Positive regulation of the Candida albicans multidrug efflux pump Cdr1p function by phosphorylation of its N-terminal extension

Abstract

Objectives: Overexpression of ATP-binding cassette (ABC) transporters is a frequent cause of multidrug resistance in cancer cells and pathogenic microorganisms. One example is the Cdr1p transporter from the human fungal pathogen Candida albicans that belongs to the pleiotropic drug resistance (PDR) subfamily of ABC transporters found in fungi and plants. Cdr1p is overexpressed in several azole-resistant clinical isolates, causing azole efflux and treatment failure. Cdr1p appears as a doublet band in western blot analyses, suggesting that the protein is post-translationally modified. We investigated whether Cdr1p is phosphorylated and the function of this modification.

Methods: Phosphorylated residues were identified by MS. Their function was investigated by site-directed mutagenesis and expression of the mutants in a C. albicans endogenous system that exploits a hyperactive allele of the Tac1p transcription factor to drive high levels of Cdr1p expression. Fluconazole resistance was measured by microtitre plate and spot assays and transport activity by Nile red accumulation.

Results: We identified a cluster of seven phosphorylated amino acids in the N-terminal extension (NTE) of Cdr1p. Mutating all seven sites to alanine dramatically diminished the ability of Cdr1p to confer fluconazole resistance and transport Nile red, without affecting Cdr1p localization. Conversely, a Cdr1p mutant in which the seven amino acids were replaced by glutamate was able to confer high levels of fluconazole resistance and to export Nile red.

Conclusions: Our results demonstrate that the NTE of Cdr1p is phosphorylated and that NTE phosphorylation plays a major role in regulating Cdr1p and possibly other PDR transporter function.

Figure 1.

Cdr1p phosphorylation. (a) Western blot analysis. Total membrane extracts prepared from strains 5674 (top panel) and STY7 (bottom panel) were treated or not with λ-phosphatase in the presence or absence of phosphatase inhibitors and separated by SDS–PAGE. Cdr1p was detected using either a specific anti-Cdr1p or a generic anti-Cdrp antibody. (b) Schematic representation of Cdr1p depicting the NTE, the two ABC core regions containing the NBDs and the transmembrane regions containing 12 TMDs organized in an NBD1–TMD1–NBD2–TMD2 topology. Cdr1p phosphorylated residues identified by MS are shown as filled circles. (c) Sequence alignment of the NTE regions of Cdr1p, Cdr2p and their orthologues in different Candida species, C. tropicalis (Ct), C. dubliniensis (Cd), C. glabrata (Cg) (www.candidagenome.org) and S. cerevisiae Pdr5p using the COBALT multiple alignment tool (www.ncbi.nlm.nih.gov/tools/cobalt/recobalt.cgi). Negatively charged residues are in bold. Phosphorylated residues identified by MS in CaCdr1p (this study) and ScPdr5p are circled. PPase, phosphatase. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2.

Expression of CDR1 in strain STY31 (Tac1p^N972D cdr1Δ/cdr1Δ cdr2Δ/cdr2Δ). (a) Schematic representation of plasmid pCDR1 containing the CDR1 integration cassette. The cassette consists of, from left to right: CDR1_up, CDR1 upstream region; the entire ORF of the CDR1 allele; SAT1 cassette (derived from pSFS2A^short); and CDR1_down, CDR1 downstream region. Abbreviations for restriction sites are: S, SacII; A, ApaI; N, NotI; and X, XhoI. The ApaI site shown in brackets was introduced by PCR. (b) CDR1 expression system. High-level expression of CDR1 is driven by the transcription factor Tac1p carrying an activating mutation (Tac1p^N972D) in the STY31 strain lacking the endogenous CDR1 and CDR2 genes. (c) Fluconazole resistance profiles of the STY31, STY7 and STY31 + CDR1 strains. Cells were incubated for 48 h at 30°C in liquid YPD medium with the indicated concentrations of fluconazole. The data are presented as the relative growth of cells in fluconazole-containing medium compared with the growth of the same strain in fluconazole-free medium, which was set at 100%. The data presented come from one representative experiment performed in duplicate. FLC, fluconazole.

Figure 3.

Expression analyses of the Cdr1p NTE phosphomutants. (a) Total membranes extracted from STY31 and strains expressing WT Cdr1p or the Cdr1p NTE phosphorylation mutants 7A and 7E were analysed by western blotting with the generic anti-Cdrp antibody (top panel). Ponceau staining of the membrane before incubation with the antibody is shown as a control for equal protein loading and transfer (bottom panel). (b) Indirect immunofluorescence detection of the indicated strains was performed using the generic anti-Cdrp antibody followed by incubation with a fluorescent secondary antibody and visualization by epifluorescence microscopy. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 4.

Fluconazole resistance profiles of the Cdr1p NTE phosphomutants. (a) The indicated strains were analysed by MIC assay. Cells were incubated for 24 and 48 h at 30°C in liquid YPD medium with the indicated concentrations of fluconazole. The data are the mean of at least three independent experiments performed in duplicate. (b) Spot assay. Serial 10-fold dilutions of the cells, starting at an OD₆₀₀ of 0.1, were spotted onto YPD plates containing fluconazole at the indicated concentrations or no drug. The plates were incubated for 48 h at 30°C. FLC, fluconazole.

Figure 5.

Nile red accumulation assay. The equivalent of 15 OD₆₀₀ units of log phase cells from the indicated strains were incubated with Nile red for 30 min at 30°C. After incubation, intracellular fluorescence was measured. Fluorescence accumulation is presented as relative fluorescence units (RFU) in which the basal levels of fluorescence in the absence of cells (PBS + Nile red) has been subtracted from the total fluorescence values. The data presented are average values from three independent experiments.