Journal Article

Alanine scanning of transmembrane helix 11 of Cdr1p ABC antifungal efflux pump of <i>Candida albicans</i>: identification of amino acid residues critical for drug efflux

Preeti Saini, Tulika Prasad, Naseem Akhtar Gaur, Suneet Shukla, Sudhakar Jha, Sneha Sudha Komath, Luqman Ahmad Khan, Qazi Mohd. Rizwanul Haq and Rajendra Prasad

in Journal of Antimicrobial Chemotherapy

Published on behalf of British Society for Antimicrobial Chemotherapy

Volume 56, issue 1, pages 77-86
Published in print July 2005 | ISSN: 0305-7453
Published online June 2005 | e-ISSN: 1460-2091 | DOI:
Alanine scanning of transmembrane helix 11 of Cdr1p ABC antifungal efflux pump of Candida albicans: identification of amino acid residues critical for drug efflux

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  • Medical Oncology
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Objectives: To investigate the role of transmembrane segment 11 (TMS11) of Candida albicans drug resistance protein (Cdr1p) in drug extrusion.

Methods: We replaced each of the 21 putative residues of TMS11 with alanine by site-directed mutagenesis. The Saccharomyces cerevisiae AD1-8u strain was used to overexpress the green fluorescent protein tagged wild-type and mutant variants of TMS11 of Cdr1p. The cells expressing mutant variants were functionally characterized.

Results: Out of 21 residues of TMS11, substitution of seven residues, i.e. A1346G, A1347G, T1351A, T1355A, L1358A, F1360A and G1362A, affected differentially the substrate specificity of Cdr1p, while 14 mutants had no significant effect on Cdr1p function. TMS11 projection in an α-helical configuration revealed with few exceptions (A1346 and F1360), a distinct segregation of mutation-sensitive residues (A1347, T1351, T1355, L1358 and G1362) towards the more hydrophilic face. Interestingly, mutation-insensitive residues seem to cluster towards the hydrophobic side of the helix. Competition of rhodamine 6G efflux, in the presence of excess of various substrates in the cells expressing native Cdr1p, revealed for the first time the overlapping binding site between azoles (such as ketoconazole, miconazole and itraconazole) and rhodamine 6G. The ability of these azoles to compete with rhodamine 6G was completely lost in mutants F1360A and G1362A, while it was selectively lost in other variants of Cdr1p. We further confirmed that fungicidal synergism of calcineurin inhibitor FK520 with azoles is mediated by Cdr1p; wherein in addition to conserved T1351, substitution of T1355, L1358 and G1362 of TMS11 also resulted in abrogation of synergism.

Conclusions: Our study for the first time provides an insight into the possible role of TMS11 of Cdr1p in drug efflux.

Keywords: transmembrane segment 11; azoles; synergy; multidrug resistance; rhodamine 6G

Journal Article.  6281 words.  Illustrated.

Subjects: Medical Oncology ; Critical Care

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