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. 2019 Jan 25;294(4):1257-1266.
doi: 10.1074/jbc.RA118.003904. Epub 2018 Dec 4.

A biosensor-based approach reveals links between efflux pump expression and cell cycle regulation in pleiotropic drug resistance of yeast

Jian Li  1 , Kristen Kolberg  1 , Ulrich Schlecht  1 , Robert P St Onge  1 , Ana Maria Aparicio  1 , Joe Horecka  1 , Ronald W Davis  1 , Maureen E Hillenmeyer  1 , Colin J B Harvey  2
Affiliations

A biosensor-based approach reveals links between efflux pump expression and cell cycle regulation in pleiotropic drug resistance of yeast

Jian Li et al. J Biol Chem. .

Abstract

Multidrug resistance is highly conserved in mammalian, fungal, and bacterial cells, is characterized by resistance to several unrelated xenobiotics, and poses significant challenges to managing infections and many cancers. Eukaryotes use a highly conserved set of drug efflux transporters that confer pleiotropic drug resistance (PDR). To interrogate the regulation of this critical process, here we developed a small molecule-responsive biosensor that couples transcriptional induction of PDR genes to growth rate in the yeast Saccharomyces cerevisiae Using diverse PDR inducers and the homozygous diploid deletion collection, we applied this biosensor system to genome-wide screens for potential PDR regulators. In addition to recapitulating the activity of previously known factors, these screens identified a series of genes involved in a variety of cellular processes with significant but previously uncharacterized roles in the modulation of yeast PDR. Genes identified as down-regulators of the PDR included those encoding the MAD family of proteins involved in the mitotic spindle assembly checkpoint (SAC) complex. Of note, we demonstrated that genetic disruptions of the mitotic spindle assembly checkpoint elevate expression of PDR-mediating efflux pumps in response to exposure to a variety of compounds that themselves have no known influence on the cell cycle. These results not only establish our biosensor system as a viable tool for investigating PDR in a high-throughput fashion, but also uncover critical control mechanisms governing the PDR response and a previously uncharacterized link between PDR and cell cycle regulation in yeast.

Keywords: biosensor; drug resistance; gene regulation; microarray; multidrug transporter.

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Conflict of interest statement

U. S., C. J. B. H., and M. E. H. own shares in Hexagon Bio

Figures

Figure 1.
Figure 1.
Construction of a biosensor system that couples PDR transporter expression and strain growth. a, schematic of the biosensor system and structures of inducing compounds used. The biosensor plasmids are outlined in Table S6. PPDR refers to promoters of PDR transporters pdr5, snq2, or yor1. Yeast strains transformed with these plasmids are grown in YNB media, without leucine and histidine. In addition, chemicals that induce the PDR response, such as parthenolide (1), cbf_5236571 (2), and FK506 (3) were added to the media. Media also contained 3-AT (4), a potent competitive inhibitor of His3 to further control the growth and dynamic range of the system. b, growth curve of JLY31, a PDR5 biosensor strain showing growth suppression by 4 (25 mm) and rescue by induction with 1 (10 μm). Curves represent the average of 4 biological replicates. c, dose-response of JLY31 under increasing concentrations of 4. Relative growth = AUC4/AUCno 4. Curves represent the best fit dose-response curve, and error bars represent S.D. (n = 4). These data are identical to Fig. S1a, whereas analogous plots for compounds 1-3 with all biosensor strains are shown in Fig. S1, b–i. d, heat map of drugs that significantly induces PDR transporters (Z ≥ 3 for one or more biosensor construct). A survey of 800 natural products on induction of the JLY31–33 biosensor strain was conducted. A Z-score was calculated to reflect the strain growth, normalized to quality control-adjusted growth of the same strain treated with DMSO.
Figure 2.
Figure 2.
Multiplexed interrogation of PDR regulators reveals multiple candidate regulators of the PDR process. a, schematic of the homozygous deletion collection screening with biosensor system. Cultures were harvested after 6 generations of growth and relative abundance of each mutant in the treatment and control conditions were quantified by microarray analysis. b, Venn diagrams representing the overlapping hits across the 3 biosensor systems for each of the three inducers (1–3). Numbers represent ORFs that were significantly up-regulated (red), down-regulated (blue), or contra-regulated (purple). c, heat map of deletion mutants for which a log2(|fc|)>0.75 and p < 0.01 for at least one condition. All data were calculated from 4 biological replicates.
Figure 3.
Figure 3.
qPCR validation of PDR transcriptional responses confirms screen hits. Strains were grown to mid-exponential phase and treated with the indicated compounds at 50 μm for 1 h. Relative induction was calculated by comparing the fold-induction of the transporter gene between hoΔ and the deletion strain. Error bars represent S.D. (n = 3). *, indicates p < 0.05, based on Student's t test on the fold-induction of the transporter gene between hoΔ and the deletion strain. Treatment with (a) 1, (b) 2, and (c) 3.
Figure 4.
Figure 4.
Disruption of spindle check point leads to elevated PDR activation. a, structures of compounds screened: cbf_5328528 (5), paf C-16 (6), k035-0031 (7), 0180-0423 (8), N,N-dimethylsphingosine (9). b, baseline corrected representative growth curve of hoΔ and mad deletion in BY4743 background under treatment with 140 μm 5. A600 were measured every 15 min over 30 h. c, dose-response of strains in b over increasing 5 concentrations. Relative growth = AUC5/AUCno drug. All wells contain the same DMSO concentration. AUC calculation was performed with a baseline corrected growth curve. The line represents best fit dose-response curve, and error bars represent S.D. (n = 3). d, baseline corrected representative growth curve of hoΔ and mad deletion in AD1–9 PDR transporter null background under 77.5 μm 5 treatment. e, dose-response of strains in d over increasing 5 concentrations. f, qPCR validation of transcription induction of all three transporters under the treatment of all five compounds in a. Methods are identical to those in Fig. 3. Error bars represent S.D. (n = 3). *, indicates p < 0.05.
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