Positive regulation of the Candida albicans multidrug efflux pump Cdr1p function by phosphorylation of its N-terminal extension
| Autor: | Christine Cameron, Elaheh Ahmadzadeh, Sandra Weber, Dominic Nehme, Martine Raymond, Sarah Tsao |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Microbiology (medical) Antifungal Agents DNA Mutational Analysis 030106 microbiology ATP-binding cassette transporter Microbial Sensitivity Tests Drug resistance Mass Spectrometry Microbiology Fungal Proteins 03 medical and health sciences chemistry.chemical_compound Drug Resistance Fungal Gene Expression Regulation Fungal Candida albicans Bone plate Humans Pharmacology (medical) Phosphorylation Fluconazole Original Research Pharmacology biology Membrane transport protein Nile red Membrane Transport Proteins biology.organism_classification Molecular biology 3. Good health Multiple drug resistance 030104 developmental biology Infectious Diseases chemistry Mutagenesis Site-Directed biology.protein Efflux Protein Processing Post-Translational |
| Zdroj: | Journal of Antimicrobial Chemotherapy. 71:3125-3134 |
| ISSN: | 1460-2091 0305-7453 |
| DOI: | 10.1093/jac/dkw252 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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