The atypical antipsychotic aripiprazole alters the outcome of disseminated Candida albicans infections

Invasive fungal infections (IFIs) impose an enormous clinical, social, and economic burden on humankind. For many IFIs, ≥ 30% of patients fail therapy with existing antifungal drugs, including the widely used azole class. We previously identified a collection of 13 approved medications that antagonize azole activity. While gain-of-function mutants resulting in antifungal resistance are often associated with reduced fitness and virulence, it is currently unknown how exposure to azole antagonistic drugs impact C. albicans physiology, fitness, or virulence. In this study, we examined how exposure to azole antagonists affected C. albicans phenotype and capacity to cause disease. We discovered that most of the azole antagonists had little impact on fungal growth, morphology, stress tolerance, or gene transcription. However, aripiprazole had a modest impact on C. albicans hyphal growth and increased cell wall chitin content. It also worsened the outcome of disseminated infections in mice at human equivalent concentrations. This effect was abrogated in immunosuppressed mice, indicating an additional impact of aripiprazole on host immunity. Collectively, these data provide proof-of-principle that unanticipated drug-fungus interactions have the potential to influence the incidence and outcomes of invasive fungal disease.

I nvasive fungal infections (IFIs) impose an enormous medical burden on humankind, causing an estimated 1.5 million deaths each year (1).Candida albicans is the most prevalent cause of IFIs in the western world, causing approximately 60,000 dissemina ted infections annually in the United States alone, with associated costs estimated at $2 billion (2)(3)(4).Three primary classes of antifungal drugs are used to treat IFIs (5).The azoles inhibit synthesis of the membrane lipid ergosterol, the polyenes kill fungi through direct interactions with ergosterol that perturb membrane integrity (6), and the echinocandins disrupt cell wall synthesis through inhibition of β−1,3 glucan synthase (7).However, therapeutic failures occur with all three classes in >30% of the patients with disseminated Candida infections and are even more common with other fungal pathogens (e.g., Aspergillus and Cryptococcus).Although acquired and intrinsic antifun gal resistance may explain some treatment failures, most infectious isolates remain sensitive, with just 0.5%-2% of C. albicans isolates resistant to the azoles and even less to the echinocandins (8)(9)(10).A variety of patient-specific factors may contribute to poor outcomes, including the severity of their immune dysfunction (11).Delays in diagnosis or in the provision of an appropriate antifungal agent are also associated with worse outcomes (11).However, many patients fail therapy for unknown reasons.
As eukaryotes, fungi, and mammals share substantial similarities in their core metabolic and signaling modules (12)(13)(14)(15).As such, the physiology of fungi residing within the human body-as part of the endogenous microbiota or as invasive patho gens-may be inadvertently affected by the medications consumed by their host.Previously, we determined that a variety of drugs approved for human use can oppose the antifungal activity of fluconazole upon C. albicans in vitro (16).Seven of these antagonists were selected for further evaluation (Table 1), including the atypical antipsychotic aripiprazole (ARI), and found to elevate the minimum inhibitory concentra tion (MIC) of fluconazole by 8-fold > 256-fold.Furthermore, the antagonistic activity of six of these drugs was dependent on the Tac1p zinc-cluster transcription factor, which activates the expression of the ATP-driven Cdr1p and Cdr2p drug efflux pumps (17).Activity of the non-steroidal anti-inflammatory drug, etofenamate, is dependent upon Upc2p, a transcription factor that activates the expression of genes encoding the enzymes required for ergosterol biosynthesis (18).Genetically encoded antimicrobial resistance, including azole-resistance in C. albicans conferred by activating mutations in the Tac1p and Upc2p transcription factors, is often associated with fitness costs (19,20).However, the impact of the fluconazole antagonistic drugs upon fungal physiology, pathogenicity, or fitness is unknown.The goal of this study was to investigate how fluconazole antagonistic medications affect the physiology, fitness, and pathogenicity of C. albicans and determine if they can influence the course of invasive infections or the efficacy of antifungal therapy.Our findings have the potential to uncover unanticipated drug-fungus interactions that may influence the incidence or outcome of IFIs.

Plasmid construction C. albicans strain construction
The CAI4 + pKE4 + NRG1 strain was kindly provided by Brian M Peters.Plasmid pLUX (22) was also kindly provided by William Fonzi (Georgetown University).The tac1Δ/Δ and derived revertant strains were constructed using the auxotrophic marker system previously described by our laboratory using the TAC1DISF and TAC1DISR primers to delete the open reading frame, and TAC1AMPF and TAC1AMPR primers to select the open reading frame and 5' promoter region for amplification into the pLUX plasmid (22).All oligonucleotides used in this study are listed in Table S8.

Drug stocks
Stock solutions of fluconazole and each compound (aripiprazole, pinaverium, prenyla mine, etofenamate, thiethylperazine, mometasone, and penbutolol) were prepared at 10 mM in dimethyl sulfoxide (DMSO) and diluted to required working concentrations.

Growth kinetic assays
Growth curve assays were set up in 96-well plates using Roswell Park Memorial Institute (RPMI) buffered to pH 7 with (3-(N-morpholino)propanesulfonic acid).C. albicans strains were grown overnight in yeast protein extract (YPD) at 30°C, and the cell density was adjusted to 1 × 10 4 cells/mL in the appropriate medium for the growth kinetic assays.An aliquot of cell suspension (100 µL) was prepared with an equal volume of twice the desired drug concentration of each antagonist.Cells were then incubated at 35°C inside a BioTek Cytation 5 plate reader shaking for 24 hours, and OD 600nm was read every 30 minutes (time).Data were then analyzed via GraphPad Prism software.These assays were repeated in technical triplicates and in biological triplicates.

Dry weight experiment
C. albicans strain SC5314 was grown overnight in YPD at 30°C.Cells were diluted 1:500 into 25 mL of RPMI-pH 7 supplemented with either 5 µM of aripiprazole, pinaverium, or 0.5% DMSO [vehicle (VEH) control] and incubated for 16 hours at 35°C.A 1 mL aliquot of each cell suspension was transferred to an Eppendorf tube and then preserved with 10% formalin for counting and to inspect morphology.Remaining cells were then filtered with a 47-mm filter using a Whatman Swin-Lok filter setup and lyophilized using a BenchTop Pro with Omnitronics before being weighed.Individual points indicate data from six independent experiments, and cross bars indicate the mean.

Phenotypic assays
C. albicans SC5314 was grown overnight in YPD at 30°C.The cell density of each strain was adjusted to 1 × 10 4 cells/mL, in RPMI-pH 7 containing twice the final drug concentra tion of each antifungal antagonist or 0.5% DMSO (vehicle control).Susceptibility to the following stressors (amount; cell target) was assayed: SDS (up to 0.1%; cell membrane), Congo Red (up to 500 µg/mL; cell wall), Caffeine (up to 10 mM; cell membrane and cell wall), FeCl 3 (up to 1 mM; electron transport chain), and NaCl (up to 1 M; ionic stress).Cells were plated, grown for 24 hours at 35°C, and scanned using an EPSON Perfection v700 flatbed scanner.Images are representatives of experiments repeated in biological duplicate.

Hyphal growth assays
C. albicans SC5314 was grown overnight in YPD at 30°C before being washed in sterile deionized water.The cell density was adjusted to 1 × 10 7 cells/mL in phosphate-buffered saline (PBS), and 2 µL of cell suspension was spotted onto M199 (2% agar plates) supplemented with 5 µM of drug or 0.5% DMSO (vehicle control).Plates were incubated for 96 hours at 37°C and imaged as above.
For liquid hyphal growth assays, C. albicans SC5314 was grown overnight in YPD at 30°C before being washed in sterile deionized water.The cell density was adjusted to 1 × 10 6 cells/mL in 5 mL of RPMI-pH 7 and incubated shaking for 6 hours at 35°C.Equal volumes of 10% formalin were added, and cells were preserved in medium + formalin for 30 minutes on ice.Cells were pelleted, supernatant was removed, and cells were resuspended in 1 mL of PBS.Cells were then imaged at 40× magnification.Pictures are representative of the assays performed in biological triplicates.

RNA sequencing analysis
SC5314 was grown in YPD medium at 30°C overnight, then sub-cultured at 1 × 10 6 cells/mL into 50 mL RPMI-pH 7 medium supplemented either with 5 µM aripiprazole, etofenamate, or mometasone or with 0.5% DMSO (vehicle control).Cells were then incubated at 35°C for 6 hours with shaking.Cells were then harvested at 4°C, superna tant was removed, and cells were frozen at −80°C.Total cellular RNA was extracted using the hot phenol method (23).Novogene provided RNA library preparation and sequencing analysis as fee-for-service.Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads.After fragmentation, the first-strand cDNA was synthesized using random hexamer primers, followed by the second-strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library.Library was checked with Qubit and real-time PCR for quantification and a bioanalyzer for size distribution detection.Libraries were then pooled and sequenced on Illumina platforms.Genes were mapped to the SC5314 haploid genome assembly 22. Drugresponsive genes were identified as those significantly up-or down-regulated compared with their respective vehicle controls, with significant gene expression identified as either > or < 2-fold (P-value < 0.05, adjusted P-value < 0.05 to account for false discovery rate).Samples were prepared in independent biological triplicates, and log 2 fold was converted to fold change, averaged, and the average value was converted to log 2 fold for the term "AVG log 2 fold."

Antifungal susceptibility testing
Antifungal susceptibility testing was performed using the broth microdilution method as described in Clinical and Laboratory Standards Institute (CLSI) document M27-A3 (24), with minor modifications.Fluconazole was diluted in DMSO, resuspended in RPMI-pH 7 at twice the final concentration, and serially diluted.C. albicans strains were grown overnight in YPD at 30°C, resuspended at 1 × 10 4 cells/mL in RPMI-pH 7, and 100 µL was transferred to wells of a round bottom 96-well plate containing an equal volume of diluted fluconazole solution.The final concentration of DMSO was 0.5% for all treat ments, with drug-free control wells having DMSO alone.Plates were incubated without shaking for 24 hours at 35°C and then scanned using an EPSON Perfection v700 Photo scanner.Experiments were performed in biological duplicates.

Cell wall staining experiments
SC5314 cultures were diluted 1:100 into 5 mL YNB-pH 7 supplemented with either 5 µM aripiprazole or 0.5% DMSO (vehicle control).Cells were incubated at 30°C for 24 hours before harvesting.Supernatant was removed, and the cells were preserved in 10% formalin for 30 minutes on ice, washed three times, and stored in PBS.Cells were then counted and diluted to a final density of 1 × 10 6 cells/mL in PBS.Cells were pelleted and supernatant was removed and stained with either concanavalin A-FITC (50 µg/mL), wheat germ agglutinin-Alexa Fluor 488 (50 µg/mL), calcofluor white (50 µg/ mL), or aniline blue (1 mg/mL) for 5 minutes, 30 minutes, or 1 hour, respectively.For each stained sample, an unstained control was also prepared.After centrifugation, cells were resuspended into 500 µL PBS.Samples were then analyzed using a NovoCyte 3000 flower cytometer, using stain-specific lasers and filter sets.Mean fluorescence intensity (MFI) was compared between vehicle or drug-treated cells.Individual points indicate data from independent biological experiments, and cross bars indicate the mean.

Macrophage challenge assays with live C. albicans
Upon recovery from cryopreservation, THP-1 cells were incubated for 3 days at 37°C, 5% CO 2 , and 90% humidity in complete RPMI 1640 medium containing 25 mM HEPES (10% heat-inactivated FBS and 100 U/mL Pen-Strep).After 3 days, THP-1 cells were counted on a Novocyte flow cytometer (Agilent) and diluted to 2.5 × 10 5 cells/mL in complete RPMI with 100 nM phorbol 12-myristate 13-acetate (PMA) (InvivoGen) to differentiate cells to a macrophage phenotype.Next, 200 µL aliquots were seeded at 5 × 10 4 cells/well in 96-well tissue-culture-treated polystyrene plates and incubated at 37°C with 5% CO 2 for 24 hours.Overnight cultures of SC5314 were subcultured 1:100 into YNB-pH 7 and incubated at 30°C for 24 hours and washed three times in sterile PBS.C. albicans suspensions were then added to a challenge medium con taining 5 µM aripiprazole or 0.5% DMSO (vehicle control) in phenol red-free RPMI-pH 7 containing 25 mM HEPES to generate multiple multiplicities of infections (MOIs).Mock-infected controls using medium alone were also included.Drug-only controls were also included with THP-1 differentiated macrophages challenged with the same drug concentrations without SC5314.A positive control for TNF-α or IL-1β release was also prepared by challenging cells with 1 µg/mL lipopolysaccharide (Escherichia coli 0111:B4; InvivoGen) for an equivalent time, followed by addition of 5 mM ATP (InvivoGen) 30 min prior to the endpoint.The cells were challenged for 4 hours, and 100 µL of culture supernatant was transferred to a polystyrene plate containing 100 µL of prediluted 1× enzyme-linked immunosorbent assay (ELISA)/enzyme-linked immunosorbent spot (ELISpot) assay buffer (eBioscience) and stored at −80°C.Culture supernatants were assessed for TNF-α or IL-1β using the Human Ready-Set-Go ELISA kit (eBioScience).ELISA optical density values from mock-infected controls were subtracted from those of Candida-challenged samples.Experiments were conducted in biological triplicates.Data are reported as the means + SEM.

Macrophage challenge assays with fixed C. albicans
THP-1 cells were recovered, incubated, and differentiated with PMA as previously described above.For challenges with fixed SC5314 yeast cells, overnight cultures were subcultured at 1:100 into YNB-pH 7 and incubated with either 0.2, 1, and 5 µM aripipra zole or 0.5% DMSO (vehicle control) and incubated at 30°C for 24 hours.For challenges with fixed SC5314 germ tubes, cells were subcultured at 1 × 10 7 cells/mL in 50 mL RPMI-pH 7 (no phenol red) and incubated at 37°C for 1.5 hours.For both challenges, fungal cells were pelleted, supernatant was removed, and cells were preserved in 10% formalin on ice for 30 minutes.C. albicans was then washed three times in sterile PBS.Macrophages were challenged with an MOI of 20:1 with either 0.2, 1, and 5 µM aripiprazole or 0.5% DMSO (vehicle control) added to the challenge media as previ ously described.Similar controls were included as described above.Plates were either incubated for 24 hours (yeast) or 8 hours (germ tube), supernatant was collected, and ELISA assays for TNF-α and IL-1β were performed as previously described.Experiments were conducted in biological triplicates.Data are reported as means + SEM.

Disseminate model of C. albicans infection
Mice were injected subcutaneously with 100 µL of either 10 mg/kg/day aripiprazole, 2 mg/kg/day aripiprazole, or vehicle twice daily, starting 3 days prior to infection.For drug formulations, aripiprazole [TCI America (98% Purity)] was prepared fresh daily as a formulation of 25% PEG400 (Sigma-Aldrich Cat.#.NC1211386), 19% Kolliphor RH40 (Sigma-Aldrich Cat.#.NC0542250), and 65% sterile water (Gibco Cat.#. 15230-147) and adjusted to 1 or 0.2 mg/mL to achieve human-equivalent steady-state concentrations.The formulation was then sonicated with a Fisher Scientific bath Sonicator (FS-30) for 15 minutes at an ultrasonic power of 130W and a frequency of 40 kHz before being adjusted to the appropriate volume of water before the final sonication to obtain a clear solution.SC5314 was grown overnight in YPD broth at 30°C with shaking, washed twice in sterile PBS, and resuspended at appropriate densities for infection of BALB/c and C57BL/6 mice (2.5 × 10 6 cells/mL) or CD-1 mice (4 × 10 6 cells/mL).Groups (n > 8 mice) were inoculated by lateral tail vein injections with 100 µL of the desired cell suspension.Mice were monitored three times a day and those experiencing severe distress were humanely euthanized.For select experiments, mice were euthanized at set time points of 48 hours post-infection (p.i) for BALB/c and C57BL/6 or 72 hours p.i. for CD-1 strains.At the endpoint, kidneys were extracted.Half of the left kidney was stored in 10% formalin for histopathology analysis, and the remaining kidneys were homogenized in pre-weighed tubes of PBS.Serial dilutions of kidney homogenates were plated on YPD agar plates containing 50 µg/mL of chloramphenicol.Fungal burdens [colony forming units (CFU)/g of kidney tissue] were determined by enumerating colonies formed after 48 hours of incubation at 30°C.
For some experiments, mice were rendered leukopenic with 150 mg/kg cyclophos phamide in 200 µL of filter-sterilized PBS via intraperitoneal injections every 3 days, beginning 2 days prior to infection.Inoculum sizes were reduced to 2.5 × 10 5 cells/mL for infections of BALB/c, or 4 × 10 5 cells/mL CD-1 mice.

Serum clinical chemistry
Blood was collected via cardiac puncture from each mouse at the time of sacrifice and aliquoted into two vials (plasma and serum separation tubes-PST and SST).Collected serum and plasma were stored at −80°C until ready for use.Serum samples were analyzed by UTHSC Regional Biocontainment Laboratory staff as fee-for-service using the DiaSys Diagnostic Systems chemical analyzer for blood urea nitrogen (BUN) and creatine.

Quantification of aripiprazole concentration in plasma
Sample preparation was done by protein precipitation using ice-cold methanol containing verapamil (25 ng/mL) as an internal standard.Each plasma sample (25 µL) was precipitated with 8 volumes (200 µL) of internal standard (IS) in methanol.Samples were vortexed for 30 seconds and filtered using 96-well Millipore sample filter plates and centrifuged at 4°C, and the filtrates were analyzed using liquid chromatrographymass spectrometry (LC-MS/MS).Chromatographic separations were carried out using a Shimadzu Nexera XR (LC-20ADXR) liquid chromatograph (Shimadzu Corporation, USA) consisting of two pumps, an online degasser, a system controller, and an autosampler.Mobile phase consisting of (i) 95% water and 5% acetonitrile with 2 mM ammonium formate buffer and 0.1% formic acid and (ii) 95% acetonitrile and 5% water with 2 mM ammonium formate buffer and 0.1% formic acid was used at a flow rate of 0.6 mL/min in gradient mode.A Phenomenex C18 2.6μ, 100 × 4.6 mm column (Phenomenex, USA) was used for separation.Samples (5 µL) were injected onto column, and the eluate was led directly into an API 4500 triple quadruple mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a turbospray ion source, which was operated in the positive ion mode.
The concentration was determined based on the peak area ratio of IS vs analyte (verapamil vs aripiprazole) against the calibration curve.A linear statistical regression model with a weighing factor of 1/X was chosen after examination of the residuals and coefficient of correlation.The lowest limit of quantitation (LLOQ) measured with acceptable accuracy and precision for aripiprazole from mouse plasma was established as 1.72 ng/mL.Appropriate dilution of samples was performed where necessary.

Mouse tissue histology
Mice were euthanized within 48 hours (p.i.), and half of the left kidney was cut cross-sec tionally and stored in 10% formalin.Kidney samples were embedded in paraffin blocks, and 5-µm slices were prepared using a microtone.Serial sections were stained with hematoxylin and eosin (H&E) or Grocott-Gomori methenamine silver (GMS).Slides were scanned at 10× and 40× using a Nano Zoom-SQ Hamamatsu digital slide scanner.

A subset of azole-antagonistic drugs impacts Candida albicans morphogene sis
We initially determined whether exposure to the seven selected azole-antagonistic drugs (Table 1) affected C. albicans growth.The SC5314 reference strain was suspen ded in RPMI medium with 5 µM of each drug (a concentration providing robust azole antagonism) ( 16) or with 0.5% DMSO (vehicle control) and incubated at 35°C.Growth was then quantified at 30-minute intervals as OD 600nm .Five out of the seven tested antagonists had no clear impact on OD 600nm (Fig. S1).Only aripiprazole and pinaverium had detectable effects on OD 600nm , with both appearing to elevate C. albicans culture density compared with drug-free control (Fig. 1A).To further examine the impact of aripiprazole and pinaverium on C. albicans biomass, dry weights were determined after cells were grown in RPMI at 35°C with either drug or 0.5% DMSO (vehicle control).Neither drug increased dry mass compared with the vehicle control.In fact, aripipra zole slightly, but reproducibly, reduced dry mass (Fig. 1B).The apparent contradiction between the drug's impact upon OD 600nm and biomass could be explained by effects upon cellular morphotype, given the capacity of C. albicans to switch between yeast, pseudohyphal and true-hyphal forms (25).To determine if any of the antagonists affect C. albicans morphogenesis, SC5314 was induced to form hyphae on M199 agar supplemen ted with 5 µM of each azole-antagonist or 0.5% DMSO (vehicle control).After 96 hours at 37°C, a prominent border of hyphae could be observed at the margin of colonies formed in the absence of drug, as well as in the presence of five drugs.However, hyphal growth was notably diminished in the presence of aripiprazole and pinaverium (Fig. 1C).Similarly, aripiprazole and pinaverium suppressed hyphal growth in liquid RPMI medium (Fig. 1D).
We next asked whether exposure to the fluconazole antagonists affects C. albicans stress tolerance.SC5314 was grown in RPMI medium containing 5 µM of each antagonist or vehicle and supplemented with varying concentrations of NaCl (ionic stress), Congo red (cell wall stress), the detergent SDS (cell membrane stress), FeCl 3 (electron-trans port chain disruptor and oxidative stress), or caffeine (cell wall, cell membrane, and DNA stressor).None of the seven drugs altered C. albicans sensitivity to NaCl, caffeine, FeCl 3 , SDS, or Congo red (Fig. S2).Collectively, these data suggest that most of the azole antagonistic medications previously reported have little impact upon C. albicans growth, stress tolerance, or morphogenesis.However, a subset including aripiprazole and pinaverium has a moderate impact on fungal physiology that is most notable during hyphal growth.

Aripiprazole, mometasone, and etofenamate regulate the transcription of a small and partially overlapping set of C. albicans genes
To provide further insight into the effect of the azole-antagonists upon C. albicans physiology, we examined how three structurally unrelated antagonists, aripiprazole, mometasone, and etofenamate alter global patterns of gene transcription.Aripipra zole and mometasone were previously determined to act at sub-micromolar concentra tions through a Tac1p-dependent mechanism (16), whereas etofenamate acts through a Upc2p-dependent mechanism (16).SC5314 was grown at 35°C in RPMI medium supplemented with 5 µM of either drug or 0.5% DMSO for 6 hours; total cellular RNA was extracted and subjected to high-throughput sequence analysis.The number of reads/kb was calculated for each gene, and transcripts increased or decreased >2-fold (P < 0.05) in the presence of drug (relative to the vehicle control) in each of three inde pendently performed experiments considered responsive.Aripiprazole, mometasone, and etofenamate significantly increased the abundance of 31, 3, and 7 transcripts, respectively (Fig. 2A and B).Notably, the abundance of two transcripts-those of CDR2 and RTA3-was elevated by all three of the azole-antagonists (Fig. 2C).CDR2 encodes a multi-drug efflux pump of the ATP-binding cassette superfamily and has an established role in facilitating genetically encoded azole resistance in C. albicans (26), whereas RTA3 encodes a phospholipid translocase also previously shown to affect azole-tolerance (27).These results are consistent with our previous data showing that the azole-antagonistic activity of aripiprazole is dependent upon the Tac1p transcription factor, which activates the transcription of CDR2 (16).Several additional transcripts known to be either induced by Tac1p or following exposure to fluphenazine (a known activator of Tac1p) (28) were identified as being elevated by one or more of the three drugs.These include CDR1, encoding a second ATP-binding cassette-type drug efflux pump that plays a major role in genetically encoded azole-resistance (25), and PDR16, which are responsive to both aripiprazole and etofenamate (Table S1).Aripiprazole, mometasone, and etofena mate decreased the abundance of 31, 22, and 10 transcripts, respectively, with HAK1, encoding a putative potassium transporter, affected by all three (Fig. 2C).Several, such as MRV2, have been reported to be either suppressed following fluphenazine exposure, by fluconazole exposure, or otherwise repressed in azole-resistant isolates (29).The relatively small sets of gene transcripts responsive to each of the azole-antagonists tested are notable and consistent with a very specific mechanism underlying their activity.Furthermore, the overlap in the transcripts regulated by more than one drug is surprising, given their structural dissimilarity and distinct mechanisms with both aripiprazole and mometasone Tac1p-dependent and etofenamate Upc2p-dependent.Additionally, at least a subset of genes affected by one or more of the antagonists are associated with virulence.For example, ICL1 (encoding isocitrate lyase) (30), ALS1 (encoding a surface adhesin) (31), and UME6 (encoding a transcription factor required to sustain hyphal growth) (32) transcripts are all suppressed by aripiprazole and have established roles in supporting C. albicans virulence (Table S5).
Together, these data suggest that the drugs identified as undermining azole antifungal activity in vitro could potentially affect the outcome of invasive C. albicans infections through either modulating therapeutic efficacy or the inherent virulence of the fungus itself.To further investigate the potential effect of the azole-antagonistic drugs upon C. albicans physiology, as well as the outcome of the infections it causes, we focused subsequent studies on aripiprazole, as we have previously shown that it exerts azole-antagonistic activity within therapeutically relevant concentrations, that is, which are achieved within the bloodstream of patients on a standard dosing regimen (150-450 ng/mL) (33).

Aripiprazole-related drugs oppose fluconazole antifungal activity through two distinct mechanisms
Aripiprazole is composed of 3,4-Dihydro-2(1H)-quinolinone and (2,3-Dichloro phenyl)piperazine moieties coupled by a butoxy linker (Fig. 3A).To determine which structure was responsible for its Tac1p-dependent azole-antagonistic activity, we evaluated the azole-antagonistic activity of two closely related drugs.Bexpiprazole has a benothiophene in place of the (2,3-Dichlorphenyl)piperazine moiety of aripiprazole and elevated the fluconazole MIC of C. albicans by ~8-fold (Fig. 3B).Cariprazine, on the other hand, possesses the (2,3-Dichlorophenyl)piperazine but has cyclohexyl and dimethylurea components in place of the 3,4-Dihydro-2(1H)-quinolinone of aripiprazole and an ethyl linker (Fig. 3A).Cariprazine also had marked fluconazole antagonistic activity on C. albicans, elevating the MIC by >256-fold (Fig. 3B).Taken alone, these data suggest that the piperazine group is likely responsible for the antagonistic activity.However, this interpretation may be oversimplified as (2,3-Dichlorophenyl)piperazine alone did not significantly affect C. albicans fluconazole sensitivity (Fig. S4B).Further more, although aripiprazole's antagonistic activity is Tac1p-dependent, bexpiprazole's appears to be both Tac1p-and Upc2p-dependent, and cariprazine is Tac1p-independent and entirely Upc2p-dependent (Fig. 3B; Fig. S3).These data are consistent with the 2-quinolinone group of aripiprazole and bexpiprazole activating Tac1p.However, neither the 3,4-Dihydro-2(1H)-quinolinone found in bexpiprazole nor the 3-cyclohexyl-1,1-dime thylurea group found in cariprazine are sufficient to decrease fluconazole sensitivity (Fig. S4C and E).Collectively, these data underscore the fact that biologically active small molecules can affect C. albicans physiology and antifungal sensitivity through multiple mechanisms and that the structure-activity relationships underlying these interactions are complex and multifaceted.

Aripiprazole does not affect the efficacy of fluconazole therapy in a mouse model of disseminated candidiasis
To determine if the antagonistic activity of aripiprazole observed in vitro affects the therapeutic efficacy of fluconazole during in vivo infection, we utilized a mouse model of disseminated candidiasis.Two groups of BALB/c mice were treated with either aripiprazole (5 mg/kg/day) or vehicle by subcutaneous injection for 3 days prior to infection with ~2.5 × 10 5 colony forming units of SC5314 via the tail vein.Daily treat ments with aripiprazole or vehicle were continued, and 24 hours post-infection, each group was sub-divided, one sub-group was treated with fluconazole (5 mg/kg/day) and the second mock-treated with vehicle alone via intraperitoneal injection.Mice were euthanized 5 days post-infection, kidneys extracted, homogenized and tissue fungal burden quantified as CFU.Fungal colonization was similar between the fluconazole and fluconazole + aripiprazole-treated groups (Fig. 4A), suggesting that aripiprazole does not antagonize fluconazole efficacy in this model.However, two mice in the aripiprazole-alone treated group unexpectedly succumbed to infection prior to the scheduled endpoint of the experiment (data not shown).Strikingly, the remaining mice in this group displayed advanced signs of morbidity.Importantly, uninfected mice treated with aripiprazole exhibited no weight loss or other signs of disease (data not shown).Although no in vivo azole antagonism was noted, these results indicated that aripiprazole may impact C. albicans virulence or the progression of invasive disease in the absence of azole therapy.

Aripiprazole exposure exacerbates the severity of disseminated C. albicans infection in mice
To further examine the effect of aripiprazole on the outcome of disseminated infection, groups of BALB/c mice were treated with either 2 or 10 mg/kg/day aripiprazole or vehicle alone, and after 3 days infected with SC5314 as described before.The mice were monitored daily for signs of disease progression; those exhibiting signs of distress were euthanized, and survival was compared using the log-rank test.Mice in both aripiprazole-treated groups succumbed to infection more rapidly than the vehicle-trea ted mice (Fig. 4B), whereas uninfected mice treated with 10 mg/kg/day of aripiprazole again remained healthy throughout the experiment.Similar results were obtained using outbred CD-1 mice (Fig. 4C), indicating these results are not mouse strain-dependent.Endpoint experiments at 48 hours post-infection revealed no significant differences in the levels of fungal colonization of kidney tissue measured as CFUs between the three treatment groups (Fig. 5B).However, foci of fungal invasion were more prominent in kidney sections of both aripiprazole-treated groups than in mock-treated animals (Fig. 5A).Additionally, the kidneys of infected aripiprazole-treated mice had substantially larger inflammatory lesions characterized by extensive neutrophil (PMN) infiltration than were observed in the mock-treated group (Fig. 5A).Analysis of serum samples obtained at 48 hours p.i. also revealed higher levels of blood urea nitrogen (BUN) than the mock-treated control mice (Fig. 5C and D), providing additional evidence that aripiprazole exposure exacerbates kidney damage and dysfunction during disseminated candidiasis.Notably, aripiprazole treatment had no effect on BUN or creatine levels in uninfected mice.Comparable results were obtained with C57BL/6 mice, further supporting that the effect of aripiprazole on the outcome of systemic C. albicans infection is mouse strain-independent (Fig. S4).Finally, we examined how aripiprazole exposure affects disease progression in severely immunosuppressed mice.Survival experiments were performed as described above, with BALB/c mice rendered leukopenic through treatment with cyclophospha mide 2 days prior to infection with 2.5 × 10 4 CFU of SC5314.Strikingly, the previously observed difference in survival between vehicle-and aripiprazole-treated groups (Fig. 4B) was eliminated (Fig. 6A).Similar findings were observed using outbred CD-1 (Fig. 6B).In a separate 48-hour endpoint experiment, kidney fungal burden and histopathology were assessed in cyclophosphamide-treated CD-1 mice.Although pockets of fungal growth were observed throughout the kidneys in both aripiprazole-and mock-treated groups, there were no obvious zones of inflammation (Fig. 6C).Quantitative fungal burdens supported the microscopy findings (Fig. 6D).Additionally, BUN and creatine levels measured in serum samples taken 48 hours p.i were similar between aripiprazoleand mock-treated groups (Fig. 6E).These data suggest that the impact of aripiprazole during systemic C. albicans infection is at least partially dependent on host immune status.

Aripiprazole modulates host response to C. albicans
Given that aripiprazole did not impact the outcome in immunosuppressed mice, we asked whether this drug alters the immunogenicity of C. albicans by modulating cell wall pathogen-associated molecular patterns (PAMPs).To determine this, C. albicans was grown in the presence or absence of 5 µM aripiprazole, fixed, and stained with aniline blue (total β-glucan), concanavalin A (total mannan), or calcofluor white (total chitin), and relative fluorescence levels quantified by flow cytometry (Fig. 7).Aripiprazole had no obvious effect on β-glucan or mannan content but did induce a significant increase in cell wall chitin content (~1.5-fold versus vehicle-treated control).Staining or 2 mg/kg/day aripiprazole or vehicle starting 3 days prior to infection, infected on day 0 with 2.5 × 10 5 CFU of SC5314 via lateral tail vein injection and monitored for 10 days.The survival of the infected drug-treated groups was compared with that of the infected mock-treated group, using a log-rank test.
(C) Groups of CD-1 mice (n = 8) were treated and infected as described in (B), and survival was monitored for 14 days.Survival was compared using a log-rank test.*P < 0.005, ***P < 0.0001.with wheat-germ agglutinin also revealed a trend toward increased chitin exposure under the conditions tested, but this did not reach statistical significance.We specula ted that changes in polysaccharide content could modulate PAMP presentation at the C. albicans cell surface and, in turn, the capacity of the mammalian host to detect or respond to the fungus.To test this hypothesis, we used an in vitro model of C. albicans-macrophage interaction.THP-1-derived macrophages were challenged with C. albicans at multiplicity of infections (MOIs) of 1:1, 2:1, 4:1, or 8:1 in the presence of either 5 µM aripiprazole or DMSO (vehicle control).After 4 hours of incubation, release of the pro-inflammatory IL-1β and TNF-α cytokines into the culture supernatant was compared by ELISA.Significantly less IL-1β (Fig. 8A) and TNF-α (Fig. 8B) were released in the presence of aripiprazole at MOIs of 2:1 or greater, indicating an altered host-fungus interaction.To determine if aripiprazole-mediated cytokine suppression was driven by its effect on host or fungal physiology, additional macrophage challenges were conducted using fixed C. albicans.First, C. albicans SC5314 was grown as yeast with 0.2, 1, or 5 µM aripiprazole or vehicle (DMSO), fixed, washed, and then applied to macrophages.Fixed yeast failed to induce robust release of either TNF-α or IL-1β (Fig. 9A).However, when C. albicans was grown as germ tubes and subsequently fixed, those cultured in the presence of aripiprazole induced significantly less TNF-α or IL-1β than vehicle-trea ted cells, with the differences most obvious at the highest concentrations (Fig. 9B).This correlated with suppression of hyphal growth in the aripiprazole-treated cells as described previously (Fig. 1D).To examine if aripiprazole also impacts host response to C. albicans, untreated fungi were grown as previously described, fixed, washed, then applied to macrophages in the presence of 0.2, 1, or 5 µM aripiprazole or vehicle.Surprisingly, macrophages co-incubated with fixed germ tubes in the presence of 5 µM aripiprazole, also released significantly less TNF-α, with a similar trend observed for IL-1β that did not reach statistical significance (Fig. 9D).In contrast, macrophages co-incubated Pictures are representative (n = 3 per group).(D) Kidneys were obtained from mice in (C), and fungal burden was quantified as CFU per gram of tissue.Data are the mean ± standard deviation, and statistical significance was calculated using a one-way ANOVA with Mann-Whitney post-test (n = 8).(E) Blood was extracted from the same mice as in (C) and serum was processed, and blood urea nitrogen (BUN) levels were quantified.Groups were compared with infected treated versus 10 mg/kg/day aripiprazole-infected mice.Data are the mean ± standard deviation, and statistical significance was calculated using a one-way ANOVA with Mann-Whitney post-test (n = 8).ns = not significant.
with fixed yeast in the presence of 5 µM of aripiprazole significantly increased the release of both TNF-α and IL-1β (Fig. 9C).Together, these results suggest that aripiprazole may alter the outcome of disseminated C. albicans infection in mice through modulation of both host and fungal physiology.

DISCUSSION
Most infectious fungi are opportunists that principally cause disease in individuals with impaired immune function.However, despite well-defined risk factors, the occurrence of IFIs in individual patients is difficult to predict, as are the clinical outcomes of subse quent therapeutic intervention.Paralleling this situation, acute and recurrent vulvovagi nal candidiasis has an idiopathic occurrence, with most affected women in otherwise good health (34)(35)(36).Each individual patient at risk of IFI presents a unique set of physiological, immunogenetic, and clinical contexts including the regimen of medica tions consumed.Our long-term goal is to better understand if, and how, medications consumed by humans for indications unrelated to fungal infection (i.e., non-antifungal drugs) influence fungal physiology and pathogenicity and if such drug-fungal interac tions have the potential to affect disease initiation, progression, or resolution.Such drug-fungus interactions are pertinent, as patients at greatest risk of IFIs typically have one or more underlying disease condition that require treatment with complex regimens of drugs tailored to individual needs.This includes those taken transiently and those intended for long-term or even life-long use, creating an additional complex and dynamic variable between at-risk individuals.Furthermore, several species of Candida are natural residents of the gastrointestinal and reproductive tracts of healthy human subjects (37,38) and, therefore, are routinely exposed to medications administered to their human host.Certainly, drugs that cause profound immune dysfunction including corticosteroids and antineoplastic agents are known to dramatically increase a patient's risk of developing an IFI (39).The use of broad-spectrum antibiotics that eliminate significant portions of the endogenous bacterial microbiota is also a significant risk factor for oral and vaginal Candida infections (40).However, aside from those that have a direct and obvious effect upon the mammalian host's primary defense mechanisms, the influence of most drugs upon the incidence and outcomes of invasive mycoses has received little attention.Several studies have sought to identify approved medications with stand-alone antifungal activity or that potentiate the activity of existing antifungal agents and can therefore potentially be repurposed to treat IFIs (12)(13)(14).Other studies have identified approved drugs that appear to antagonize or oppose the activity of antifungal therapeutics (6,15,41,42).However, the rudimentary outcomes of these studies (e.g., growth inhibition) are unlikely to identify those that have subtle or nuanced effects upon fungal physiology, pathogenicity, or immunogenicity that could potentially alter fungal fitness or interaction with the mammalian host and therefore the initiation, progression, or outcome of infection.Given the fundamental similarity of fungi and mammals at the molecular level, it is likely that many drugs designed to induce a biological response in human cells have an analogous impact upon fungal physiology.However, the consequences of exposing commensal and pathogenic fungi to most medications approved for human use remain unknown.This specific study assessed the impact of several drugs we previously found to oppose the antifungal activity of the azole class of antifungals upon C. albicans (16).Genetically encoded azole resistance in C. albicans is often associated with point mutations in one or more key transcription factors that regulate the expression of the target enzyme Erg11p or of multi-drug efflux pumps.However, mutations that confer antifungal resistance are often associated with fitness costs in the absence of the selecting agent (43,44), including point mutations that activate either Tac1p or Upc2p transcription factors (20,45).Therefore, it is surprising that despite six of the seven antagonists investigated in this study acting in a Tac1p-dependent manner and the seventh in an Upc2p-dependent manner, only two had an obvious impact on C. albicans filamentous growth or phenotype at concentrations sufficient to substantially elevate azole MIC.Thus, azole-antagonistic activity of a drug or xenobiotic is not necessarily or inherently associated with significant detrimental impacts on C. albicans fitness.Consistent with our phenotypic data, the transcription of a surprisingly small number of genes was affected by all three of the antagonists examined, indicating that they affect a very specific aspect of fungal physiology.Furthermore, many of the drug-respon sive genes identified are consistent with a Tac1p-dependent mechanism, including etofenamate, which we have previously shown to act largely through an Upc2p-depend ent mechanism.It is therefore possible that the majority of the remaining 13 drugs we previously identified as fluconazole antagonists (16), also act through Tac1p-and/or Upc2p-dependent mechanisms, despite dissimilarity in structural, chemical, and physical properties.Viewed from this perspective, it appears that C. albicans has evolved systems to specifically sense and respond to exogenous xenobiotics, which incidentally are adept at sensing medicinal compounds.One possibility is that as C. albicans has co-evolved with its mammalian host, these sensing mechanisms arose to detect host hormones or other signals that help the fungus regulate its physiology to favor commensalism or tissue invasion.A related prospect is that as part of a large and complex community of endogenous microbes residing within non-sterile body sites, xenobiotic detection systems evolved to respond to competing microbes that release antifungal molecules.In these circumstances, the seemingly promiscuous nature of the Tac1p-and Upc2p-based xenobiotic response modules, as well as the broad-substrate specificities of the drugefflux pumps regulated by Tac1p, would be advantageous to respond to a multitude of distinct threats.Much of this study focused upon the atypical antipsychotic aripiprazole to estab lish proof of principle that previously unanticipated drug-fungus interactions have the potential to influence the outcome of invasive fungal disease.Although its azole-antag onistic activity observed in vitro did not translate to reduced antifungal efficacy in the mouse model of disseminated infection, it expedites the progression of infection in the absence of azole therapy.Intuitively, this could be due to effects on the fungus that alter its inherent virulence or upon the mammalian host that impact its intrinsic resistance to infection or capacity to mount an effective immune response.Based on the observa tion that aripiprazole suppressed hyphal formation under in vitro conditions and the importance of yeast-hypha morphogenesis to C. albicans virulence (46), worse outcomes in aripiprazole-treated mice infected with C. albicans are surprising.However, we did not observe any obvious effect of aripiprazole treatment upon C. albicans morphotype in tissue sections from infected mice.Thus, given the multitude of signaling pathways able to activate yeast-hypha morphogenesis (25), it is possible that aripiprazole's effect upon hyphal growth is less pronounced in the context of the robust filamentation cues encountered within mammalian tissue.An alternative possibility is that by delaying the transition of the yeast form provided in the initial inoculum into the more tissue-invasive form, aripiprazole enhances the dispersal of infectious particles from the initial foci of infection, leading to a greater number of active infection sites within the kidney and other tissues.Either way, the tissue lesions observed in C. albicans-infected mice treated with aripiprazole are much larger, levels of kidney damage sustained are greater, and the lesions are associated with elevated levels of immune cell infiltration compared with the mock-treated controls.To further examine this relationship, BALB/c mice were treated with aripiprazole as described previously, then infected with ~4 × 10 5 cells of a "yeast-locked" C. albicans strain that overexpresses NRG1, which encodes a repressor of hyphal growth.Although this strain did not induce lethality, aripiprazole treatment increased morbidity and kidney weight without increasing fungal burden (Fig. S5).These data suggest that aripiprazole's effect on disease progression is independent of hyphal morphogenesis.
One possible explanation for this finding is that aripiprazole altered C. albicans immunogenicity in such a way as to promote a vigorous and potentially damaging host response.This could explain why the effects of aripiprazole were lost in the immunosup pressed model of disseminated infection.Notably, exposing C. albicans to sub-growth inhibitory concentrations of the echinocandin antifungals increases β-glucan exposure and immune recognition both in vitro and in vivo (47,48) (49,50).We were able to detect differences in the levels of chitin in aripiprazole-treated C. albicans.However, chitin exposure is typically associated with fungal tolerance (49), and our in vitro studies indicate that fixed C. albicans cells pre-treated with aripiprazole stimulate less proinflammatory cytokine release from macrophages than untreated cells.
Although these studies were initiated following our observation that aripiprazole affects C. albicans physiology, we were not able to discount that the drug directly or indirectly affects the host's response to infection, resulting in a less effective defense.Curiously, aripiprazole also appears to affect the human macrophage respon ses, dampening the release of two key pro-inflammatory cytokines in response to fixed C. albicans.Thus, it is possible aripiprazole may exert both drug-fungus and drug-host interactions that impact the outcome of invasive C. albicans infections.Indeed, aripiprazole use in humans has also been associated with decreased pro-inflammatory cytokine levels (51).Based on previous studies, both TNF-α and IL1-β are expected to have a protective role in disseminated C. albicans infection (52)(53)(54); thus, it is not surprising that suppression of their release from key cells of the innate immune response would have a detrimental impact on the outcome of infection in mice.Nonetheless, at first glance, the suppression of key pro-inflammatory responses by macrophages is inconsistent with the apparently hyperinflammatory lesions observed in aripiprazoletreated mice infected with C. albicans.These findings could be reconciled by the inherent complexity and multi-faceted nature of the mechanisms by which mammals detect and respond to infectious fungi.Hematopoietic, epithelial, and endothelial cells all make important contributions to host defense against pathogenic fungi (49), with each responding to a discrete cohort of PAMPs and often producing qualitatively or quantita tively distinct responses.For example, macrophages preferentially respond to C. albicans hyphal over yeast forms to produce pro-inflammatory cytokines (55); peripheral blood mononuclear cells (PBMCs), on the other hand, respond more vigorously to the yeast form (56). Thus, it is possible that the very same aripiprazole-induced changes in PAMP presentation that suppress macrophage responses could hyper-stimulate pro-inflammatory responses in other host cell types.Either way, both our in vitro and in vivo data clearly establish that aripiprazole changes the nature of the C. albicans-host interaction in ways that can affect disease progression.
In a broader context, this study raises the issue of biologically active molecules including the medications consumed by colonized or infected individuals disrupting either mammalian or fungal physiology and thus potentially the equilibrium of the host-pathogen interaction.Drug-fungus interactions, for example, could alter the outcome of infection by: (i) causing profound physiological dysfunction that compromi ses the viability, fitness, or pathogenicity of the fungus in vivo, (ii) changing the surface characteristics of the fungus in ways that alter its immunogenicity, binding/activation of complement, or sensitivity to host-derived antimicrobial peptides, or (iii) altering fungal sensitivity to antifungal drugs.It is also important to consider the effects of each drug at therapeutically relevant drug concentrations, which, in the context of disseminated infection, most investigators will take as unbound blood serum concentra tions from patients on a standard dosing regimen.However, Candida species naturally colonize the gastrointestinal (GI) tract, where the concentration of orally administered drugs (or those excreted via the GI tract) is potentially much higher.Additionally, drug concentrations within specific tissues can differ markedly from serum, and extensive tissue dissolution within foci of infection could have further effects.We observed C. albicans-infected mice had significantly higher serum concentrations of aripiprazole (~1,500 ng/mL) than uninfected mice treated with the same dose (~450 ng/mL) (Fig. S6).This is most likely because aripiprazole is almost entirely removed by metabolism in the liver, and often, genes encoding drug-metabolizing enzymes are downregulated by systemic inflammation and similar stress (like acute infection) (57)(58)(59)(60).Aripiprazole is extensively metabolized by the CYP450 enzymes CYP2D6 and CYP3A4, and fluconazole is also a moderate CYP3A4 inhibitor.This could lead to reduced drug metabolism and thus increased plasma concentration.Such effects can further modulate host response as well as fungal physiology.
The effect of any individual medication on the outcome of IFIs is unlikely to be immediately apparent in the clinical setting due to the intricacies of each infected individual's circumstances that include complex and highly varied drug regimens provided and relatively small numbers of infected patients treated with any given medication.Additionally, the effects of drug-fungus or even drug-host interactions upon the course of an IFI are also likely to be context-dependent, with many factors such as the causative species, specific nature and severity of a patient's immune deficiency, site of infection, and genetic factors being key determinants.Thus, it remains to be determined how many drugs approved for human use can modulate the outcome of IFIs.Nonetheless, the findings of this study can serve to raise awareness of the potential influence of non-antifungal medications on the outcome of invasive fungal infections.

FIG 1
FIG 1 Aripiprazole and pinaverium alter C. albicans cellular morphology.(A) C. albicans strain SC5314 was subcultured at 1 × 10 4 cells/mL in RPMI-pH 7 with 5 µM of aripiprazole, pinaverium, or 0.5% DMSO (vehicle) and incubated at 35°C for 48 hours.Growth was monitored as OD 600nm every 30 minutes.Experiments were performed in biological triplicates, and plots indicate the mean ± standard deviation for the OD 600nm value at each time point.(B) SC5314 was subcultured at 1 × 10 6 cells/mL in RPMI-pH 7 with 5 µM of either drug or vehicle and incubated at 35°C for 16 hours.Cells were harvested on filters, lyophilized, and weighed.Individual points indicate data from four independent experiments, and cross bar indicates the mean.Significance was calculated using a one-way analysis of variance (ANOVA) with Dunnet's post-test (C).SC5314 was suspended at 1 × 10 7 cells/mL in PBS, and 2 µL was spotted on M199 agar supplemented with 5 µM of either drug or vehicle.Colonies were imaged after 96 hours of incubation at 37°C.(D).SC5314 was suspended in RPMI-pH 7 with either drug or vehicle and incubated at 35°C for 16 hours and fixed in 10% formalin, and cellular morphology examined microscopically with a 40× objective.*P < 0.05.ns = not significant.

FIG 2
FIG 2 Azole-antagonistic drugs alter the transcription of a small and partially overlapping set of C. albicans genes.SC5314 was subcultured into RPMI-pH 7 supplemented with 5 µM of aripiprazole, mometasone, etofenamate, or 0.5% DMSO (vehicle) and cultured for 6 hours at 35°C.Total RNA was extracted, and transcriptional profiling was performed.Drug-responsive genes were defined as those increased or decreased by >2-fold (adjusted P < 0.05) compared with vehicle-treated samples in each of three independent biological replicates.(A and B) Venn diagram showing the number of gene transcripts for significantly upregulated genes (A) or downregulated genes (B).(C) Genes identified that were responsive to all three drugs.

FIG 3
FIG 3 Aripiprazole-related compounds also possess azole antagonistic activity.(A) Structures of aripiprazole and structurally related drugs, bexpiprazole and cariprazine.Shared sub-structures are designated as follows: (2,3-Dichlorophenyl)piperazine (black square) and 3,4-Dihydro-2(1H)-quinoline (gray square).(B) Fluconazole antagonistic activity was determined by CLSI susceptibility assays conducted with C. albicans SC5314 as well as strains lacking either Tac1p or Upc2p in RPMI-pH 7 with 5 µM of each compound or 0.5% DMSO (vehicle control).Minimum inhibitory concentrations (MICs) were evaluated at 24 hours for each compound relative to the vehicle control.Experiments were run in biological duplicates.

FIG 4
FIG 4 Aripiprazole exposure exacerbates the severity of disseminated C. albicans infection in mice.(A) Female BALB/c mice (n = 6 per group) were treated daily with 5 mg/kg/day aripiprazole or vehicle starting 3 days prior to infection.On day 0, mice were infected intravenously with ~2.5 × 10 5 CFU of SC5314.Fluconazole (5 mg/kg/day -FLUC) was administered 24 hours post infection, and mice were euthanized 5 days p.i. Kidneys were extracted and homogenized, and fungal burden quantified as colony forming units per gram of tissue.Data are the mean ± standard deviation, and statistical significance was calculated using a one-way ANOVA with Kruskal-Wallis post-test.†Only four mice plotted for ARI/VEH group as 2 succumbed prior to the endpoint.(B) Groups of BALB/c mice (n > 11) were treated daily with 10

FIG 5 Full
FIG 5 Aripiprazole treatment increases fungal invasion and kidney damage.(A) BALB/c mice were treated daily with either 10 or 2 mg/kg/day aripiprazole or vehicle starting 3 days prior to infection.Mice were then infected with 2.5 × 10 5 CFU of SC5314 or PBS (uninfected control).At the 48 hour endpoint, mice were euthanized; kidneys were extracted and stained with H&E (top panel) or GMS (bottom panel) for each condition.Pictures are representative (n = 3 per group).(B) Fungal burden was quantified as colony forming units per gram of kidney tissue of mice described for panel (A).Data are the mean ± standard deviation, and statistical significance was calculated using a one-way ANOVA with Kruskal-Wallis post-test (n > 11).(C and D) Blood was extracted from mice described in (A).Serum was processed, and blood urea nitrogen (BUN) (C) or creatinine (D) levels were quantified.Groups were compared with uninfected vehicle.Data are the mean ± standard deviation, and significance was determined using a one-way ANOVA with Kruskal-Wallis post-test (n > 11).*P < 0.05, **P < 0.005.ns = not significant.

FIG 6
FIG 6 Aripiprazole does not affect the outcome of disseminated C. albicans infection in cyclophosphamide-treated mice.(A) BALB/c mice (n = 8 per group) were treated with either 10 mg/kg/day aripiprazole or vehicle daily starting 3 days prior to infection.They were also administered cyclophosphamide (150 mg/kg) 2 days prior to infection and every 3 days thereafter.Mice were then infected with 2.5 × 10 4 CFU of C. albicans SC5314 and monitored.The survival of the infected drug-treated groups was compared with the infected mock-treated group using the log-rank test.(B) Groups of CD-1 mice (n = 8) were treated as described in (A) and infected with 5 × 10 4 CFU of C. albicans SC5314, and survival monitored for 10 days.Survival was compared using a log-rank test.(C) CD-1 mice were treated and infected as described in (B), and mice were euthanized 72 hours p.i. Kidneys were extracted and stained with either H&E (top panel) or GMS (bottom panel).

FIG 7
FIG 7 Aripiprazole modulates C. albicans cell wall chitin content.SC5314 was cultured in YNB-pH 7 with either 5 µM of aripiprazole or 0.5% DMSO (vehicle) at 30°C for 24 hours.Cells were then fixed in formalin, washed and stained for mannan (concanavalin A-FITC conjugate) (A), chitin content (calcofluor white) (B), chitin exposure (wheat germ agglutinin-Alexa Fluor 488 conjugate) (C), or β-glucan content (aniline blue) (D).Fluorescence intensity was measured by flow cytometry, and drug-treated groups were normalized to the paired vehicle control.Each data point represents the median fluorescence of 20,000 events, and data are represented as the mean ± standard deviation of six independent experiments.Statistical significance was calculated using 2-tail unpaired Student's t-test.*P < 0.05.ns = not significant.

FIG 9
FIG 9 Aripiprazole alters C. albicans-macrophage interaction.(A) C. albicans strain SC5314 was grown in YNB-pH 7 medium supplemented with 0.2-5 µM aripiprazole or 0.5% DMSO (vehicle control) for 24 hours at 30°C to achieve yeast form cells before being fixed in 10% formalin.Macrophages were co-incubated with fixed yeast at an MOI of 20:1 at 37°C for 24 hours, and supernatant was collected.ELISA assays were performed to quantify TNF-α or IL-1β.Experiments were conducted in technical quadruplicates and performed independently in biological triplicates.Data are depicted as the mean ± standard deviation.Statistical significance was calculated using one-way ANOVA test with Dunnet's post-test.(B) SC5314 was grown in RPMI-pH 7 medium supplemented with the same aripiprazole concentrations or vehicle control for 1.5 hours at 37°C to encourage germ-tube formation, then fixed as described in (A).Macrophages were co-incubated with fixed germ-tube cells atan MOI of 20:1 at 37°C for 8 hours before supernatant was collected.ELISA assays were performed as described in (A).(C and D) SC5314 was grown as yeast (C) or germ tubes (D) and fixed as described above.Cells were added to macrophages at an MOI of 20:1 in the presence of 0.2-5 µM ARI or 0.5% DMSO.Macrophages were co-incubated as described in (A) or (B).TNF-α and IL-1β release were quantified by ELISA.Data are the mean ± standard deviation.Statistical significance was calculated using one-way ANOVA with Dunnet's post-test.*P < 0.01, **P < 0.001, ****P < 0.0001.ns = not significant.