AAC Accepts, published online ahead of print on 16 September... Antimicrob. Agents Chemother. doi:10.1128/AAC.01504-13

Transcription

AAC Accepts, published online ahead of print on 16 September 2013
Antimicrob. Agents Chemother. doi:10.1128/AAC.01504-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Multidrug Resistant Transporter Mdr1p Mediated Uptake of a Novel
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Antifungal Compound
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Affiliations:
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1 Georgetown University Medical Center, Department of Microbiology & Immunology,
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Washington, DC, 20057, USA.
2 Department of Biostatistics, Bioinformatics and Biomathematics, Lombardi Comprehensive
Cancer Center, Georgetown University Medical Center, Washington
DC, 20057, USA.
3 Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology,
Institute of Microbiology, Beijing, 100190, China.
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By
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Nuo Sun1, Dongmei Li1, William Fonzi1, Xin Li2, Lixin Zhang3, and Richard Calderone1*
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*To whom correspondence should be addressed. E-mail: [email protected].
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Running title: Overcoming MDR resistance in Candida albicans
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Key words: Efflux, fluconazole, resistance, MDR, candidiasis
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Abstract
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The activity of many anti-infectious drugs has been compromised by the evolution of
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multidrug resistant (MDR) pathogens. For life-threatening fungal infections, such as those
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caused by Candida albicans, overexpression of MDR1, which encodes an MDR efflux pump of
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the major facilitator superfamily (MFS), often confers resistance to chemically unrelated
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substances, including the most commonly used azole antifungals. As the development of new
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and efficacious antifungals has lagged far behind the growing emergence of resistant strains, it is
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imperative to develop strategies to overcome multidrug resistance. Previous advances have been
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made mainly to deploy combinational therapy to restore azole susceptibility, which, however,
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requires coordination of two or more compounds. We observed a unique phenotype in which
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Mdr1p facilitates the uptake of a specific class of compounds. Among them, we describe a novel
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antifungal small molecule, Bis [1,6-a:5',6'-g] quinolizinium 8-methyl-salt (BQM) (US Patent
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Application Serial No.61/793,090,2013) that has potent and broad antifungal activity. Notably,
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BQM exploits the MDR phenotype in C. albicans to promote the inhibitory effect. Rather than
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causing an antagonism of MDR strains, it exhibits a highly potentiated activity against a
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collection of clinical isolates and lab strains that overexpress MDR1. The activity of BQM
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against MDR1 overexpressed isolates is due to its facilitated intracellular accumulation.
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Microarray comparisons showed an extensive upregulation of MDR1 as well as polyamine
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transporters in a fluconazole resistant strain. We then demonstrated that the polyamine
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transporters augment the accumulation of BQM. Importantly, BQM had greater activity than
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fluconazole and itraconazole against various fungal pathogens, including MDR A. fumigatus.
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Thus, our findings offer a paradigm shift to overcome MDR and the promise of improving
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antifungal treatment, especially in MDR pathogens.
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Introduction
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Fungi that cause invasive infections are now referred to as the “hidden killers” (1). Mortality
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due to these infections is similar to drug resistant TB and exceeds malaria (1). Of these
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pathogens, Candida species have emerged among the top three causes of microbial nosocomial
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infectious diseases in humans, resulting in 46-75% mortality, and A. fumigatus is the most deadly
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and frequent mold infection of humans (1, 2). The incidence of candidiasis has increased sharply
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over the past few decades primarily due to hospital interventions such as cancer chemotherapy,
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surgery, organ/bone marrow transplantation, and indwelling devices (3). The cost in the US of
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treating candidiasis infections is ~$2.0 billion per year (4, 5). However, current therapies have
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led to only modest success in reducing the high mortality rates of invasive fungal infections, in
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part due to drug toxicity (amphotericin B), a narrow spectrum of activity (echinocandins), or, in
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the case of the fungistatic triazoles, the selection of CDR1,2, MDR1, or ERG11 over expression
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or ERG11 mutations in isolates caused by an overdependence of these therapies (6-8).
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Reduced intracellular accumulation of drugs by genes encoding drug transporters is a
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prominent mechanism of resistance in Candida strains. Drug transporters, such as the ABC
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(ATP-binding cassette) transporters, CDR1, CDR2 (Candida Drug Resistance) and an MFS
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transporter, MDR1 (Multi-Drug Resistance), play key roles in azole resistance as deduced by
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their high level of expression in the majority of azole-resistant clinical C. albicans isolates (9-
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11). While ABC transporters depend on ATP as an energy source, MDR1, encoding Mdr1p, is a
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member of the MFS transporters that use the proton gradient across the cytoplasmic membrane
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to supply energy for transport (12-14). Mdr1p exports a variety of structurally unrelated
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compounds, such as fluconazole, benomyl, cerulenin, and brefeldin A (9, 14-16). In drug-
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susceptible C. albicans strains, MDR1 is expressed at low levels. However, clinical isolates
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consistently overexpressed MDR1 when the patient was continuously treated with fluconazole (8,
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9, 17). Deletion of MDR1 in these resistant isolates reversed the drug resistance phenotype (18,
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19). Moreover, engineered overexpression of MDR1 increased resistance (19, 20). In C. albicans,
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MDR1 overexpression results mainly from gain-of-function mutations in the transcription factor
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Mrr1p (14, 21).
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The development of new antifungal therapeutics is crucial. Unfortunately, the antifungal
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pipeline has slowed down considerably. Most new triazoles are remodeled older versions that do
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not address the emergence of resistance. With an already narrow list of antifungal drugs,
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alternate choices become problematic if resistance to triazoles develops during therapy. We
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believe there is a great need for paradigm shift to exploit the resistant mechanisms for more
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efficient therapy. In part, our data address this need. We hypothesized that the MDR phenotype
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conferred by caMDR1 overexpression could be harnessed through the use of MDR1-dependent
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cytotoxic agents for effective antifungal strategies.
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Materials and Methods
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Strains, strain maintenance, and plasmids
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All strains used in the present study are listed in Table S1 and were maintained as frozen stocks
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in 96-well plates and propagated on yeast extract-peptone-dextrose (YPD) agar when needed
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(1% yeast extract, 2% peptone, 2% glucose, 2% agar).
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Antifungal susceptibility testing
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BQM (NSC156627) and all other NSC compounds were provided by the Developmental
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Therapeutics Program of NIH/NCI. Drug susceptibility testing was carried out in flat bottom, 96-
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well microtiter plates (Greiner Bio One) using the broth microdilution protocol according to the
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Clinical and Laboratory Standards Institute M-27A methods. Growth inhibition of all strains was
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evaluated in the presence of drugs and reported as a % inhibition of untreated cells as previously
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described (22). In brief, overnight cultures were prepared in YPD, washed and ~103 cells/100µl
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were inoculated into microtiter wells. Growth was evaluated by measuring cell density OD595
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after 24h of incubation at 30°C. Experiments were repeated at least three times. Data were
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averaged and statistical significance among treatments determined. MIC determinations were
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also done with lab stock cultures of azole susceptible and resistant strains. For these experiments,
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the compounds were tested at the indicated concentrations. Strains were grown as described
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above. Relative growth was calculated based on OD595 data and visualized using a heatmap.
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Drop plate assays
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Growth inhibition was visualized by plating 5 µl of ten-fold serial dilutions of cells onto YPD
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agar plates containing BQM at the indicated concentrations. Cells were grown overnight in YPD
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broth at 30°C, washed with saline, and standardized by hemocytometer counts. Plates were
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photographed and evaluated after 48 h of incubation at 30°C.
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Accumulation assays of BQM
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All C. albicans strains were grown at 30°C overnight in YPD medium and washed twice with
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phosphate-buffered saline (PBS) (pH 7.0). Cells were diluted with RPMI 1640 medium (2%
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glucose without bicarbonate and buffered with 0.165 M MOPS to pH 7.0) to 108 cells/ml, as
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determined by hemocytometer and confirmed by plate count. BQM was added at the
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concentrations indicated in Tables or Figures. 1-ml of each sample was removed after incubation
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at 200 rpm (30°C) at 0, 15, 30, 45 and 60 min, centrifuged, washed, and suspended in 1ml PBS.
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200 l of cell suspensions were transferred into 96-well microplates with clear bottoms (Greiner,
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Germany) to measure fluorescence intensity of BQM. To measure requirement for proton
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gradients, one set of cell suspensions was treated with 20 g/ml CCCP or not treated for 30min
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before introducing BQM.
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RNA preparation and microarray
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All C. albicans strains were grown at 30°C overnight in YPD medium and washed twice with
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PBS (pH 7.0). Cells were suspended with RPMI 1640 medium. As previously described (23),
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total RNA was extracted and, the integrity and purity of total RNA were assessed using an
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Agilent Bioanalyzer (Aglient Technologies) and OD260/280. One-color microarray-based gene
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expression analysis was done using the Agilent Low Input Quick Amp Labeling kit. C. albicans
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cDNA synthesis was carried out using 100 ng total RNA, and all other methods were carried out
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using the manufacturer’s instructions. Hybridization was carried out for 17 h in an Agilent
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SureHyb hybridization chamber and the microarrays were scanned with an Agilent SCAN
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Microarray Scanner System using the AgilentHD_GX_1-color 5 M protocol. The microarrays
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used in this study were designed from assembly 21 of the C. albicans genome using eArray from
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Agilent Technologies (design ID 017942). A total of 6101 genes (including 12 mitochondrial
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genes) are represented by two sets of probes, both spotted in duplicate. Probes are randomly
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distributed. Eight copies of each array were printed on a single slide (8×15,000) and hybridized
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individually. For each microarray analysis, three independent biological replicates are
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performed. Tiff format image files were analyzed by Agilent Feature Extraction software.
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Cyanine 3 intensities were then logarithmically transformed and statistically normalized. BRB
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Array-Tools was then used for the differential test. In this analysis, we adopted the cutoff for the
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parametric p-value <0.05 and [fold change] > 2 to determine the significant gene lists, and using
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FDR as a reference. Genes that were up- or downregulated 2.0 fold were selected and considered
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to be differentially expressed. Gene ontology analysis was performed at the Candida genome
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database (CGD, www.candidagenome.org).
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Quantitative real-time PCR
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We used previously published methods for these experiments (23, 24). Approximately 1 µg of
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the total RNA was subjected to first-strand cDNA synthesis (QuantiTect Reverse Transcription,
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Qiagen). Real-time PCR assays were performed with 20-µl reaction volumes that contained 1x
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iQ SyBR green Supermix (Bio-Rad), including a 0.2 µM concentration of each primer and 8 µl
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of a 1:8 dilution of each cDNA from each strain. The transcription level of each gene was
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normalized to 18S rRNA levels. Data are presented as the means ± standard deviations (SD)
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from three biological replicates. The 2–
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was used to determine the fold change in gene transcription. The primers used for real-time PCR
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expression analysis were the following: for 18S rRNA, CGCAAGGCTGAAACTTAAAGG
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(forward) and AGCAGACAAATCACTCCACC (reverse); For MRR1, ACA CCC AGG GCT
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AGT ATA GAC (forward) and ACG ACA TCT CCA GAA ACA GAC (reverse).
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Evaluation of BQM in G. mellonella assays
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Injection of fungal pathogens and antifungal drugs was performed as described (25, 26). In brief,
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larvae were obtained from Vanderhorst, Inc.. Sixteen larvae (330±20 mg) were used per group.
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Each injection of 10 l of C. albicans cells, BQM, or control was performed via a distinct proleg.
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Larvae were incubated at 37°C, and the number of dead larvae was scored daily. C. albicans
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inoculum was prepared from overnight cultures grown in YPD. Cells were washed three times in
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PBS. Cell densities were determined by hemocytometer. Kill curves were plotted and analyzed
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by the Kaplan-Meier method (GraphPad Prism).
CT
(where CT is the threshold cycle) method of analysis
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Results
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C. albicans overexpressing MDR1 exhibit highly increased susceptibility to a novel small
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molecule compound, Bis [1,6-a:5',6'-g] quinolizinium 8-methyl-salt with acetic acid (BQM)
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Clinical isolates consistently overexpressing MDR1 generally demonstrated a multidrug
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resistant phenotype to azoles including fluconazole as well as an Mdr1p specific substrate,
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cerulenin (14, 19, 27). However, we have observed that the MDR1 overexpressed strains,
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compared to drug susceptible isolates, exhibit highly increased susceptibility (~20 fold) to a class
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of isoquinoline derivatives (Figure S1). Among them, we identified BQM with the most potent
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antifungal activity (Figure S1). To validate if BQM is active against clinically drug resistant
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strains, we initially screened 47 isolates of C. albicans listed in Table S1, many of which have
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mutations in their ERG11 azole target gene or overexpress drug efflux transporters. As expected,
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isolates with ERG11 mutations or overexpression of transporter (drug efflux) genes, either
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CDR1/2 and/or MDR1, showed high levels of fluconazole resistance (Figure 1). These strains
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grew at 32 g/ml fluconazole or higher (Figure 1). Meanwhile, most isolates were susceptible to
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micafungin at 0.02-0.08 µg/ml. BQM had potent antifungal activity against most of the clinical
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isolates at a dosage of 3.2µg/ml. Interestingly, we found that many fluconazole-resistant C.
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albicans strains failed to grow even at 0.2µg/ml of BQM (Figure 1). Consistent with our
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observation, those strains that were inhibited strongly by BQM uniformly displayed fluconazole
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resistance associated with MDR1 overexpression. This data indicated that BQM may have an
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MDR1-overexpression-selective inhibitory property.
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The hypersusceptibility of these strains to BQM seems counterintuitive as CaMdr1p is a
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multidrug transporter that causes efflux of a variety of structurally unrelated compounds (14, 19).
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Conversely, our data indicate that efflux of BQM by CaMdr1p seemed unlikely since MDR
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strains were hypersusceptible to BQM. We hypothesized that enhanced antifungal activity
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resulted from increased intracellular accumulation of BQM in MDR1 overexpressed isolates. To
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test this hypothesis, we compared intracellular levels of BQM with its MIC50 values of all
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isolates described in Figure 1. After a 1 h incubation with 2 g/ml BQM, isolates that showed
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increased susceptibility to BQM had accumulated more intracellular BQM, suggesting a
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significant correlation (R2=0.8962) between BQM uptake and susceptibility (Figure 2).
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BQM activity is facilitated by MDR1 overexpression and regulated by the Mrr1p
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transcription factor
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We then determined if BQM activity and uptake were MDR1-dependent by comparing the
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drug susceptibility of a matched pair of fluconazole-susceptible (strain CaS) and fluconazole-
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resistant (CaMDR) clinical C. albicans isolates. CaS has minimal levels of MDR1 and was
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initially isolated from an HIV patient with oral candidiasis, while strain CaMDR was isolated
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from the same patient after a two-year treatment with fluconazole and overexpressed MDR1 (14,
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19, 27). CaMDR demonstrated a multidrug resistant phenotype. It was resistant to both
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fluconazole (Figure 3A) and cerulenin (Figure 3B) due to high levels of MDR1 expression.
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Deletion of MDR1 from CaMDR, strain Camdr , reversed the fluconazole and cerulenin
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resistant phenotypes (Figure 3A and 3B), while deletion of MRR1 (Camrr ), an activated
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transcription factor conferring MDR1 overexpression in CaMDR, lead to a greater fluconazole
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and cerulenin susceptibility (Figure 3A and 3B), consistent with previous findings that MDR1
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overexpression due to MRR1 gain of expression contributes to fluconazole resistance in CaMDR
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(14, 18). Both null mutants were susceptible to fluconazole, whereas resistance was partially
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recovered in the MDR1 reconstituted strain (19).
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In addition, MDR1 transporter activity
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correlated well with susceptibility to cerulenin, a Camdr1p specific substrate, as Camdr
and
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Camrr
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BQM susceptibility was the precise inverse. BQM showed significantly elevated activity against
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strains CaMDR and Camdr +MDR compared to CaS (Figure 3C).
completely lost their resistance to cerulenin (Figure 3B). Intriguingly, the pattern of
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To determine if the hypersusceptibility is associated with intracellular accumulation in these
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strains, the accumulation of BQM was evaluated in each strain over time. We observed that
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CaMDR and Camdr +MDR accumulated 70% more BQM than strains deleted of MDR1 or
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MRR1 (Figure 4). As CaMdr1p utilizes the proton gradient across the plasma membrane as
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energy for transport (28, 29), we also explored whether BQM uptake was driven by a proton
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gradient.
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chlorophenylhydrazone (CCCP) significantly reduced the accumulation of BQM by CaMDR
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(Figure S2), suggesting that the proton gradient played an important role in the uptake of BQM.
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Moreover, BQM accumulation was concentration-dependent in CaMDR (Figure S3).
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Collectively, while MDR transporters expel substrates like fluconazole and cerulenin, we found
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that BQM had greater activity against MDR C. albicans. The observation correlated with a much
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higher accumulation of BQM in strains that overexpress MDR1.
Exposure
to
20 g/ml
of
the
uncoupling
agent
carbonyl-cyanide-m-
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MRR1 regulation of MDR1 and polyamine transporters confer the hypersusceptibility to
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BQM
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It is noteworthy that in the preceding experiments, disruption of MDR1 did not completely
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restore BQM susceptibility to strain CaS (Figure 3C). However, strain Camrr was similarly
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susceptible as CaS to BQM (Figure 3C). Also, like CaS, Camrr accumulated 20% less BQM
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than Camdr (Figure 4), indicating that in addition to MDR1, MRR1 may regulate other genes
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that contribute to BQM susceptibility. Gain-of-function mutations in MRR1 are the major
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mechanism of MDR1 overexpression in fluconazole-resistant strains (14). Transcriptional
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profiling of in vivo DNA binding studies showed that a constitutively active Mrr1p binds to and
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upregulates numerous direct target genes in addition to MDR1 (14). Indeed, CaMDR had a
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higher level of MRR1 expression compared to CaS and Camrr
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expression of the activated allele of MRR1 is sufficient to confer resistance to fluconazole
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(Figure 6A) and cerulenin (Figure 6B), but resulted in hypersusceptibility to BQM (Figure 6C),
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indicating that Mrr1p plays a central role in BQM activity. Besides MDR1, other genes are
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regulated by Mrr1p (14, 30). Therefore, it is more than likely that mechanisms in addition to the
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MDR1 transporter are involved in the regulation of BQM activity.
(Figure 5). In addition, the
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To address the role of additional genes in BQM susceptibility, we compared CaS and CaMDR
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strains by microarray analysis. A total of 409 genes were downregulated in CaMDR compared to
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CaS (Figure 7 and Table S2), while upregulation of 452 genes was detected in CaMDR,
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including MDR1 and MRR1 (Figure 8 and Table S3) (cut-off of 2.0 fold, P-value <0.05 and
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FDR<0.2, n=3). Consistent with previous data (14, 30), a large number of genes with
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oxidoreductive functions (15%, GOID: 16491, P-value 1.17×10-10), such as IFD6, orf19.7306
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and CSH1 (all belonging to the aldo-keto reductase family), were upregulated together with
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MDR1. These genes are involved in the regulation of intracellular redox homeostasis and
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intracellular levels of reactive oxygen species (ROS). However, the precise function of these
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genes is currently unknown, and their potential involvement in an oxidative stress response
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remains speculative (14).
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Moreover, fifty genes in addition to MDR1 with transmembrane transporter activity were
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upregulated (11%, GOID: 22804, P-value 9.38 × 10-8) (Figure 8). Strikingly, among these
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transporters, amine/polyamine transmembrane transporters were highly enriched (Figure 8), the
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majority of which serve as importers for essential nutrients. Notably, many of the
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amine/polyamine transporters are regulated by Mrr1p (30).
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Polyamines (such as putrescine, spermidine, and spermine) are essential organic cations
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required for protein and nucleic acid synthesis and therefore cell growth (31). Fungal cells tightly
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regulate polyamine homeostasis with polyamine transport (both uptake and efflux) (31).
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However, much less is known about polyamine transporter proteins and their regulation in C.
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albicans compared to Saccharomyces cerevisiae, in which the TPO (Transporter of POlyamines)
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genes have important roles in detoxification and polyamine excretion (32). It is noteworthy that
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the four TPO genes belong to the Drug: H+ Antiporter-1 (12-transmembrane Spanner; DHA1)
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family of MFS (32). Remarkably, the CaMDR1 ortholog, ScFLR1 is structurally closely related
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to the TPO family members, TPO1-TPO4; therefore, caMDR1 and polyamine transporters are
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very likely related (32). In fact, ten of the S. cerevisiae DHA1 genes have C. albicans orthologs,
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including the TPO1-TPO4 (Table 1). Polyamine uptake is repressed by high intracellular levels
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of polyamines (31). Growth in the presence of spermidine led to reduced activity and uptake of
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BQM (Figure 9), implicating polyamine transporters in the uptake of BQM. In addition,
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spermine uptake in yeast is thought to be regulated by phosphorylation and dephosphorylation of
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serine/threonine protein kinases such as scPTK1 and scPTK2 (polyamine transport kinases 1 and
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2) (33, 34). We found that the C. albicans null mutant of PTK2 conferred increased resistance to
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BQM (Figure 10). However the ptk2∆ remained susceptible to fluconazole (data not shown).
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We anticipate that CaMDR1 will exhibit similar functional characteristics with TPO genes in
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polyamine transport in spite of the fact that substrate specificity varies. In fact, CaMDR was
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more resistant to spermine compared to Camdr (Figure S4), suggesting that CaMdr1p may act
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in the efflux of excess polyamines. In addition, the ortholog of MDR1 in Schizosaccharomyces
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pombe
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(http://www.pombase.org). Collectively, these results indicate that activated Mrr1p promotes
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expression of MDR1 and multiple polyamine transmembrane transporter genes, which together
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lead to hypersusceptibility of CaMDR to BQM. However, whether the CaMdr1p transports BQM
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directly remains to be determined. Additional BQM importers deserve further investigation.
(SPAC17C9.16c)
encodes
a
putative
polyamine
transport
protein
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BQM potently inhibits clinical fungal pathogens
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Although C. albicans is the leading cause of invasive candidiasis, infections caused by other
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Candida species have been increasing. These include C. glabrata, C. krusei, and C. parapsilosis,
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which are inherently more resistant to trizoles. Moreover, A. fumigatus is the most deadly and
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frequent mold infection of humans (1). We measured antifungal activity of BQM and its
286
derivatives on a range of yeast pathogens, including C. albicans, clinical isolates of C.
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guilliermondii, C. glabrata, C. tropicalis, C. parapsilosis, C. lusitaniae, C. apicola, C. krusei,
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and two C. neoformans isolates (Table 2). We determined that BQM showed potent activity
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against these common invasive fungal pathogens, including a C. krusei strain, which is highly
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resistant to fluconazole (Table 2). Markedly, these compounds strongly inhibited a number of
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itraconazole resistant A. fumigatus, the chief cause of invasive aspergillosis (IA). A. fumigatus is
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intrinsically resistant to fluconazole, while drug-resistant A. fumigatus strains are resistant to
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itraconazole MIC> 100 g/ml (Table 2). The therapeutic index of BQM activity against our
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panel of pathogens is about 130-215-fold more active than against mammalian cell lines,
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implying the mammalian cells may tolerate BQM. Included in our assays are related compounds
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that also were effective against pathogens but less so against mammalian cell lines. Furthermore,
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consistent with the in vitro data, BQM demonstrated a stronger protective effect against CaMDR
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infection than CaS (Figure S5) in the wax moth Galleria mellonella infection model (25, 35). A
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major translational implication of these data is applying BQM as a lead compound to develop
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antifungal agents in treatment of life-threatening fungal infections as drug resistance is common.
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Our study reveals a novel function of MDR1 in raising the susceptibility of drug-resistant
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fungal pathogens to some compounds, such as BQM. Thus, the drug resistance phenotype
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conferred by a gain-of-function mutation of the transcription factor MRR1 and therefore MDR1
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overexpression could be harnessed through the use of MDR1-facilitated cytotoxic agents like
305
BQM for effective antifungal strategies. The findings reported here may represent a novel
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strategy to overcome multidrug resistance, not only in fungal pathogens but also perhaps in
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bacterial pathogens or even human diseases such as drug resistant cancers.
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Discussion
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The emergence of multidrug resistance is a global problem that renders current drugs
311
ineffective, which is exacerbated by the shrinking pipeline of antimicrobial agents. Extensive
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studies on mechanisms of drug resistance indicate difficulties that are not easy to overcome
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therapeutically. Towards solving the current drug resistance problem, a straightforward option
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that could extend the usefulness of antimicrobials such as the triazoles is to develop compounds
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that reverse drug resistance. In fact, the search for inhibitors of MDR pumps has been suggested
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previously against bacterial and fungal pathogens and human cancers (36-39). Although we
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found BQM could exploit the drug transporter Mdr1p to increase accumulation, we do not know
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if efflux pumps are inhibited by BQM.
14
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An important point resides in the possibility that Mdr1p mediates the direct influx of BQM.
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ScTPO1, encoding a polyamine transporter of the DHA1 family catalyzes the uptake of
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polyamines at high pH and excretion at lower pH (40). It is possible that Mdr1p mediates bi-
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directional transport according to different substrates. However, crystal structure information and
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detailed functional studies of this transporter are needed to validate this idea. Several indirect
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effects of overexpression or deletion of CaMdr1p can be imagined such as co-expression of other
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genes regulated by Mrr1p, modification of membrane permeability, and induction of BQM-
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influx symporters. Transcriptional profiling of C. albicans indicated that transporter genes were
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upregulated in an MDR isolate. A closer look at these data revealed that upregulation also
328
included the family of polyamine transporters. Like CaMDR1, these proteins are fungal-specific
329
(41) and amendable to development of new drug targets. Our data provide a unique paradigm to
330
explore the function of distinct drug importers, such as the polyamine transporter, which has
331
been shown to be involved in histatin 5 uptake (31). Interestingly, our data demonstrate that
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while the ptk2∆ is resistant to BQM, it is susceptible to fluconazole. Compounds directed
333
against these transporters, like BQM should be identified in drug discovery approaches.
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A final important point resides in the mechanism of action of BQM. We observed that BQM
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interacted with mitochondria, and induced ROS production. We have also observed that
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fluorescent BQM co-accumulates in mitochondria with the mitochondrial stain, mitotracker (data
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not shown). We also observed that several mitochondrial mutants, such as the goa1 and ndh51
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null mutants (22, 42, 43) showed increased resistance to BQM compared to the wild type control
339
(data not shown). The role of mitochondria in fluconazole susceptibility among Candida spp has
340
been discussed (22, 44). Mitochondrial mutants of Candida glabrata are either resistant or have
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increased susceptibility to azoles. Thus, it appears that specific mitochondrial mutations
15
342
determine the levels of resistance/susceptibility. The requirements of several mitochondrial
343
proteins in growth, morphogenesis and virulence of C. albicans suggest a promising avenue for
344
further research on their exploitation as drug targets (23, 24, 42, 43, 45). Further mechanistic
345
studies on mitochondria should attract great interest.
346
347
348
349
Figure legends
350
Figure 1. Clinical isolates of Candida albicans overexpressing MDR1 exhibit highly
351
increased susceptibility to BQM. The activity of BQM is compared to fluconazole in 47
352
isolates of C. albicans, many with drug resistance phenotypes. The relative growth was
353
calculated by normalizing cultures to an OD595 after 24 h and compared to the DMSO only
354
control wells. Susceptibility profiles are indicated as color changes from no growth (black) to
355
growth (yellow) for each inhibitor (average of three independent experiments). MDR1
356
overexpressed isolates (in red rectangle) are hypersusceptible to BQM although resistant to
357
fluconazole. Right: strains are clustered according to their susceptibility, source, and/or
358
resistance mechanisms.
359
Figure 2. A scatter plot of intracellular BQM accumulation and MIC50 values of 47 clinical
360
isolates. The x axis represents the MIC50 values of each isolate in g/ml, and the y axis indicates
361
relative accumulation of BQM (measured by its fluorescence) normalized with CaMDR (average
362
of three independent experiments). The MDR1 overexpressing strains are indicated in red. Each
363
point in the scatter plot represents one isolate. R square represents the Pearson correlation of
364
MIC50 and accumulation.
16
365
Figure 3. The susceptibility to BQM and its accumulation in MDR1 overexpressing C.
366
albicans strains is partially MDR1 dependent and regulated by MRR1. (A). MDR1
367
overexpression confers fluconazole resistance. Relative growth of strains CaMDR (MDR1
368
overexpression), and Camdr +MDR (reconstituted from Camdr
369
resistant to fluconazole, while CaS, Camdr
370
(the regulator of MDR1, MRR1 null derived from CaMDR) are more susceptibile to fluconazole.
371
Relative growth is calculated by normalizing cultures to an OD595 after 24 h and compared to the
372
no drug control wells (Mean ± s.d. of three independent experiments). (B) Strain CaMDR and to
373
a lesser extent, Camdr +MDR, are resistant to cerulenin compared to CaS, Camdr , and
374
Camrr . All strains were grown in the presence of varying concentrations of cerulenin and
375
growth recorded as a % of control cultures (mean ± s.d., n=3). (C) Fluconazole-resistant strains
376
are, conversely, hypersusceptible to BQM. Data are presented as the percentage of growth
377
compared with untreated cells (mean ± s.d. of three independent experiments).
378
Figure 4. Increased accumulation of BQM in the MDR1 overexpressing strain CaMDR is
379
abolished in the MRR1 knockout, a null strain lacking the MRR1 gain-of-function allele that is a
380
known regulator of MDR1. Cell samples were removed at 0, 15, 30, 45, and 60 min and each was
381
normalized to an equivalent number of CaMDR cells at 60 min. The value of CaMDR at 60 min
382
was designated as 100%. Mean values from three independent experiments are shown. Error bars
383
indicate standard deviation.
384
Figure 5. Relative expression levels of MRR1 by qRT-PCR measurements (Mean ± s.d. of three
385
independent experiments). N.D., not detected.
386
Figure 6. (A) MRR1gain-of-function confers fluconazole resistance. The strain (mrr1+MRR1*)
387
contains the constitutively activated MRR1 (G997V) and is resistant to fluconazole, while the
with MDR1), which are
(MDR1 null derived from CaMDR), and Camrr
17
388
wild type (MRR1) and mrr1
are susceptible to fluconazole. (B) A Gain-of-function in MRR1
389
confers cerulenin resistance. Data are presented as the percentage growth of strains. Strain
390
mrr1 +MRR* (containing a mutated and overexpressed MRR1) is resistant to cerulenin
391
compared to the mrr1 and a wild type strain containing the non-mutated MRR1 (mean ± s.d. of
392
three independent experiments).
393
BQM. Data are presented as the percentage of growth compared with untreated cells (mean ± s.d.
394
of three independent experiments).
395
Figure 7. Microarray analysis of CaMDR/CaS. Data are presented as a pie chart of functional
396
gene categories (Gene Ontology Term analysis) of downregulated genes in CaMDR compared to
397
CaS. A total of 409 genes were downregulated, defined by a minimum 2-fold decrease of gene
398
expression (cut-off of 2.0 fold, P-value <0.05 and FDR<0.2 of three independent experiments).
399
Figure 8. 452 upregulated genes in CaMDR presented as a pie chart including transmembrane
400
transporters (11%), of which, 5% are polyamine transporters.
401
Figure 9. Spermidine (2mM), a substrate for polyamine transporters reduces BQM accumulation
402
and leads to reduced activity. Spermidine increases the resistance of C. albicans SC5314 to
403
BQM presented as the percentage of growth (left) and reduces the accumulation of BQM (right).
404
Data represent the mean ± s.d. of three independent experiments.
405
Figure 10. The ptk2 null mutant is resistant to BQM, compared to wild type C. albicans SC5314.
406
Relative growth and accumulation shows the PTK2 mutant (PTK2 ) is resistant to BQM (mean
407
± s.d., n=3). Lower panel, drop plate assays (ten-fold serial dilutions of each strain). A
408
representative graph of three independent experiments is shown.
(C) MRR1 gain-of-function confers hypersusceptibility to
409
410
18
411
Table 1. The polyamine transporter family of S. cerevisiae (left) and C. albicans. Orthologs of
412
the latter species are indicated.
S. cerevisiae
C.albicans orthologs
Systematic Name
Standard Name
YBR008C
FLR1
YBR043C
QDR3
orf19.136
YBR180W
DTR1
orf19.553
YHR048W
YHK8
NONE
YIL120W
QDR1
orf19.6992, orf19.508 (QDR1)
YIL121W
QDR2
NONE
YGR138C
TPO2
YLL028W
TPO1
orf19.7148 (TPO2), orf19.341, orf19.6577 (FLU1)
YNL065W
AQR1
orf19.6992, orf19.508 (QDR1)
YNR055C
HOL1
orf19.2517, orf19.1582, orf19.2991 (HOL1), orf19.4889
YOR273C
TPO4
orf19.473 (TPO4)
YPR156C
TPO3
orf19.4737(TPO3), orf19.7148(TPO2)
orf19.5604 (MDR1)
orf19.4737 (TPO3), orf19.6577
413
414
415
416
417
418
419
19
420
Table 2. BQM is active against various fungal pathogens with low toxicity in human cells. MIC
421
values of indicated compounds are shown against a variety of fungal pathogens, including
422
itraconazole-resistant A. fumigatus (lower part of the table). Toxicity is evaluated with three
423
human cells lines. FLC, fluconazole; ITC, itraconazole.
Species
FLC
ITC
403831 BQM
156624 157108 160459
MIC ( g/ml)
C.albicans (SC5314)
0.25
1
0.4
2
1
4
C. guilliermondii
2
1
0.4
2
2
2
C. glabrata
2
0.25
<0.2
1
0.25
0.5
C. tropicalis
0.5
1
0.4
2
2
2
C. parapsilosis
1
2
0.8
4
2
4
C. lusitaniae
2
0.5
<0.2
1
0.5
1
C. apicola
0.25
1
0.4
2
1
2
C.krusei
32
2
0.4
2
2
2
C. neoformans (H99)
4
0.5
0.8
4
1
4
C. neoformans (JEC-21)
2
0.5
<0.2
1
0.25
0.5
A.fumigatus (H11-20)
0.5
1
0.2
2
1
2
A.fumigatus (AF293)
0.5
1
0.2
2
1
2
MDR A.fumigatus RIT2
>100
1
0.2
2
1
2
>100
1
0.2
2
1
2
>100
1
0.2
2
1
2
>100
1
0.2
2
1
2
>100
1
0.2
2
1
2
>100
1
0.2
2
1
2
>100
1
0.2
2
1
2
HepG2 liver cell
80
26
75
64
52
NIH/3T3 Fibroblast cell
96
43
>100
90
80
293T kidney cell
80
24
75
70
60
424
20
425
Acknowlegements
426
We thank Stephen White and the Developmental Therapeutics Program (DTP) of NIH/NCI for
427
providing the research compounds. The authors wish to thank Joachim Morschauser, Theodore
428
White, Dominique Sanglard, Patrice LePape, David Perlin and the Fungal Genetics Stock Center
429
for providing the C. albicans and A. fumigatus strains. We thank David Goerlitz, of the
430
Microscopy & Imaging and the flow cytometry facility at the Georgetown University Lombardi
431
Cancer Center for technical help. This research was supported in part by bridge funds provided
432
by the Georgetown University Biomedical Graduate Organization (BGRO). N.S received a
433
Georgetown University Medical Center (GUMC) Graduate Student Organization (MCGSO)
434
grant to support this research.
435
436
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572
23
Figure 1
BQ M
Figure 1. C linical isolates of C andida albicans overexpressing M D R 1
exhibit highly increased susceptibility to B Q M . The activity of BQM is
compared to fluconazole in 47 isolates of C . albicans, many with drug
resistance phenotypes. The relative growth was calculated by normalizing
cultures to an OD595 after 24 h and compared to the DMSO only control wells.
Susceptibility profiles are indicated as color changes from no growth (black) to
growth (yellow) for each inhibitor (average of three independent experiments).
M D R 1 overexpressed isolates (in red rectangle) are hypersusceptible to BQM
although resistant to fluconazole. Right: strains are clustered according to their
susceptibility, source, and/or resistance mechanisms.
R elative accum ulation
ofBQ M (% )
Figure 2
100
R 2= 0.8962
50
0
0.1
1
BQ M M IC 50 (g/m l)
10
Figure 2. A scatter plot of intracellular BQM accumulation and MIC50 values of
47 clinical isolates. The x axis represents the MIC50 values of each isolate in
g/ml, and the y axis indicates relative accumulation of BQM (measured by its
fluorescence) normalized with CaMDR (average of three independent
experiments). The M D R 1 overexpressing strains are indicated in triangles. Each
point in the scatter plot represents one isolate. R square represents the Pearson
correlation of MIC50 and accumulation.
Figure 3A
R elative grow th (% ofC ontrol)
Fluconazole
C aS
C aM D R
C am dr
100
C am dr+M D R
C am rr
50
0
0.1
1
10
100
1000 ( g/m l)
Figure 3. The susceptibility to BQM and its accumulation in MDR1 overexpressing C.
albicans strains is partially MDR1 dependent and regulated by MRR1. (A). MDR1
overexpression confers fluconazole resistance. Relative growth of strains CaMDR (MDR1
overexpression), and C
MDR (reconstituted from C
resistant to fluconazole, while CaS, C
C
with MDR1), which are
(MDR1 null derived from CaMDR), and
(the regulator of MDR1, MRR1 null derived from CaMDR) are more susceptibile
to fluconazole. Relative growth is calculated by normalizing cultures to an OD595 after 24
h and compared to the no drug control wells (Mean ± s.d. of three independent
experiments).
Figure 3B
C erulenin
C aS
C aM D R
100
C am dr
C am dr+M D R
C am rr
50
0
0.1
1
10
100
1000 ( g/m l)
(B) Strain CaMDR and to a lesser extent, C
compared to CaS, C
and C
MDR are resistant to cerulenin
. All strains were grown in the presence
of varying concentrations of cerulenin and growth recorded as a % of control
cultures (mean ± s.d., n=3).
Figure 3C
BQ M
C aS
100
C aM D R
C am dr
C am dr+M D R
C am rr
50
0
0.01
0.1
1
10 ( g/m l)
(C) Fluconazole-resistant strains are, conversely, hypersusceptible to BQM. Data
are presented as the percentage of growth compared with untreated cells (mean ±
s.d. of three independent experiments).
Figure 4
ofBQ M (% )
C aS
C aM D R
100
C am dr
C am dr+M D R
C am rr
50
0
0
15
30
45
Tim e (m in)
60
Figure 4. Increased accumulation of BQM in the M D R 1 overexpressing strain
CaMDR is abolished in the M R R 1 knockout, a null strain lacking the M R R 1
gain-of-function allele that is a known regulator of M D R 1. Cell samples were
removed at 0, 15, 30, 45, and 60 min and each was normalized to an
equivalent number of CaMDR cells at 60 min. The value of CaMDR at 60 min
was designated as 100%. Mean values from three independent experiments
are shown. Error bars indicate standard deviation.
C
am
am
aM
C
am
dr
+
M
C
C
R
rr

D
dr

R
aS
D
C
R elative expression
Figure 5
10
M RR1
5
N.D.
0
Figure 5. Relative expression levels of M R R 1 by qRT-PCR measurements
(Mean ± s.d. of three independent experiments). N.D., not detected.
Figure 6A
Fluconazole
M RR1
100
m rr1
m rr1+M R R 1*
50
0
0.1
1
10
100
1000 ( g/m l)
Figure 6. (A) M R R 1gain-of-function confers fluconazole resistance. The
strain (m rr1+M R R 1*) contains the constitutively activated M R R 1 (G997V)
and is resistant to fluconazole, while the wild type (M R R 1) and mrr1 are
susceptible to fluconazole.
Figure 6B
C erulenin
100
M RR1
m rr1
m rr1+M R R 1*
50
0
0.1
1
10
100
1000 ( g/m l)
(B) A Gain-of-function in M R R 1 confers cerulenin resistance. Data are
presented as the percentage growth of strains. Strain mrr1 +MRR* (containing
a mutated and overexpressed M R R 1) is resistant to cerulenin compared to the
mrr1 and a wild type strain containing the non-mutated M R R 1 (mean ± s.d. of
three independent experiments).
Figure 6C
BQ M
M RR1
100
m rr1
m rr1+M R R 1*
50
0
0.01
0.1
1
10 ( g/m l)
(C) M R R 1 gain-of-function confers hypersusceptibility to BQM. Data are
presented as the percentage of growth compared with untreated cells (mean ±
s.d. of three independent experiments).
Figure 7
Figure 7.Microarray analysis of CaMDR/CaS. Data are presented as a pie chart
of functional gene categories (Gene Ontology Term analysis) of downregulated
genes in CaMDR compared to CaS. A total of 409 genes were downregulated,
defined by a minimum 2-fold decrease of gene expression (cut-off of 2.0 fold, Pvalue <0.05 and FDR<0.2 of three independent experiments).
Figure 8
Figure 8. 452 upregulated genes in CaMDR presented as a pie chart
including transmembrane transporters (11%), of which, 5% are polyamine
transporters.
Figure 9
B Q MM
BBH
100
SC 5314
100
SC 531 
2m M sperm idine
50
0
0.01
ofBBH
B Q MM (% )
P<0.0001
50
0
0.1
1
10 ( g/m l)
SC 5314
SC 5314+
2m M sperm idine
Figure 9. Spermidine (2mM), a substrate for polyamine transporters reduces
BQM accumulation and leads to reduced activity. Spermidine increases the
resistance of C . albicans SC5314 to BQM presented as the percentage of
growth (left) and reduces the accumulation of BQM (right). Data represent the
mean ± s.d. of three independent experiments.
Figure 10
BBBH
Q MM
w ild type
100
PTK 2
50
0
0.01
0.1
10 ( g/m l)
1
100
ofBBH
B Q MM (% )
P<0.001
50
0
w ild type
C aM D R
PTK 2
YPD
B Q M 8 g/ml
w ild type
PTK 2
Figure 10. The ptk2 nullmutant is resistant to BQM, compared to wild type C .
albicans SC5314. Relative growth and accumulation shows the PTK2 mutant
(PTK2 ) is resistant to BQM (mean ± s.d., n=3). Lower panel, drop plate assays
(ten-fold serial dilutions of each strain). A representative graph of three
independent experiments is shown.

AAC Accepts, published online ahead of print on 16 September... Antimicrob. Agents Chemother. doi:10.1128/AAC.01504-13

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