Abstract
Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.
Highlights
Transmembrane peptide mimics (TMPMs) may be useful to prevent helix-helix interaction of multidrug transporter proteins
Localization of FITC-tagged TMPMs in Cdr1p-overexpressing Cells—To determine whether the synthesized TMPMs interacted with Cdr1p, we used Saccharomyces cerevisiae cells in which Cdr1p was stably overexpressed from a genomic PDR5 locus in a mutant AD1-8uϪ strain that lacks seven ABC transporters [30]
We have previously shown that overexpression of Cdr1p leads to expression levels that are sufficient for the biochemical characterization of the transporters [12]. To determine whether these TMPMs bind to yeast cells overexpressing Cdr1p, we tagged the TMPMs with FITC, a well known fluorescent dye, which is highly sensitive, stable, and widely used in fluorescence microscopy [31]
Summary
Transmembrane peptide mimics (TMPMs) may be useful to prevent helix-helix interaction of multidrug transporter proteins. A rationally designed hydrophobic peptide mimic against TMH4 of the small multidrug-resistant protein of Halobacterium salinarum was shown to block the drug transporter [9]. It was observed that FITC-tagged TMPMs bound to the cell surface of yeast cells and blocked the efflux of trapped fluorescent substrates, such as Nile red (NR) and Rhodamine 6G (R6G), in yeast cells overexpressing Cdr1p. These TMPMs chemosensitized AR clinical isolates of Candida. We provide evidence that selective peptide mimics of TMHs block the efflux of fluorescent substrates of a yeast multidrug transporter and provide a novel template for optimizing TMPM development as potent nontoxic inhibitors
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
