HOLMES AR, HULL C, NIIMI K, LAMPING E, CANNON RD, MONK BC; Interscience Conference on Antimicrobial Agents and Chemotherapy (42nd : 2002 : San Diego, Calif.).
Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 2002 Sep 27-30; 42: abstract no. M-227.
University of Otago, Dunedin, New Zealand
BACKGROUND: Candida infections that are refractory to treatment, due to the development of azole-resistance, are an increasing clinical problem. Drug resistance most frequently results from expression of energy-dependent drug efflux pumps in the yeast cell membrane. The goal of this study was to identify specific inhibitors of the drug efflux pumps. METHODS: A heterologous membrane protein expression system in a Saccharomyces cerevisiae host strain (AD1-8u[-]), depleted of seven endogenous ABC-type multidrug efflux pumps, was used to investigate the S. cerevisiae Pdr5p multidrug efflux pump and its homologue, Cdr1p, from Candida albicans. Both Pdr5p and Cdr1p conferred resistance to azole drugs when over-expressed in the host strain. Pumping activity was assayed by spectrofluorimetry using rhodamine 6G as a substrate. This assay was used to assess the effect on rhodamine efflux by synthetic peptide leads from a 1.8-million-member combinatorial library. RESULTS: Rhodamine 6G was accumulated in starved yeast cells by diffusion. Energy-dependent efflux of the rhodamine 6G was observed for the recombinant strains, but was undetectable in the host strain. The efflux of rhodamine 6G was potassium ion and growth-phase dependent, and was inhibited in the presence of fluconazole at sub-MIC concentrations. The D-octapeptide KN20 and the D-decapeptide BM2, which were shown to chemosensitise azole-resistant yeast strains to fluconazole, inhibited rhodamine 6G efflux from the Pdr5p and Cdr1p over-expressing strains in a dose-dependent manner. KN20 inhibited the ATPase activity of Pdr5p whereas BM2 inhibited the plasma membrane proton pumping ATPase, Pma1p. CONCLUSIONS: Rhodamine 6G and fluconazole compete for efflux by Pdr5p and Cdr1p, confirming that both compounds are substrates for efflux by these membrane proteins. The synthetic peptide inhibitors KN20 and BM2 were identified as useful leads in the development of chemosensitisers that directly and indirectly affect multidrug efflux by yeast.
Publication Types:
Keywords:
- Adenosine Triphosphatases
- Candida albicans
- Cell Membrane
- Fluconazole
- Membrane Proteins
- Microbial Sensitivity Tests
- Peptides
- Rhodamines
- Saccharomyces cerevisiae
- Saccharomyces cerevisiae Proteins
- rhodamine 6G
Other ID:
UI: 102267583
From Meeting Abstracts