Specificity of drug transport mediated byCaMDR1: A major facilitator ofCandida albicans

Abstract

CaMDR1 encodes a major facilitator superfamily (MFS) protein in Candida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p in Sf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance in C. albicans, were independently expressed in a common hypersensitive host JG436 of Saccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.

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).…”

Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p

“…For example, it has been shown that intracellular [ 3 H]fluconazole levels decreased when CDR1 (20,33) and MDR1 (37) were overexpressed in S. cerevisiae and also that fluconazole MICs increased when CDR1, CDR2 (36,53), and MDR1 (31) were overexpressed in C. albicans. The levels to which CDR1, CDR2, and MDR1 were expressed in C. albicans in the present study did not approach the expression levels that others achieved in S. cerevisiae or C. albicans; this may have been due to our use of episomal rather than integrating vectors and our use of the ACT1 and GAL1 promoters instead of fluconazole-inducible promoters.…”

Drug Susceptibilities of Yeast Cells Are Affected by Membrane Lipid Composition

“…The absorbance of the supernatant was measured at 527 nm (23). The passive diffusion of FLC was checked by using radiolabeled [ 3 H]FLC (24,47). The deenergized cells were incubated with 100 nM [ 3 H]FLC (0.7 TBq mmol Ϫ1 ); and aliquots were removed at various times, rapidly filtered, and washed three times with ice-cold buffer A containing an equimolar concentration of cold drug on a Millipore manifold filtration assembly with 0.45-m-pore-size cellulose nitrate filter disks (Millipore, Bedford, Mass.).…”

“…The fold increase in resistance to each drug was calculated as the difference in resistance between the nontransformant and transformant pair for each cell type. MTX is the substrate for CaMdr1p (24), and the fold change in resistance is much higher for CaMDR1-transformed erg mutant cells than for CDR1-transformed erg mutant cells. Note the change in the x axis for fold MTX resistance.…”