Candida Drug Resistance Protein 1, a Major Multidrug ATP Binding Cassette Transporter of Candida albicans, Translocates Fluorescent Phospholipids in a Reconstituted System

Abstract

Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that approximately 70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With approximately 6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was approximately 15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function.

Relative Contributions of the Candida albicans ABC Transporters Cdr1p and Cdr2p to Clinical Azole Resistance

“…Synergism between transporters has previously been reported for bacterial multidrug resistance pumps (18) and also members of the P-type ATPase and ABC transporter families involved in regulating the phospholipid composition of the membrane of erythrocytes in mammals (7). Cdr1p and Cdr2p have been shown to function as phospholipid floppases, being able to translocate fluorescent phospholipids across the membrane lipid bilayer in an ATPdependent fashion (36,39), and it is possible that the abrogation of Cdr1p and Cdr2p activity may cause a major alteration of the asymmetrical distribution of phospholipids in the cell membrane which would exacerbate the cell response to drugs. Since it was not observed with AMB ( Fig.…”

The ABCs of Candida albicans Multidrug Transporter Cdr1

“…Cdr1p, which itself prefers to be localized within microdomains enriched with sphingolipids and ergosterol, is also responsible for the asymmetric distribution of phosphoglycerides in C. albicans plasma membranes (53,54). The floppase activity of Cdr1p has also been shown with purified and functionally reconstituted protein (55). Similar to steroid transport, phosphoglyceride translocation could be outcompeted by certain drugs, which again not only points to the polyspecificity of the transporter but also highlights the overlapping binding sites within the large, flexible drug binding cavity.…”

Molecular mechanisms underlying the tetrandrine-mediated reversal of the fluconazole resistance ofCandida albicans

“…In fluconazole-sensitive CA-3, the expression levels of CDR1 and CDR2 increased in the presence of FLC or TET alone, whereas these expression levels decreased in cells that were treated with FLC þ TET; in fluconazole-resistant CA-16, the expression levels of CDR1 and CDR2 decreased in the presence of FLC or TET alone, whereas these expression levels significantly decreased in cells that were treated with FLC þ TET. As Cdr1p and Cdr2p require ATP to provide the energy for the active transport of drugs (Shukla et al, 2007), we examined the intracellular ATP content of C. albicans and found that the cellular ATP supply decreased after the FLC þ TET treatment.…”

“…The ABC transporters Cdr1p and Cdr2p require ATP to provide energy for the active transport of drugs, and efflux pump functionality depends not only on the number of the transporter proteins, but also on the cellular ATP supply (Shukla et al, 2007). Therefore, we examined the molecular mechanism of TET effects by detecting the expression levels of drug resistance genes and the intracellular ATP content of C. albicans.…”