Deletion of CDR1 reveals redox regulation of pleiotropic drug resistance in Candida glabrata

Journal Pre-proof Deletion of CDR1 reveals redox regulation of pleiotropic drug resistance in Candida glabrata Kseniia V. Galkina, Michiyo Okamoto, Hiroji Chibana, Dmitry A. Knorre, Susumu Kajiwara PII:

S0300-9084(19)30360-8

DOI:

https://doi.org/10.1016/j.biochi.2019.12.002

Reference:

BIOCHI 5803

To appear in:

Biochimie

Received Date: 25 September 2019 Accepted Date: 9 December 2019

Please cite this article as: K.V. Galkina, M. Okamoto, H. Chibana, D.A. Knorre, S. Kajiwara, Deletion of CDR1 reveals redox regulation of pleiotropic drug resistance in Candida glabrata, Biochimie, https:// doi.org/10.1016/j.biochi.2019.12.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Deletion of CDR1 reveals redox regulation of pleiotropic drug resistance in Candida glabrata Kseniia V. Galkina1,2, Michiyo Okamoto3, Hiroji Chibana3, Dmitry A. Knorre3,4*, Susumu Kajiwara5* 1

Faculty of Bioengineering and Bioinformatics, Moscow State University, Leninskiye Gory 1–

73, Moscow, 119991, Russia 5

2

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskiye

Gory 1–40, Moscow, 119991, Russia 3

Medical Mycology Research Center, Chiba University 1-8-1 Inohana, Chuo-ku, Chiba 260-

8673, Japan 4

Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Moscow,

10

119991, Russia 5

School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-

cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan

*Corresponding authors: 15

Dmitry Knorre, Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskiye Gory 1–40, Moscow, 119991, Russia e-mail: [email protected] Susumu Kajiwara, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan

20

e-mail: [email protected]

Abstract Microbial cells sense the presence of xenobiotics and, in response, upregulate genes involved in pleiotropic drug resistance (PDR). In yeast, PDR activation to a major extent 25

relies on the transcription factor Pdr1. In addition, many xenobiotics induce oxidative stress, which may upregulate PDR independently of Pdr1 activity. Mitochondria are important sources of reactive oxygen species under stressful conditions. To evaluate the relevance of

this redox pathway, we studied the activation of PDR in the yeast Candida glabrata, which we 30

treated

with

a

mitochondrially

targeted

antioxidant

plastoquinonyl-decyl-

triphenylphosphonium and dodecyltriphenylphosphonium (C12TPP) as a control. We found that both compounds induced activation of PDR genes and decreased the intracellular concentration of the PDR transporter substrate Nile red. Interestingly, the deletion of PDR transporter gene CDR1 inhibited the decrease in Nile red accumulation induced by antioxidant plastoquinonyl-decyl-triphenylphosphonium but not that by C12TPP. Moreover,

35

antioxidant alpha-tocopherol inhibited C12TPP-mediated activation of PDR in ∆cdr1 but not in the wild-type strain. Furthermore, pre-incubation of yeast cells with low concentrations of hydrogen peroxide induced a decrease in the intracellular concentration of Nile red in ∆cdr1 and ∆pdr1 as well as in control cells. Deletion of PDR1 inhibited the C12TPP-induced activation of CDR1 but not that of FLR1, which is a redox-regulated PDR transporter gene. It

40

appears that disruption of the PDR1/CDR1 regulatory circuit makes auxiliary PDR regulation mechanisms crucial. Our data suggest that redox regulation of PDR is dispensable in wildtype cells because of redundancy in the activation pathways, but is manifested upon deletion of CDR1.

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Keywords: multiple drug resistance; antifungals; signalling; oxidative stress

1 Introduction Mild stresses have been shown to induce yeast cell tolerance to a wide range of 50

stressful conditions [1–3] as a result of an orchestrated environmental stress response [4,5]. Some stresses induce oxidative stress in the cytosol or mitochondria by increasing hydrogen peroxide generation or abrogating the function of antioxidant enzymes. For example, a high concentration of ethanol [6,7], heat shock [8,9] or genotoxic stress [10] increases superoxide and hydrogen peroxide levels in yeast cells. This endogenous secondary oxidative stress

55

plays a role in cell survival which is not currently well understood. On the one hand, reactive oxygen species modify macromolecules and alter their functions [11,12]. On the other hand, mild oxidative stress activates an adaptive response. The preconditioning of yeast cells by pro-oxidants induces tolerance to NaCl [13], ethanol [14,15] and genotoxic agents [16]. Antioxidants prevent and hydrogen peroxide facilitates the activation of heat-shock stress

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response [8]. Treatment of yeast cells with low doses of xenobiotics, as with environmental

stresses, activates the adaptive response. Specifically, the binding of xenobiotics to the Pdr1 transcription factor [17] induces the transcription of pleiotropic drug resistance (PDR) transporter genes [18,19]. PDR transporters have broad substrate specificity and are 65

responsible for the efflux of toxic compounds from the cytoplasm at the cost of ATP hydrolysis (ABC transporters) or proton translocation into the cytoplasm (MFS transporters) [20,21]. Some xenobiotics induce secondary oxidative stress [22–25] (as reviewed by [26,27]). However, the role of secondary oxidative stress in the regulation of xenobiotic resistance is still not well understood. ABC transporters with broad substrate specificity are

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absent from a list of genes regulated by the hydrogen-peroxide-sensitive transcription factor Yap1 [28–30]. Genome-wide microarray studies [4] or Ribo-seq [31] of hydrogen-peroxidetreated Saccharomyces cerevisiae cells did not reveal an increase in the levels of mRNA of major PDR ABC transporters such as PDR5 and SNQ2. The yeast Candida glabrata is a common human opportunistic pathogen that causes

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invasive diseases and frequently shows or evolves drug resistance upon prolonged antifungal therapy [32,33]. Candida glabrata is closely related to the well-studied yeast Saccharomyces cerevisiae than to other pathogenic Candida species (e.g. C. albicans) [34]. While S. cerevisiae is considered as a reliable model for the research of azole antifungals [35], there are still some differences in drug resistance and stress response mechanisms. In

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particular, C. glabrata shows high resistance to exogenous hydrogen peroxide [36] as well as accelerated dynamic activation of stress response genes [37]. According to YEASTRACT+, a manually curated database of yeast transcription regulatory associations, CDR1 (the C. glabrata orthologue of PDR5) and SNQ2 are also absent in the list of experimentally confirmed CgYap1 targets [38]. Microarray analysis of yeast genes

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upregulated in response to benomyl revealed CgYap1 regulation of CDR2, an orthologue of S. cerevisiae ABC transporter gene PDR15 [37]. Benomyl is a microtubule depolymerising agent that interferes with multiple cellular processes and induces, in particular, oxidative stress response [29]. However, the ChiP-seq analysis of CgYap transcription factors binding sites did not detect SNQ2 as a direct target for CgYap1 [39]. Taken together, this data

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implies the absence of a strong connection between PDR and oxidative stress response in both yeast species. On the contrary, other studies have implied the presence of crosstalk between the PDR activation and hydrogen peroxide signalling pathways [40–42]. It has been shown that YAP1 overexpression can lead to PDR1-dependent diazaborine resistance [41]. Yap1p has

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been shown to mediate the expression of PDR5 and SNQ2 under heat-shock conditions [40]. YAP1 can confer resistance to antifungals via the upregulation of the PDR MFS transporter FLR1 in S. cerevisiae [43] and C. glabrata [44]. Finally, it has been recently shown that the deletion of a catalase gene CTT1 in S. cerevisiae increased azole tolerance

[42]. 100

It is tempting to decrease pathogenic yeast resistance to antifungals by supplementation with antioxidants [45]. In this study, we compared the effect of two lipophilic cations: dodecyltriphenylphosphonium (C12TPP) and a mitochondria-targeted antioxidant, plastoquinonyl-decyl-triphenylphosphonium (SkQ1). The chemical structures of the compounds are shown in Figure 1A. SkQ1 inhibits the production of mitochondrial ROS in

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yeast [46] and higher eukaryotes [47]. We used C. glabrata cells as a model and monitored PDR activation by the accumulation of the fluorescent PDR transporter substrate Nile red in cells. We found that both cations upregulated PDR in C. glabrata, but the deletion of the CDR1 gene specifically inhibited the effect of SkQ1. Our data suggest that secondary oxidative stress upregulates drug resistance, but this effect is dispensable in wild-type cells

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because of redundancy in the activation mechanisms.

2 Materials and methods 2.1 Growth conditions and strains We used a CBS138 C. glabrata strain [48] as the wild-type, and a KUE100 strain was used for the construction of gene disruption mutants: ∆cdr1 (deletion of CAGL0M01760g) 115

and ∆pdr1 (deletion of CAGL0A00451g) [49]. To isolate these mutants, each gene in the KUE100 strain was replaced by a DNA cassette with the CgHIS3 gene. Cells were grown on a yeast peptone dextrose (YPD, 1% yeast extract, 2% peptone, 2% dextrose and 2% agar) agar medium.

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2.2 Drug susceptibility assay Drug susceptibility and drug X drug interaction assays were performed in liquid YPD medium in 96-well microtiter plates. We inoculated yeast in YPD medium to a final OD600 of 0.005. Then, plates were incubated with shaking at 37°C for 24 h. The minimum inhibitory concentrations (MIC) and optical density (OD) of the cultures under different concentrations

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of lipophilic cations and miconazole were measured at a wavelength of 600 nm using a VARIOSKAN LUX™ (Thermo Scientific) microplate reader. Miconazole, α-tocopherol and Nile red were obtained from Wako Chemical (Osaka, Japan). C12TPP and SkQ1 (bromine salts) were originally synthesised at A.N.Belozersky Institute, Moscow State University [50].

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2.3 Flow cytometry Yeast cells were grown overnight in YPD medium and then resuspended to an OD600 of 0.2 in liquid-rich growth medium. After 1 h of pre-incubation with the inhibitors or solvent at

37°C, Nile red was added to a final concentration of 7 µM. The accumulation of Nile red was assessed using an EC800 (Sony) flow cytometer with an excitation wavelength of 488 nm on 135

the emission filter (585/42 nm). At least 10,000 events were analysed in each experiment. 2.4 RNA extraction and RT-PCR Yeast cells were grown overnight in solid YPD medium, resuspended to an OD600 of 0.5 in 6 mL of liquid-rich growth medium and incubated with the inhibitors or solvents at

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37°C for 1 h. RNA was isolated from the yeast cells using the hot formamide extraction method described by Shedlovskiy and colleagues [51]. RNA samples were isolated independently four times on separate days. RNA quality and quantity were assessed by electrophoresis using a GeneQuant100 spectrophotometer (GE healthcare). cDNA was synthesised by annealing 1.7 µg of RNA with ReverTra Ace® qPCR Master Mix with gDNA

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Remover (Toyobo). All primers used in this study are listed in Table S1. Real-time PCR was performed using THUNDERBIRD® SYBR® qPCR Mix (Toyobo) according to the manufacturer’s instructions. The thermal profile for SYBR Green RT-qPCR included an initial heat-denaturing step at 95°C for 10 min, 42 cycles. Each cycle comprised a denaturation step at 95°C for 15 s, an annealing step at a custom temperature for each primer for 30 s

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and a DNA synthesis step at 72°C for 30 s and was coupled with fluorescence measurements. Each sample was analysed in triplicate, and a non-template control was added to each run. Target mRNA levels were normalised to the reference gene ACT1.

3 Results Deletion of the PDR ABC transporter genes inhibited S. cerevisiae yeast growth in 155

the presence of the toxic substrates of these transporters [52]. We found that deletion of PDR1 and CDR1 decreased C. glabrata tolerance to the lipophilic cations C12TPP and mitochondria-targeted antioxidant SkQ1 (Figure 1B). PDR1 encodes a transcription factor (TF) homologous to S. cerevisiae PDR1/PDR3 TFs [53], and CDR1 is a major ABC transporter regulated by PDR1. Deletion of PDR1 induced a more pronounced effect than

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the deletion of CDR1. These observations suggest that C12TPP and SkQ1 are exported by Cdr1p as well as by other Pdr1-regulated ABC transporters. PDR transporter substrates can competitively inhibit the efflux of other xenobiotics by these transporters. At the same time, PDR transporter substrates can activate the expression of PDR transporter genes. Thus, it is usually unclear whether a specific

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compound can increase or decrease the efflux of other ABC transporter substrates. To investigate this issue, we used a Nile red accumulation assay, which we have previously

applied to S. cerevisiae. Nile red is a fluorescent dye, and its concentration in cells is negatively correlated to the activity of PDR transporters [54]. We have found that the majority of C. glabrata cells accumulated a high amount of Nile red, and a small 170

subpopulation showed low Nile red levels (Figure 2A). Supplementation with C12TPP increased the concentration of Nile red in the cells of this subpopulation (Figure 2A). This effect indicates a competitive inhibition of Nile red efflux by C12TPP. Pre-incubation of yeast cells with C12TPP or SkQ1 for 60 min prevented an accumulation of Nile red in yeast cells (Figures 2B–D). It should be noted that for the Nile red accumulation experiments, we used

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denser cell suspensions (OD600 = 0.2) than that used in the growth experiments in Figure 1 (initial OD600 = 0.005). At the same time, yeast cells absorb lipophilic cations [55]; thus, the inhibitory concentration should strongly depend on the cell density of the suspension. Thus, it was expected that yeast cells preserved the ability to prevent Nile red accumulation up to 7.5 µM of C12TPP (Figure 2C), whereas the same concentration prevented growth of a low-

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cell density cell suspension (Figure 1B). We used miconazole (2.5 µM) as a positive control in the Nile red accumulation assay. Taken together, these experiments (Figure 1, Figure 2) indicate that the lipophilic cations C12TPP and SkQ1 are competitive inhibitors of PDR and the activators of PDR in C. glabrata, whereas the contribution of each effect depends on the incubation time.

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We then tested the role of CDR1 in PDR activation by lipophilic cations using the same Nile red accumulation assay. SkQ1 and C12TPP share similar physico-chemical properties. For this reason, C12TPP is commonly used as a control in experiments with SkQ1 to discriminate between its antioxidant and non-specific effects [56]. We found that both lipophilic cations induced a decrease in Nile red levels in wild-type strains (Figure 2 and

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Figure 3A). The PDR activation of mitochondria-targeted antioxidant SkQ1 was much weaker in the ∆cdr1 strain than the effect of C12TPP lacking redox active groups (Figure 3A). Both compounds were able to induce a decrease in Nile red concentration in ∆pdr1 cells (Figure 3A). These results can be explained if there is a mechanism of Nile red efflux activation which is independent of PDR1 and CDR1. To test this possibility, we measured

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the activation of PDR by C12TPP in the presence and absence of a non-mitochondrial antioxidant α-tocopherol. α-tocopherol (vitamin E) is a hydrophobic antioxidant that prevents the propagation of the chain reaction of lipid peroxidation and interference with redoxsignalling pathways [57]. As with SkQ1, the supplementation of α-tocopherol partially prevented the effect of C12TPP in ∆cdr1 cells, but not in wild-type cells (Figure 3B).

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Since α-tocopherol inhibits C12TPP-induced PDR activation in ∆cdr1 cells, we hypothesised that pro-oxidants might upregulate PDR. We found that pre-incubation of yeast cells with hydrogen peroxide upregulated PDR in all strains tested (Figure 4A). The activation of PDR by C12TPP was enhanced by the application of 2 mM of hydrogen

peroxide (Figure 4B). To demonstrate that the decrease of Nile red concentration in cells 205

was mediated by the activation of PDR genes, we measured the mRNA level of some PDR transporter genes. We selected CDR1, CDR2 and SNQ2, all of which are regulated by Pdr1, and FLR1, which is regulated by the hydrogen-peroxide-sensitive TF YAP1 [44]. We found that C12TPP and SkQ1 induced the expression of CDR1, CDR2 and FLR1 (Figure 5), and the deletion of PDR1 prevented the upregulation of CDR1 and CDR2. Hydrogen peroxide

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did not increase the expression levels of these genes, but it did upregulate FLR1. Lipophilic cations induced FLR1 in the ∆pdr1 strain as well as in the wild-type (Figure 5D). This observation shows that FLR1 can be activated even when the Pdr1p-regulatory circuit is not functional. Since lipophilic cations are both competitive inhibitors and activators of PDR, they

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can either increase or decrease the MIC of other toxic compounds. To discriminate between these possibilities, we assessed the combined effects of C12TPP and miconazole. We measured increases in OD for wild-type and ∆pdr1 yeast strains under incremental concentrations in C12TPP, miconazole and combinations in 96-well microplates. We found that C12TPP inhibits the azole cytostatic effects in WT and ∆cdr1 strains (Figure 6A).

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However, we were not able to identify antagonism between SkQ1 and miconazole, even while using small incremental steps of concentration (Figure 6B). Non-toxic concentrations of SkQ1 enhanced miconazole toxicity (Figure 6B). We examined the effect of SkQ1 preincubation on PDR induction by low concentrations of miconazole (Figure 6C). We found that miconazole pre-treatment prevented the accumulation of Nile red; that is, it induces

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PDR in wild-type and ∆cdr1, but the effect was significantly reduced in the ∆pdr1 strain. In the ∆cdr1 strain, mitochondria-targeted antioxidant SkQ1 was less efficient in azole resistance upregulation than its non-antioxidant counterpart. Co-supplementation of SkQ1 inhibited the miconazole-induced PDR activation in ∆cdr1 cells (Figure 6C). In contrast to SkQ1, antioxidant α-tocopherol did not alleviate miconazole toxicity (Figure 6D).

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4 Discussion The widespread use of antimicrobial compounds drives the evolution of drug resistance and decreases the efficiency of the antimicrobial compounds [58]. Antimycotics and antibiotics also induce short-term non-inherited adaptation that can make a pathogen resistant to a wide range of compounds. The inhibition of acclimation is a promising strategy

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against drug-resistant strains of pathogenic yeasts [59]. Our results suggest that these shortterm adaptation mechanisms are redundant. We showed that antioxidant the molecule αtocopherol interferes with the activation of Nile red efflux in C. glabrata cells, but this

interference is manifested only in the strain in which the major PDR transporter CDR1 is deleted. Moreover, in ∆cdr1 cells, but not in wild-type strain, mitochondria-targeted 240

antioxidant SkQ1 was much less prominent in PDR activation than chemically a similar lipophilic cation C12TPP, which lacks a redox active group (Figure 3). These observations imply that a transporter gene can mediate the sensing of the xenobiotics or PDR activation. We suggest that, in wild-type strains, a negative feedback loop inhibits the accumulation of the xenobiotic in the cytoplasm (Figure 7). This loop relies on functional CDR1 and makes

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the contribution of other regulatory mechanisms dispensable. However, in ∆cdr1 cells, the role of alternative mechanisms become more pronounced. For example, the accumulation of the xenobiotic in the cytoplasm above a certain concentration threshold could be necessary for the activation of a PDR-independent redox-signalling pathway. The MFS transporter FLR1 is activated by C12TPP and SkQ1 in ∆pdr1 strains (Figure

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5). C12TPP were shown earlier to induce oxidative stress-mediated mitochondrial network remodelling in S. cerevisiae [60]. Given that FLR1 gene is regulated by YAP1 [39,44], we suppose that both lipophilic cations did induce oxidative stress in C. glabrata. We expected that the antioxidant properties of SkQ1 could prevent an amplification of signalling mediated by reactive oxygen species and consequent decrease in FLR1 activation. However, we did

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not find significant differences between the expression level of FLR1 in SkQ1-pre-treated and C12TPP-pre-treated strains (Figure 5D). These results appear to be inconsistent with the Nile red accumulation and drug × drug interaction experimental results (Figure 2 and Figure 6). The discrepancy can be explained if there are other PDR gene targets other than FLR1 regulated by secondary oxidative stress.

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We demonstrated that lipophilic cations are substrates and activators of PDR transporters in Candida glabrata. The contribution of the inhibitory and activating effects of lipophilic cations depended on incubation time. At the first moment, after the addition, lipophilic cations increase the concentration of the PDR substrate in yeast cells; however, 1 hour of pre-incubation reversed their effects (Figure 2). Meanwhile, exogenous hydrogen

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peroxide also induced PDR (Figure 4) and that this effect was independent from PDR1 and CDR1 genes, the major components of PDR in yeast. Importantly, reactive oxygen species production is a phagocyte-mediated host response that opposes to pathogenic fungi [61,62]. We speculate that this host response as well as the activation of secondary endogenous oxidative stress could upregulate PDR in C. glabrata.

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5 Conclusions Taken together, our data suggest that adaptive response to oxidative stress provides

an alternative route for PDR activation and makes drug resistance activation mechanisms in yeast redundant. Our findings reveal an additional layer of robustness of drug resistance regulation mechanisms in pathogenic yeasts.

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6 Authors contributions DAK and SK designed the study; SK acquired funding; MO and HC provided mutant strains; KVG performed the experiments and formal analysis of the data; KVG and DAK prepared the illustrations; DAK drafted the manuscript; KVG, HC, DAK and SK edited the manuscript. All authors read and approved the final version of the manuscript.

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7 Acknowledgement This work was supported by Inter-University Exchange Project, Ministry for Education, Culture, Sports, Science and Technology (MEXT) (Japan).

8 Conflict of interest statement Nothing to declare

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Figure legends 490

Figure 1. Deletions of PDR genes CDR1 and PDR1 inhibit yeast growth in the presence of lipophilic cations C12TPP and SkQ1. (A) Chemical structures of C12TPP and SkQ1. (B) Growth of wild-type (WT), ∆cdr1 and ∆pdr1 C. glabrata strains in the presence of C12TPP and SkQ1. Heat maps represent the relative growth of yeast cells in 96-well plates (OD after 24 h of incubation) compared to an untreated control.

495 Figure 2. Lipophilic cations act as both inhibitors (A) and activators (B–D) of PDR in Candida glabrata cells depending on the pre-incubation time. (A) Simultaneous supplementation of Nile red and xenobiotics reveals competitive inhibition of their efflux and increases the intracellular concentration of Nile red. Scheme of the experiment and 500

representative results of flow cytometry analysis. (B–D) Pre-incubation of yeast cells with lipophilic cations induces a decrease in Nile red accumulation levels. (B) Scheme of the PDR activation assay and representative flow cytometry histograms. (C) Quantification of the results with various concentrations of C12TPP and miconazole (MNZ, 2.5 µM). (D) Quantification of the results for SkQ1. The bar plots show mean Nile red fluorescence ± SE.

505

The number of biological replicates is indicated in the basement of the bar plots for each condition. The P values were calculated using unpaired Mann–Whitney tests. Figure 3. Antioxidants inhibit PDR activation in Candida glabrata ∆cdr1 strain. (A) Quantification of flow cytometry data for yeast cells (wild-type, WT; ∆cdr1 and ∆pdr1) pre-

510

incubated (1 h) with C12TPP (2.5 µM), SkQ1 (10 µM) and miconazole (MNZ, 2.5 µM). Mean

± S.E., n = 5–15. * P value < 0.006 according to unpaired Mann–Whitney tests. (B) Quantification of flow cytometry data for wild-type and ∆cdr1 yeast cells pre-incubated (1 h) with lipophilic cation C12TPP (2.5 µM) with or without supplementation of antioxidant αtocopherol. Mean ± S.E., n = 6. P value was calculated according to the unpaired Mann– 515

Whitney test.

Figure 4. Hydrogen peroxide decreases Nile red accumulation in Candida glabrata yeast cells with deleted PDR genes. (A) Yeast cells were pre-incubated for 1 h with indicated concentrations of hydrogen peroxide and then stained with Nile red. Mean ± S.E., 520

n = 6. (B) Hydrogen peroxide (2 mM) modulates C12TPP-induced PDR activation. C12TPP concentration is 0.5 µM (mean ± S.E., n = 6). Figure 5. Lipophilic cations and hydrogen peroxide increase mRNA levels of major PDR transporter genes. Quantitative PCR experiments for (A) CDR1, (B) CDR2, (C) SNQ2

525

and (D) FLR1. All mRNA levels were normalised to the ACT1 mRNA level. The equivalent ratio of target gene mRNA to ACT1 mRNA level was set at 1. C12TPP concentration is 5 µM, SkQ1 concentration is 12.5 µM, and H2O2 concentration is 10 mM (mean ± S.E., n = 4–8). * P < 0.05; ** P < 0.01 according to Mann–Whitney test compared to untreated control.

530

Figure 6. SkQ1 inhibits activation of PDR by miconazole and increases its cytostatic effect. (A, B) Drug × drug interaction plot for C12TPP × miconazole (MNZ) and SkQ1 × miconazole (MNZ). (C) PDR activation in wild-type and PDR-deficient C. glabrata cells pretreated with SkQ1 (10 µM) and/or miconazole (MNZ, 2.5 µM); mean ± S.E., n = 6. P value was calculated using unpaired Mann–Whitney tests. (D) Drug × drug interaction plot for

535

miconazole × α-tocopherol. Figure 7. Hypothetical scheme of drug resistance activation in Candida glabrata. An increase in xenobiotic concentration induces PDR transporter genes and inhibits further xenobiotic accumulation. Together, sensing and efflux form a negative feedback loop. The

540

deletion of the main PDR transporter gene CDR1 reveals a redox regulation loop of pleiotropic drug resistance, which is sensitive to antioxidants and hydrogen peroxide.

A

OH CH3

P

CH 3

CH3

P OH

Redox group

n=3

1

50

45

40

35

Relative Growth 30

25

20

10

5

2.5

0

10

8.75

7.5

SkQ1 7

6.5

6

5.5

5

3.75

C12TPP 2.5

0

B

WT

WT

Δcdr1

Δcdr1

Δpdr1

Δpdr1

n=3

0.5 0

10 min

FLOW CYTOMETRY

Solvent + Fluorescent PDR-pump substrate

Count

1 hour in YPD

Unstained control Control 10μM C12TPP

100

Xenobiotic + Fluorescent PDR-pump substrate

Efflux competitive inhibition assay

A

50

0

100

102

101

103

105

104

Nile red levels, a.u.

B

Solvent

10 min

Count

FLOW CYTOMETRY

Fluorescent PDR-pump substrate

1 hour

PDR activation assay

10 min

Xenobiotic

50

0

100

101

D 1 200 1 000 800 600 400 200 3

3

6

3

3

3

0.5

1

2.5

5

7.5

10

C12TPP, µM

5

MNZ

9

Control

0

103

105

104

1 600 1 400 1 200 1 000 800 600 400 200 0

9

Control

p=0.0018

1 400

102

Nile red levels, a.u.

p=0.0018

Nile red levels, a.u.

C Nile red levels, a.u.

Unstained control Control 2.5μM C12TPP 10μM SkQ1

100

3

2.5

3

5

3

6

4

4

3

7.5 10 12.5 15 20

SkQ1, µM

1 000

WT

Δcdr1

Control C12TPP SkQ1 MNZ

MNZ

Control C12TPP SkQ1

0

C12TPP SkQ1 MNZ

500

Δpdr1

p=0.005

N.S.

2 500 2 000 1 500 1 000 500 0

--

10 50 10 50 0

+ +

WT

+

Control

*

3 000

Control

2 000 1 500

B Nile red levels, a.u.

2 500

Control

Nile red levels, a.u.

A

*

* *

3 000

--

10 50 10 50 0 Tocopherol, μM

+ +

Δcdr1

+ C12TPP

A

B 3 000

2,500 2,000

Δpdr1 1,500

Δcdr1 1,000

WT

500 0

2

4 6 H2O2, mM

8

10

12

Nile red levels, a.u.

Nile red levels, a.u.

3,000

2 500 2 000 1 500 1 000 500 0

-- -+ ++ +- -- -+ ++ +- -- -+ ++ +WT

Δcdr1

Δpdr1

H 2 O2 C12TPP

0.1

0.06

0.04

0.02

D WT

0.08

0.05

0.04

0

WT * **

0.06

**

Δpdr1 0.07

**

Δcdr1 Δpdr1

**

WT 0 Control SkQ1 C12TPP H 2 O2 Control SkQ1 C12TPP H 2 O2 Control SkQ1 C12TPP H 2 O2

*

CDR2/ACT1 level

1

Control SkQ1 C12TPP H 2 O2 Control SkQ1 C12TPP H 2 O2 Control SkQ1 C12TPP H 2 O2

0.08

FLR1/ACT1 level

1.5

**

0

Δcdr1

Control SkQ1 C12TPP H 2 O2

WT

B **

C 0 Control SkQ1 C12TPP H 2 O2 Control SkQ1 C12TPP H 2 O2 Control SkQ1 C12TPP H 2 O2

0.5

Control SkQ1 C12TPP H 2 O2 Control SkQ1 C12TPP H 2 O2

CDR1/ACT1 level 2

**

SNQ2/ACT1 level

A 0.2

0.15

0.1

0.05

Δcdr1

Δcdr1

Δpdr1

0.09

*

0.03

0.02

0.01

Δpdr1

WT n=3

Δcdr1 n=3

Miconazole, mg/L 0 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16

WT 100 75

2 000

Relative Growth 1.0 0.8 0.6 0.4 0.2 0

p=0.008

1 500 1 000

5 0

Miconazole, mg/L

Δcdr1

WT

+ + - SkQ1 - + + MNZ + + + +

Δcdr1

Control

- + + + +

Control

100

Control

0

25 10

75 50 25 10 5 0

Δpdr1

α-tocopherol, μM

500

50

α-tocopherol, μM

2 500

MNZ, mg/L

0 7.5 10 12.5 13.8 15 16.3 17.5 18.8 19.3 20 0 0.03 0.06 0.09 0.12 0.18 0.25

MNZ, mg/L

0 0.12 0.18 0.25 0.38 0.5 0.75

D

3 000

Nile red levels, a.u.

0 7.5 10 12.5 13.8 15 16.3 17.5 18.8 19.3 20

0 1 1.25 1.5 1.75 2 2.5 3 3.5 4 4.5

Δcdr1 n=5

SkQ1, μM

0 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16

C

0 0.03 0.06 0.09 0.12 0.18 0.25

MNZ, mg/L

0 0.12 0.18 0.25 0.38 0.5 0.75

WT n=5

B

C12 TPP, μM

Relative 0 growth 1.0

SkQ1, μM

MNZ, mg/L

C12 TPP, μM

0 1 1.75 2 2.25 2.5 3.75 5 5.5 6 6.5

A

Relative 0 growth 1.0