Inhibitor-Resistant Mutants Give Important Insights into Candida albicans ABC Transporter Cdr1 Substrate Specificity and Help Elucidate Efflux Pump Inhibition.

Content Provider	Europe PMC
Author	Niimi, Masakazu Niimi, Kyoko Tanabe, Koichi Cannon, Richard D. Lamping, Erwin
Copyright Year	2022
Abstract	Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin, and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor-resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated 20 Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (8 mutants), enniatin B (8), or beauvericin (4). The 20 mutations were in just 9 residues at the centers of transmembrane segment 1 (TMS1) (6 mutations), TMS4 (4), TMS5 (4), TMS8 (1), and TMS11 (2) and in A713P (3), a previously reported FK506-resistant “hot spot 1” mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) variants were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25-resistant mutants were altered in four amino acids as follows: G521R, M639I, A713P, and T1355N (TMS11). These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N possibly cause inhibitor resistance by altering TMS contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.
Page Count	24
ISSN	00664804
Journal	Antimicrobial Agents and Chemotherapy
Volume Number	66
PubMed Central reference number	PMC8765293
Issue Number	1
PubMed reference number	34780272
e-ISSN	10986596
DOI	10.1128/AAC.01748-21
Language	English
Publisher	American Society for Microbiology
Publisher Date	2021-11-15
Publisher Place	N.W., Washington, DC
Access Restriction	Open
Rights License	This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Copyright © 2022 Niimi et al.
Subject Keyword	Candida albicans Cdr1 ABC transporter PDR transporter drug resistance efflux pump inhibitors transport cycle kinetics
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Pharmacology Pharmacology (medical)