by DOX treatment.Although such phenotypes were also observed in serum-containingmedia by DOX treatment, they were alleviated. Furthermore, themutant could grow in DOX-treated mice without a severe reductionin the number of cells. Thus, depleting the expression of theERG11 gene lowered the number of CFU by only 1/10 due to the accumulationof 4,14-demethylzymosterol in vitro, and it did not result inthe defective growth of fungal cells in mice. These results suggestedthat Erg11p is not an ideal target molecule of antifungals forC. 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.

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

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

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

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