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. 1 Multidrug Resistant Transporter Mdr1p Mediated Uptake of a Novel 2 Antifungal Compound 3 4 Affiliations: 5 1 Georgetown University Medical Center, Department of Microbiology & Immunology, 6 7 8 9 10 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. 11 12 By 13 Nuo Sun1, Dongmei Li1, William Fonzi1, Xin Li2, Lixin Zhang3, and Richard Calderone1* 14 *To whom correspondence should be addressed. E-mail: [email protected] 15 16 17 Running title: Overcoming MDR resistance in Candida albicans 18 Key words: Efflux, fluconazole, resistance, MDR, candidiasis 19 20 21 Abstract 22 The activity of many anti-infectious drugs has been compromised by the evolution of 23 multidrug resistant (MDR) pathogens. For life-threatening fungal infections, such as those 24 caused by Candida albicans, overexpression of MDR1, which encodes an MDR efflux pump of 25 the major facilitator superfamily (MFS), often confers resistance to chemically unrelated 26 substances, including the most commonly used azole antifungals. As the development of new 27 and efficacious antifungals has lagged far behind the growing emergence of resistant strains, it is 28 imperative to develop strategies to overcome multidrug resistance. Previous advances have been 29 made mainly to deploy combinational therapy to restore azole susceptibility, which, however, 30 requires coordination of two or more compounds. We observed a unique phenotype in which 31 Mdr1p facilitates the uptake of a specific class of compounds. Among them, we describe a novel 32 antifungal small molecule, Bis [1,6-a:5',6'-g] quinolizinium 8-methyl-salt (BQM) (US Patent 33 Application Serial No.61/793,090,2013) that has potent and broad antifungal activity. Notably, 34 BQM exploits the MDR phenotype in C. albicans to promote the inhibitory effect. Rather than 35 causing an antagonism of MDR strains, it exhibits a highly potentiated activity against a 36 collection of clinical isolates and lab strains that overexpress MDR1. The activity of BQM 37 against MDR1 overexpressed isolates is due to its facilitated intracellular accumulation. 38 Microarray comparisons showed an extensive upregulation of MDR1 as well as polyamine 39 transporters in a fluconazole resistant strain. We then demonstrated that the polyamine 40 transporters augment the accumulation of BQM. Importantly, BQM had greater activity than 41 fluconazole and itraconazole against various fungal pathogens, including MDR A. fumigatus. 42 Thus, our findings offer a paradigm shift to overcome MDR and the promise of improving 43 antifungal treatment, especially in MDR pathogens. 2 44 Introduction 45 Fungi that cause invasive infections are now referred to as the “hidden killers” (1). Mortality 46 due to these infections is similar to drug resistant TB and exceeds malaria (1). Of these 47 pathogens, Candida species have emerged among the top three causes of microbial nosocomial 48 infectious diseases in humans, resulting in 46-75% mortality, and A. fumigatus is the most deadly 49 and frequent mold infection of humans (1, 2). The incidence of candidiasis has increased sharply 50 over the past few decades primarily due to hospital interventions such as cancer chemotherapy, 51 surgery, organ/bone marrow transplantation, and indwelling devices (3). The cost in the US of 52 treating candidiasis infections is ~$2.0 billion per year (4, 5). However, current therapies have 53 led to only modest success in reducing the high mortality rates of invasive fungal infections, in 54 part due to drug toxicity (amphotericin B), a narrow spectrum of activity (echinocandins), or, in 55 the case of the fungistatic triazoles, the selection of CDR1,2, MDR1, or ERG11 over expression 56 or ERG11 mutations in isolates caused by an overdependence of these therapies (6-8). 57 Reduced intracellular accumulation of drugs by genes encoding drug transporters is a 58 prominent mechanism of resistance in Candida strains. Drug transporters, such as the ABC 59 (ATP-binding cassette) transporters, CDR1, CDR2 (Candida Drug Resistance) and an MFS 60 transporter, MDR1 (Multi-Drug Resistance), play key roles in azole resistance as deduced by 61 their high level of expression in the majority of azole-resistant clinical C. albicans isolates (9- 62 11). While ABC transporters depend on ATP as an energy source, MDR1, encoding Mdr1p, is a 63 member of the MFS transporters that use the proton gradient across the cytoplasmic membrane 64 to supply energy for transport (12-14). Mdr1p exports a variety of structurally unrelated 65 compounds, such as fluconazole, benomyl, cerulenin, and brefeldin A (9, 14-16). In drug- 66 susceptible C. albicans strains, MDR1 is expressed at low levels. However, clinical isolates 3 67 consistently overexpressed MDR1 when the patient was continuously treated with fluconazole (8, 68 9, 17). Deletion of MDR1 in these resistant isolates reversed the drug resistance phenotype (18, 69 19). Moreover, engineered overexpression of MDR1 increased resistance (19, 20). In C. albicans, 70 MDR1 overexpression results mainly from gain-of-function mutations in the transcription factor 71 Mrr1p (14, 21). 72 The development of new antifungal therapeutics is crucial. Unfortunately, the antifungal 73 pipeline has slowed down considerably. Most new triazoles are remodeled older versions that do 74 not address the emergence of resistance. With an already narrow list of antifungal drugs, 75 alternate choices become problematic if resistance to triazoles develops during therapy. We 76 believe there is a great need for paradigm shift to exploit the resistant mechanisms for more 77 efficient therapy. In part, our data address this need. We hypothesized that the MDR phenotype 78 conferred by caMDR1 overexpression could be harnessed through the use of MDR1-dependent 79 cytotoxic agents for effective antifungal strategies. 80 81 Materials and Methods 82 83 Strains, strain maintenance, and plasmids 84 All strains used in the present study are listed in Table S1 and were maintained as frozen stocks 85 in 96-well plates and propagated on yeast extract-peptone-dextrose (YPD) agar when needed 86 (1% yeast extract, 2% peptone, 2% glucose, 2% agar). 87 Antifungal susceptibility testing 88 BQM (NSC156627) and all other NSC compounds were provided by the Developmental 89 Therapeutics Program of NIH/NCI. Drug susceptibility testing was carried out in flat bottom, 96- 4 90 well microtiter plates (Greiner Bio One) using the broth microdilution protocol according to the 91 Clinical and Laboratory Standards Institute M-27A methods. Growth inhibition of all strains was 92 evaluated in the presence of drugs and reported as a % inhibition of untreated cells as previously 93 described (22). In brief, overnight cultures were prepared in YPD, washed and ~103 cells/100µl 94 were inoculated into microtiter wells. Growth was evaluated by measuring cell density OD595 95 after 24h of incubation at 30°C. Experiments were repeated at least three times. Data were 96 averaged and statistical significance among treatments determined. MIC determinations were 97 also done with lab stock cultures of azole susceptible and resistant strains. For these experiments, 98 the compounds were tested at the indicated concentrations. Strains were grown as described 99 above. Relative growth was calculated based on OD595 data and visualized using a heatmap. 100 Drop plate assays 101 Growth inhibition was visualized by plating 5 µl of ten-fold serial dilutions of cells onto YPD 102 agar plates containing BQM at the indicated concentrations. Cells were grown overnight in YPD 103 broth at 30°C, washed with saline, and standardized by hemocytometer counts. Plates were 104 photographed and evaluated after 48 h of incubation at 30°C. 105 Accumulation assays of BQM 106 All C. albicans strains were grown at 30°C overnight in YPD medium and washed twice with 107 phosphate-buffered saline (PBS) (pH 7.0). Cells were diluted with RPMI 1640 medium (2% 108 glucose without bicarbonate and buffered with 0.165 M MOPS to pH 7.0) to 108 cells/ml, as 109 determined by hemocytometer and confirmed by plate count. BQM was added at the 110 concentrations indicated in Tables or Figures. 1-ml of each sample was removed after incubation 111 at 200 rpm (30°C) at 0, 15, 30, 45 and 60 min, centrifuged, washed, and suspended in 1ml PBS. 112 200 l of cell suspensions were transferred into 96-well microplates with clear bottoms (Greiner, 5 113 Germany) to measure fluorescence intensity of BQM. To measure requirement for proton 114 gradients, one set of cell suspensions was treated with 20 g/ml CCCP or not treated for 30min 115 before introducing BQM. 116 RNA preparation and microarray 117 All C. albicans strains were grown at 30°C overnight in YPD medium and washed twice with 118 PBS (pH 7.0). Cells were suspended with RPMI 1640 medium. As previously described (23), 119 total RNA was extracted and, the integrity and purity of total RNA were assessed using an 120 Agilent Bioanalyzer (Aglient Technologies) and OD260/280. One-color microarray-based gene 121 expression analysis was done using the Agilent Low Input Quick Amp Labeling kit. C. albicans 122 cDNA synthesis was carried out using 100 ng total RNA, and all other methods were carried out 123 using the manufacturer’s instructions. Hybridization was carried out for 17 h in an Agilent 124 SureHyb hybridization chamber and the microarrays were scanned with an Agilent SCAN 125 Microarray Scanner System using the AgilentHD_GX_1-color 5 M protocol. The microarrays 126 used in this study were designed from assembly 21 of the C. albicans genome using eArray from 127 Agilent Technologies (design ID 017942). A total of 6101 genes (including 12 mitochondrial 128 genes) are represented by two sets of probes, both spotted in duplicate. Probes are randomly 129 distributed. Eight copies of each array were printed on a single slide (8×15,000) and hybridized 130 individually. For each microarray analysis, three independent biological replicates are 131 performed. Tiff format image files were analyzed by Agilent Feature Extraction software. 132 Cyanine 3 intensities were then logarithmically transformed and statistically normalized. BRB 133 Array-Tools was then used for the differential test. In this analysis, we adopted the cutoff for the 134 parametric p-value <0.05 and [fold change] > 2 to determine the significant gene lists, and using 135 FDR as a reference. Genes that were up- or downregulated 2.0 fold were selected and considered 6 136 to be differentially expressed. Gene ontology analysis was performed at the Candida genome 137 database (CGD, www.candidagenome.org). 138 Quantitative real-time PCR 139 We used previously published methods for these experiments (23, 24). Approximately 1 µg of 140 the total RNA was subjected to first-strand cDNA synthesis (QuantiTect Reverse Transcription, 141 Qiagen). Real-time PCR assays were performed with 20-µl reaction volumes that contained 1x 142 iQ SyBR green Supermix (Bio-Rad), including a 0.2 µM concentration of each primer and 8 µl 143 of a 1:8 dilution of each cDNA from each strain. The transcription level of each gene was 144 normalized to 18S rRNA levels. Data are presented as the means ± standard deviations (SD) 145 from three biological replicates. The 2– 146 was used to determine the fold change in gene transcription. The primers used for real-time PCR 147 expression analysis were the following: for 18S rRNA, CGCAAGGCTGAAACTTAAAGG 148 (forward) and AGCAGACAAATCACTCCACC (reverse); For MRR1, ACA CCC AGG GCT 149 AGT ATA GAC (forward) and ACG ACA TCT CCA GAA ACA GAC (reverse). 150 Evaluation of BQM in G. mellonella assays 151 Injection of fungal pathogens and antifungal drugs was performed as described (25, 26). In brief, 152 larvae were obtained from Vanderhorst, Inc.. Sixteen larvae (330±20 mg) were used per group. 153 Each injection of 10 l of C. albicans cells, BQM, or control was performed via a distinct proleg. 154 Larvae were incubated at 37°C, and the number of dead larvae was scored daily. C. albicans 155 inoculum was prepared from overnight cultures grown in YPD. Cells were washed three times in 156 PBS. Cell densities were determined by hemocytometer. Kill curves were plotted and analyzed 157 by the Kaplan-Meier method (GraphPad Prism). CT (where CT is the threshold cycle) method of analysis 158 7 159 Results 160 C. albicans overexpressing MDR1 exhibit highly increased susceptibility to a novel small 161 molecule compound, Bis [1,6-a:5',6'-g] quinolizinium 8-methyl-salt with acetic acid (BQM) 162 Clinical isolates consistently overexpressing MDR1 generally demonstrated a multidrug 163 resistant phenotype to azoles including fluconazole as well as an Mdr1p specific substrate, 164 cerulenin (14, 19, 27). However, we have observed that the MDR1 overexpressed strains, 165 compared to drug susceptible isolates, exhibit highly increased susceptibility (~20 fold) to a class 166 of isoquinoline derivatives (Figure S1). Among them, we identified BQM with the most potent 167 antifungal activity (Figure S1). To validate if BQM is active against clinically drug resistant 168 strains, we initially screened 47 isolates of C. albicans listed in Table S1, many of which have 169 mutations in their ERG11 azole target gene or overexpress drug efflux transporters. As expected, 170 isolates with ERG11 mutations or overexpression of transporter (drug efflux) genes, either 171 CDR1/2 and/or MDR1, showed high levels of fluconazole resistance (Figure 1). These strains 172 grew at 32 g/ml fluconazole or higher (Figure 1). Meanwhile, most isolates were susceptible to 173 micafungin at 0.02-0.08 µg/ml. BQM had potent antifungal activity against most of the clinical 174 isolates at a dosage of 3.2µg/ml. Interestingly, we found that many fluconazole-resistant C. 175 albicans strains failed to grow even at 0.2µg/ml of BQM (Figure 1). Consistent with our 176 observation, those strains that were inhibited strongly by BQM uniformly displayed fluconazole 177 resistance associated with MDR1 overexpression. This data indicated that BQM may have an 178 MDR1-overexpression-selective inhibitory property. 179 The hypersusceptibility of these strains to BQM seems counterintuitive as CaMdr1p is a 180 multidrug transporter that causes efflux of a variety of structurally unrelated compounds (14, 19). 181 Conversely, our data indicate that efflux of BQM by CaMdr1p seemed unlikely since MDR 8 182 strains were hypersusceptible to BQM. We hypothesized that enhanced antifungal activity 183 resulted from increased intracellular accumulation of BQM in MDR1 overexpressed isolates. To 184 test this hypothesis, we compared intracellular levels of BQM with its MIC50 values of all 185 isolates described in Figure 1. After a 1 h incubation with 2 g/ml BQM, isolates that showed 186 increased susceptibility to BQM had accumulated more intracellular BQM, suggesting a 187 significant correlation (R2=0.8962) between BQM uptake and susceptibility (Figure 2). 188 189 BQM activity is facilitated by MDR1 overexpression and regulated by the Mrr1p 190 transcription factor 191 We then determined if BQM activity and uptake were MDR1-dependent by comparing the 192 drug susceptibility of a matched pair of fluconazole-susceptible (strain CaS) and fluconazole- 193 resistant (CaMDR) clinical C. albicans isolates. CaS has minimal levels of MDR1 and was 194 initially isolated from an HIV patient with oral candidiasis, while strain CaMDR was isolated 195 from the same patient after a two-year treatment with fluconazole and overexpressed MDR1 (14, 196 19, 27). CaMDR demonstrated a multidrug resistant phenotype. It was resistant to both 197 fluconazole (Figure 3A) and cerulenin (Figure 3B) due to high levels of MDR1 expression. 198 Deletion of MDR1 from CaMDR, strain Camdr , reversed the fluconazole and cerulenin 199 resistant phenotypes (Figure 3A and 3B), while deletion of MRR1 (Camrr ), an activated 200 transcription factor conferring MDR1 overexpression in CaMDR, lead to a greater fluconazole 201 and cerulenin susceptibility (Figure 3A and 3B), consistent with previous findings that MDR1 202 overexpression due to MRR1 gain of expression contributes to fluconazole resistance in CaMDR 203 (14, 18). Both null mutants were susceptible to fluconazole, whereas resistance was partially 204 recovered in the MDR1 reconstituted strain (19). 9 In addition, MDR1 transporter activity 205 correlated well with susceptibility to cerulenin, a Camdr1p specific substrate, as Camdr and 206 Camrr 207 BQM susceptibility was the precise inverse. BQM showed significantly elevated activity against 208 strains CaMDR and Camdr +MDR compared to CaS (Figure 3C). completely lost their resistance to cerulenin (Figure 3B). Intriguingly, the pattern of 209 To determine if the hypersusceptibility is associated with intracellular accumulation in these 210 strains, the accumulation of BQM was evaluated in each strain over time. We observed that 211 CaMDR and Camdr +MDR accumulated 70% more BQM than strains deleted of MDR1 or 212 MRR1 (Figure 4). As CaMdr1p utilizes the proton gradient across the plasma membrane as 213 energy for transport (28, 29), we also explored whether BQM uptake was driven by a proton 214 gradient. 215 chlorophenylhydrazone (CCCP) significantly reduced the accumulation of BQM by CaMDR 216 (Figure S2), suggesting that the proton gradient played an important role in the uptake of BQM. 217 Moreover, BQM accumulation was concentration-dependent in CaMDR (Figure S3). 218 Collectively, while MDR transporters expel substrates like fluconazole and cerulenin, we found 219 that BQM had greater activity against MDR C. albicans. The observation correlated with a much 220 higher accumulation of BQM in strains that overexpress MDR1. Exposure to 20 g/ml of the uncoupling agent carbonyl-cyanide-m- 221 222 MRR1 regulation of MDR1 and polyamine transporters confer the hypersusceptibility to 223 BQM 224 It is noteworthy that in the preceding experiments, disruption of MDR1 did not completely 225 restore BQM susceptibility to strain CaS (Figure 3C). However, strain Camrr was similarly 226 susceptible as CaS to BQM (Figure 3C). Also, like CaS, Camrr accumulated 20% less BQM 227 than Camdr (Figure 4), indicating that in addition to MDR1, MRR1 may regulate other genes 10 228 that contribute to BQM susceptibility. Gain-of-function mutations in MRR1 are the major 229 mechanism of MDR1 overexpression in fluconazole-resistant strains (14). Transcriptional 230 profiling of in vivo DNA binding studies showed that a constitutively active Mrr1p binds to and 231 upregulates numerous direct target genes in addition to MDR1 (14). Indeed, CaMDR had a 232 higher level of MRR1 expression compared to CaS and Camrr 233 expression of the activated allele of MRR1 is sufficient to confer resistance to fluconazole 234 (Figure 6A) and cerulenin (Figure 6B), but resulted in hypersusceptibility to BQM (Figure 6C), 235 indicating that Mrr1p plays a central role in BQM activity. Besides MDR1, other genes are 236 regulated by Mrr1p (14, 30). Therefore, it is more than likely that mechanisms in addition to the 237 MDR1 transporter are involved in the regulation of BQM activity. (Figure 5). In addition, the 238 To address the role of additional genes in BQM susceptibility, we compared CaS and CaMDR 239 strains by microarray analysis. A total of 409 genes were downregulated in CaMDR compared to 240 CaS (Figure 7 and Table S2), while upregulation of 452 genes was detected in CaMDR, 241 including MDR1 and MRR1 (Figure 8 and Table S3) (cut-off of 2.0 fold, P-value <0.05 and 242 FDR<0.2, n=3). Consistent with previous data (14, 30), a large number of genes with 243 oxidoreductive functions (15%, GOID: 16491, P-value 1.17×10-10), such as IFD6, orf19.7306 244 and CSH1 (all belonging to the aldo-keto reductase family), were upregulated together with 245 MDR1. These genes are involved in the regulation of intracellular redox homeostasis and 246 intracellular levels of reactive oxygen species (ROS). However, the precise function of these 247 genes is currently unknown, and their potential involvement in an oxidative stress response 248 remains speculative (14). 249 Moreover, fifty genes in addition to MDR1 with transmembrane transporter activity were 250 upregulated (11%, GOID: 22804, P-value 9.38 × 10-8) (Figure 8). Strikingly, among these 11 251 transporters, amine/polyamine transmembrane transporters were highly enriched (Figure 8), the 252 majority of which serve as importers for essential nutrients. Notably, many of the 253 amine/polyamine transporters are regulated by Mrr1p (30). 254 Polyamines (such as putrescine, spermidine, and spermine) are essential organic cations 255 required for protein and nucleic acid synthesis and therefore cell growth (31). Fungal cells tightly 256 regulate polyamine homeostasis with polyamine transport (both uptake and efflux) (31). 257 However, much less is known about polyamine transporter proteins and their regulation in C. 258 albicans compared to Saccharomyces cerevisiae, in which the TPO (Transporter of POlyamines) 259 genes have important roles in detoxification and polyamine excretion (32). It is noteworthy that 260 the four TPO genes belong to the Drug: H+ Antiporter-1 (12-transmembrane Spanner; DHA1) 261 family of MFS (32). Remarkably, the CaMDR1 ortholog, ScFLR1 is structurally closely related 262 to the TPO family members, TPO1-TPO4; therefore, caMDR1 and polyamine transporters are 263 very likely related (32). In fact, ten of the S. cerevisiae DHA1 genes have C. albicans orthologs, 264 including the TPO1-TPO4 (Table 1). Polyamine uptake is repressed by high intracellular levels 265 of polyamines (31). Growth in the presence of spermidine led to reduced activity and uptake of 266 BQM (Figure 9), implicating polyamine transporters in the uptake of BQM. In addition, 267 spermine uptake in yeast is thought to be regulated by phosphorylation and dephosphorylation of 268 serine/threonine protein kinases such as scPTK1 and scPTK2 (polyamine transport kinases 1 and 269 2) (33, 34). We found that the C. albicans null mutant of PTK2 conferred increased resistance to 270 BQM (Figure 10). However the ptk2∆ remained susceptible to fluconazole (data not shown). 271 We anticipate that CaMDR1 will exhibit similar functional characteristics with TPO genes in 272 polyamine transport in spite of the fact that substrate specificity varies. In fact, CaMDR was 273 more resistant to spermine compared to Camdr (Figure S4), suggesting that CaMdr1p may act 12 274 in the efflux of excess polyamines. In addition, the ortholog of MDR1 in Schizosaccharomyces 275 pombe 276 (http://www.pombase.org). Collectively, these results indicate that activated Mrr1p promotes 277 expression of MDR1 and multiple polyamine transmembrane transporter genes, which together 278 lead to hypersusceptibility of CaMDR to BQM. However, whether the CaMdr1p transports BQM 279 directly remains to be determined. Additional BQM importers deserve further investigation. (SPAC17C9.16c) encodes a putative polyamine transport protein 280 281 BQM potently inhibits clinical fungal pathogens 282 Although C. albicans is the leading cause of invasive candidiasis, infections caused by other 283 Candida species have been increasing. These include C. glabrata, C. krusei, and C. parapsilosis, 284 which are inherently more resistant to trizoles. Moreover, A. fumigatus is the most deadly and 285 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. 287 guilliermondii, C. glabrata, C. tropicalis, C. parapsilosis, C. lusitaniae, C. apicola, C. krusei, 288 and two C. neoformans isolates (Table 2). We determined that BQM showed potent activity 289 against these common invasive fungal pathogens, including a C. krusei strain, which is highly 290 resistant to fluconazole (Table 2). Markedly, these compounds strongly inhibited a number of 291 itraconazole resistant A. fumigatus, the chief cause of invasive aspergillosis (IA). A. fumigatus is 292 intrinsically resistant to fluconazole, while drug-resistant A. fumigatus strains are resistant to 293 itraconazole MIC> 100 g/ml (Table 2). The therapeutic index of BQM activity against our 294 panel of pathogens is about 130-215-fold more active than against mammalian cell lines, 295 implying the mammalian cells may tolerate BQM. Included in our assays are related compounds 296 that also were effective against pathogens but less so against mammalian cell lines. Furthermore, 13 297 consistent with the in vitro data, BQM demonstrated a stronger protective effect against CaMDR 298 infection than CaS (Figure S5) in the wax moth Galleria mellonella infection model (25, 35). A 299 major translational implication of these data is applying BQM as a lead compound to develop 300 antifungal agents in treatment of life-threatening fungal infections as drug resistance is common. 301 Our study reveals a novel function of MDR1 in raising the susceptibility of drug-resistant 302 fungal pathogens to some compounds, such as BQM. Thus, the drug resistance phenotype 303 conferred by a gain-of-function mutation of the transcription factor MRR1 and therefore MDR1 304 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 306 strategy to overcome multidrug resistance, not only in fungal pathogens but also perhaps in 307 bacterial pathogens or even human diseases such as drug resistant cancers. 308 309 Discussion 310 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 312 studies on mechanisms of drug resistance indicate difficulties that are not easy to overcome 313 therapeutically. Towards solving the current drug resistance problem, a straightforward option 314 that could extend the usefulness of antimicrobials such as the triazoles is to develop compounds 315 that reverse drug resistance. In fact, the search for inhibitors of MDR pumps has been suggested 316 previously against bacterial and fungal pathogens and human cancers (36-39). Although we 317 found BQM could exploit the drug transporter Mdr1p to increase accumulation, we do not know 318 if efflux pumps are inhibited by BQM. 14 319 An important point resides in the possibility that Mdr1p mediates the direct influx of BQM. 320 ScTPO1, encoding a polyamine transporter of the DHA1 family catalyzes the uptake of 321 polyamines at high pH and excretion at lower pH (40). It is possible that Mdr1p mediates bi- 322 directional transport according to different substrates. However, crystal structure information and 323 detailed functional studies of this transporter are needed to validate this idea. Several indirect 324 effects of overexpression or deletion of CaMdr1p can be imagined such as co-expression of other 325 genes regulated by Mrr1p, modification of membrane permeability, and induction of BQM- 326 influx symporters. Transcriptional profiling of C. albicans indicated that transporter genes were 327 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 332 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. 334 A final important point resides in the mechanism of action of BQM. We observed that BQM 335 interacted with mitochondria, and induced ROS production. We have also observed that 336 fluorescent BQM co-accumulates in mitochondria with the mitochondrial stain, mitotracker (data 337 not shown). We also observed that several mitochondrial mutants, such as the goa1 and ndh51 338 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 341 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 MDR A.fumigatus RIT3 >100 1 0.2 2 1 2 MDR A.fumigatus RIT5 >100 1 0.2 2 1 2 MDR A.fumigatus RIT8 >100 1 0.2 2 1 2 MDR A.fumigatus RIT10 >100 1 0.2 2 1 2 MDR A.fumigatus RIT11 >100 1 0.2 2 1 2 MDR A.fumigatus RIT14 >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. 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Antimicrob Agents Chemother 54:1476-1483. Richards TS, Oliver BG, White TC. 2008. Micafungin activity against Candida albicans with diverse azole resistance phenotypes. J Antimicrob Chemother 62:349-355. 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). R elative grow th (% ofC ontrol) 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). R elative grow th (% ofC ontrol) 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 R elative accum ulation 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. R elative grow th (% ofC ontrol) 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. R elative grow th (% ofC ontrol) 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). R elative grow th (% ofC ontrol) 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 R elative accum ulation ofBBH B Q MM (% ) R elative grow th (% ofC ontrol) 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 R elative accum ulation ofBBH B Q MM (% ) R elative grow th (% ofC ontrol) 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.
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