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Antimicrobial Agents and Chemotherapy, January 2008, p. 146-156, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.01072-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Tetracycline-Inducible Expression of Individual Secreted Aspartic Proteases in Candida albicans Allows Isoenzyme-Specific Inhibitor Screening{triangledown}

Peter Staib,1,2 Ulrich Lermann,1 Julia Blaβ-Warmuth,1 Björn Degel,3 Reinhard Würzner,4 Michel Monod,2 Tanja Schirmeister,3 and Joachim Morschhäuser1*

Institut für Molekulare Infektionsbiologie, Universität Würzburg, Würzburg, Germany,1 Department of Dermatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland,2 Institut für Pharmazie und Lebensmittelchemie, Universität Würzburg, Würzburg, Germany,3 Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Innsbruck, Austria4

Received 14 August 2007/ Returned for modification 2 October 2007/ Accepted 16 October 2007

The yeast Candida albicans possesses a gene family that encodes secreted aspartic proteases (Saps), which are important for the virulence of this human fungal pathogen. Inhibitors of the Saps could therefore be used as novel antimycotic agents for the treatment of C. albicans infections. In the present study, we established a bioassay which allows testing of the activity of potential protease inhibitors against specific Sap isoenzymes by their ability to inhibit protease-dependent growth of C. albicans. In a medium containing bovine serum albumin (BSA) as the sole source of nitrogen, C. albicans specifically expresses the Sap2p isoenzyme, which degrades the BSA and thereby enables the fungus to grow. As the other SAP genes are not significantly expressed under these conditions, mutants lacking SAP2 are unable to utilize BSA as a nitrogen source and cannot grow in such a medium. To investigate whether forced expression of SAP genes other than SAP2 would also allow growth on BSA, we constructed a set of strains expressing each of the 10 SAP genes from a tetracycline-inducible promoter in a sap2{Delta} mutant background. Expression of Sap1p, Sap2p, Sap3p, Sap4p, Sap5p, Sap6p, Sap8p, and a C-terminally truncated, secreted Sap9p restored the growth of the sap2{Delta} mutant with different efficiencies. This set of strains was then used to test the activities of various aspartic protease inhibitors against specific Sap isoenzymes by monitoring growth on BSA in the presence of the inhibitors. While pepstatin blocked the activity of all of the Saps tested, the human immunodeficiency virus protease inhibitors ritonavir and saquinavir inhibited growth of the strains expressing Sap1p to Sap3p and Sap1p, respectively, but not that of strains expressing other Saps. Therefore, the strain set can be used to test the activity of new protease inhibitors against individual C. albicans Sap isoenzymes by their ability to block the growth of the pathogen.

* Corresponding author. Mailing address: Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. Phone: 49-931-31 21 52. Fax: 49-931-31 25 78. E-mail: joachim.morschhaeuser{at}mail.uni-wuerzburg.de

{triangledown} Published ahead of print on 22 October 2007.

Antimicrobial Agents and Chemotherapy, January 2008, p. 146-156, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.01072-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lermann, U., Morschhauser, J. (2008). Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans. Microbiology 154: 3281-3295 [Abstract] [Full Text]  


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