Jpn. J. Infect. Dis., 58, 1-7, 2005
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Invited Review
Functional analysis of fungal drug efflux transporters by heterologous expression in Saccharomyces cerevisiae
Masakazu Niimi*, Shun-ichi Wada, Koichi Tanabe, Aki Kaneko, Yukie Takano, Takashi Umeyama, Nozomu Hanaoka, Yoshimasa Uehara, Erwin Lamping1, Kyoko Niimi1, Sarah Tsao1, Ann R. Holmes1, Brian C. Monk1 and Richard D. Cannon1
Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo 162-8640, Japan and 1Molecular Microbiology Laboratory, Department of Oral Sciences, University of Otago, Dunedin, New Zealand
(Received October 8, 2004)
*Corresponding author: Mailing address: Department of Bioactive
Molecules, National Institute of Infectious Diseases, Toyama 1-23-1,
Shinjuku-ku, Tokyo 162-8640, Japan. Tel: +81-3-5285-1111, Fax:
+81-3-5285-1272, E-mail: niimi@nih.go.jp
This article is an Invited Review based on a lecture presented at the 13th Symposium of the National Institute of Infectious Diseases, Tokyo, 21 May 2003.
Contents:
1. Introduction
2. Azole resistance in pathogenic fungi
3. Expression of multidrug efflux pumps in S. cerevisiae
4. Functional analysis of efflux pumps
5. Post-translational regulation of drug efflux pumps
6. Screening of inhibitors of fungal drug efflux pumps
7. Conclusion
SUMMARY: Clinically important resistance of fungal pathogens to azole antifungal drugs is most frequently caused by the over-expression of energy-dependent drug efflux pumps. These pumps usually belong to either the ATP-binding cassette (ABC) family or the Major Facilitator Superfamily (MFS) class of membrane transporter. Little is known about how these pumps work and there is an urgent need to develop pump antagonists that circumvent azole resistance. We have developed a protein hyper-expression system to facilitate functional analysis of efflux pumps based on a Saccharomyces cerevisiae host which has been deleted in seven major ABC transporters to reduce the background of endogenous efflux activity. Plasmid pABC3 was engineered to allow functional hyper-expression of foreign proteins in this host. The main advantages of the system include its ease of directional cloning and the use of homologous recombination to stably integrate single copy constructs into the host genome under the control of a highly active transcriptional regulator. The system has been used to clone and functionally hyper-express genes encoding drug efflux pumps from several pathogenic fungi. Furthermore, the protein hyper-expression system has been used to screen for pump inhibitors and study the structure and function of heterologous membrane proteins.