Functional Expression of Candida albicans Drug Efflux Pump Cdr1p in a Saccharomyces cerevisiae Strain Deficient in Membrane Transporters

Abstract

Analysis of the transport functions of individual Candida albicans plasma membrane drug efflux pumps is hampered by the multitude of endogenous transporters. We have stably expressed C. albicans Cdr1p, the major pump implicated in multiple-drug-resistance phenotypes, from the genomic PDR5 locus in a Saccharomyces cerevisiae mutant (AD1-8u ؊ ) from which seven major transporters of the ATP-binding cassette (ABC) family have been deleted. High-level expression of Cdr1p, under the control of the S. cerevisiae PDR5 promoter and driven by S. cerevisiae Pdr1p transcriptional regulator mutation pdr1-3, was demonstrated by increased levels of mRNA transcription, increased levels of nucleoside triphosphatase activity, and immunodetection in plasma membrane fractions. S. cerevisiae AD1-8u؊ was hypersensitive to azole antifungals (the MICs at which 80% of cells were inhibited [MIC 80 s] were 0.625 g/ml for fluconazole, <0.016 g/ml for ketoconazole, and <0.016 g/ml for itraconazole), whereas the strain (AD1002) that overexpressed C. albicans Cdr1p was resistant to azoles (MIC 80 s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 g/ml, respectively). Drug resistance correlated with energy-dependent drug efflux. AD1002 demonstrated resistance to a variety of structurally unrelated chemicals which are potential drug pump substrates. The controlled overexpression of C. albicans Cdr1p in an S. cerevisiae background deficient in other pumps allows the functional analysis of pumping specificity and mechanisms of a major ABC transporter involved in drug efflux from an important human pathogen.Candida albicans is an asexual diploid fungus that causes opportunistic infections commonly seen in immunocompromised and debilitated patients (9, 30). An estimated 33 to 55% of patients with human immunodeficiency virus infection and AIDS contract oropharyngeal candidosis (34), and the synthetic triazole fluconazole has been the mainstay of their treatment. The widespread use of prolonged fluconazole therapy has increased the incidence of treatment failure due to fluconazole-resistant C. albicans (3,14,21,34,42). A number of studies have identified the major azole resistance mechanisms (1,20,38,41,42,(44)(45)(46). These include overexpression of, or mutations in, the drug target, 14␣-sterol demethylase; mutations in other parts of the sterol biosynthesis pathway; and, most commonly, overexpression of drug efflux proteins.C. albicans possesses transporters such as Cdr1p and Cdr2p with homology to proteins of the ATP-binding cassette (ABC) family (10,16,18,19,31), as well as Ben r p, which has homology to the major facilitator superfamily (MFS) class of drugproton antiport efflux pumps (1,5,36,46). The BEN r gene encodes a transporter associated with resistance to benomyl and methotrexate when it is expressed in Saccharomyces cerevisiae. The C. albicans CDR1 gene is a homologue of S. cerevisiae PDR5, which encodes a multidrug efflux pump, and CDR1 is the gene most often associated with energy-dependent drug efflux in fluconazole-r...

Chimeras of the ABC drug transporter Cdr1p reveal functional indispensability of transmembrane domains and nucleotide-binding domains, but transmembrane segment 12 is replaceable with the corresponding homologous region of the non-drug transporter Cdr3p

“…In order to check the localization of chimeric proteins, each construct, at the C-terminus, was also tagged with the green fluorescent protein (GFP). For functional analysis of the CDR1/CDR3 chimeras, a heterologous hyper-expression system, where Cdr1p is stably overexpressed from a genomic PDR5 locus in a S. cerevisiae mutant AD1-8u 2 , was used (Nakamura et al, 2002). The host AD1-8u 2 , from which seven major ABC transporters have been deleted, was derived from a Pdr1-3 mutant strain with a gain-of-function mutation in the transcription factor Pdr1p, resulting in a constitutive hyperinduction of the PDR5 promoter.…”

The roles of CDR1, CDR2, and MDR1 in kaempferol-induced suppression with fluconazole-resistant Candida albicans

“…Interestingly, KAE in combination with FLC could synergistically inhibit all the strains after 48 h of incubation in the T-K test (Figure 1) including C. albicans z2003, indicating that it was useful for T-K test to further assess the interpretations acquired in a checkerboard assay when FICI was a little higher than 0.5. The upregulations of multidrug efflux pump controlled by Cdr1p, Cdr2p belonging to ATP-binding cassette superfamily (APC transporter) and Mdr1p, a member of major facilitator superfamily (MFS) were implicated in most fluconazole-resistant C. albicans strains (Niimi et al 2004;Holmes et al 2012;Prasad and Rawal 2014) as FLC was a substrate for CDR1, CDR2, and MDR1 (Kohli et al 2001;Nakamura et al 2001). Rhodamine 6G was the most common used fluorescent dye of ABC pump substrate requiring ATP as energy to observe transporter-mediated FLC resistance (Lamping et al 2007;Peralta et al 2012).…”

Conserved Asp327 of Walker B Motif in the N-Terminal Nucleotide Binding Domain (NBD-1) of Cdr1p ofCandida albicansHas Acquired a New Role in ATP Hydrolysis

“…The mutations were introduced into plasmid pPSCDR1-GFP according to manufacturer's instructions, and the mutated plasmid pPSCDR1-GFP linearized with XbaI was used to transform AD1-8u -cells by the lithium acetate transformation protocol exploiting uracil prototrophy (26,27).…”