Confirmation of three predicted disulphide bonds in Candida albicans Cdr1.

Objectives: Candida albicans is the most common cause of superficial fungal infections in the oral cavity. However, these infections can become life-threatening invasive infections in immunocompromised patients. Fluconazole (FLC) is frequently used to treat Candida infections. The increase in FLC-resistant C. albicans clinical isolates, often caused by the overexpression of the ATP-binding-cassette multidrug efflux transporter Cdr1, and the paucity of alternative treatment options pose serious clinical concerns. Six conserved extracellular cysteine residues are predicted to form disulphide bonds and contribute to the structural and functional integrity of Cdr1.

The aim of this project was to experimentally verify three predicted disulphide bonds between six conserved cysteines in the extracellular domain of Cdr1.
Methods: Disulphide bonds CC1 (C712-C732), CC2 (C1418-C1441) and CC3 (C1402-C1444) were introduced separately, and in combination (i.e. CC1/3, CC1/2/3), into Cdr1-CED - that had all six extracellular cysteines replaced with serines, but remained fully functional because of a point mutation (A1207T). The five Cdr1-CED-variants were overexpressed in the heterologous host Saccharomyces cerevisiae AD1-8u^- with a C-terminal green fluorescent protein (GFP) tag. Genomic DNA extraction, PCR amplification and DNA sequencing confirmed the AD/Cdr1-CED-GFP-variants. Plasma-membrane (PM) localisation, efflux pump function and expression levels were determined by confocal-microscopy, measuring FLC-susceptibilities and determining in-gel GFP-fluorescence levels of isolated PM proteins separated by SDS-PAGE.
Results: The FLC-susceptibilities of the five Cdr1-CED-GFP variants were identical to AD/CDR1-CED-GFP (128mg/l; CC2 and CC3), halved (64mg/l; CC1), or two-fold (256mg/l; CC1/3) and four-fold (512mg/l; CC1/2/3) increased. The expression levels were halved (CC1/3), unchanged (CC2 and CC3) or doubled (CC1 and CC1/2/3). The Cdr1-CED CC1/3 and CC1/2/3 variants exhibited improved PM localisation. These three disulphide bonds are now being independently investigated by mass spectrometry of Cdr1.
Conclusions: Three extracellular disulphide bonds are critical for proper folding, PM localisation, and efflux pump function of Cdr1.