Inhibition of Biofilm Formation by Candida albicans and Polymicrobial

May 5, 2019 - Candida albicans is an opportunistic pathogenic yeast and is responsible for candidiasis. It readily colonizes host tissues and implant ...
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Article Cite This: ACS Infect. Dis. 2019, 5, 1177−1187

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Inhibition of Biofilm Formation by Candida albicans and Polymicrobial Microorganisms by Nepodin via Hyphal-Growth Suppression Jin-Hyung Lee,†,⊥ Yong-Guy Kim,†,⊥ Sagar Kiran Khadke,† Aki Yamano,‡ Akio Watanabe,*,§ and Jintae Lee*,† †

School of Chemical Engineering, Yeungnam University, 280 Daehak-Ro, Gyeongsan 38541, Republic of Korea Okinawa Research Center Company, Ltd., 12-75 Ulumasi, Okinawa 904-2234, Japan § Research Institute for Biological Functions, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan

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ABSTRACT: Candida albicans is an opportunistic pathogenic yeast and is responsible for candidiasis. It readily colonizes host tissues and implant devices, and forms biofilms, which play an important role in pathogenesis and drug resistance. In this study, the antibiofilm, antihyphal, and antivirulence activities of nepodin, isolated from Rumex japonicus roots, were investigated against a fluconazole-resistant C. albicans strain and against polymicrobial-microorganism-biofilm formation. Nepodin effectively inhibited C. albicans biofilm formation without affecting its planktonic cell growth. Also, Rumex-root extract and nepodin both inhibited hyphal growth and cell aggregation of C. albicans. Interestingly, nepodin also showed antibiofilm activities against Candida glabrata, Candida parapsilosis, Staphylococcus aureus, and Acinetobacter baumannii strains and against dual biofilms of C. albicans and S. aureus or A. baumannii but not against Pseudomonas aeruginosa. Transcriptomic analysis performed by RNA-seq and qRT-PCR showed nepodin repressed the expression of several hypha- and biofilm-related genes (ECE1, HGT10, HWP1, and UME6) and increased the expression of several transport genes (CDR4, CDR11, and TPO2), which supported phenotypic changes. Moreover, nepodin reduced C. albicans virulence in a nematode-infection model and exhibited minimal cytotoxicity against the nematode and an animal cell line. These results demonstrate that nepodin and Rumex-root extract might be useful for controlling C. albicans infections and multispecies biofilms. KEYWORDS: dual biofilms, Candida albicans, hyphae, nepodin, Staphylococcus aureus, Acinetobacter baumannii

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Candida albicans is one of the most common fungal pathogens, causing systemic and invasive infections. It is also responsible for life-threatening candidiasis.7 C. albicans colonizes host tissues, catheters, and indwelling medical devices8,9 and develops biofilms on biotic and abiotic surfaces that are often tolerant to conventional antifungal therapeutics and the host immune system.10 C. albicans biofilms are composed of yeast, hyphal, and pseudohyphal elements,10 and the transition of yeast cells to hyphae appears to control biofilm formation. In fact, hyphal transition is considered a

iofilms play important roles in infectious diseases and in a variety of device-related infections.1,2 Because of their greater tolerance to antimicrobial treatments, biofilms formed by pathogens can pose serious problems to human health.3 Furthermore, several pathogenic microorganisms often form multispecies biofilms, which further increase tolerance to antimicrobial agents.4,5 Therefore, novel strategies are required to control single and multispecies pathogenic biofilms and to identify nontoxic approaches. Unlike strategies based on antibiotics or antifungal agents that aim to inhibit cell growth, it is important that we identify biofilm inhibitors that do not inhibit planktonic cell growth in order to reduce the risk of drug resistance.6 © 2019 American Chemical Society

Received: January 28, 2019 Published: May 5, 2019 1177

DOI: 10.1021/acsinfecdis.9b00033 ACS Infect. Dis. 2019, 5, 1177−1187

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Figure 1. Antibiofilm activity of nepodin against C. albicans. (A) Antibiofilm activity of nepodin against C. albicans DAY185 in PDB medium determined after incubation for 24 h. The chemical structure of nepodin is shown. (B,C) Planktonic cell growth of C. albicans DAY185 in the presence of nepodin (B) under shaking at 250 rpm in a 250 mL flask and (C) without shaking in 96-well plates. (D) Biofilm formation by C. albicans on polystyrene plates in the presence of nepodin at concentrations of 2 and 10 μg/mL as observed by confocal laser microscopy. Scale bars represent 100 μm. (E,F) Biofilm formation quantified by (E) COMSTAT analysis and (F) biofilm dry weight. *P < 0.05 vs nontreated controls. “None” indicates the nontreated control.

crucial virulence factor in Candida infections.11 However, many clinical C. albicans strains exhibit drug resistance against commercial antifungals (e.g., azoles and polyenes), which often have chemical-toxicity issues.12−14 Therefore, novel antibiofilm and antivirulence drugs are required to suppress C. albicans biofilm formation and virulence. Plants and phytochemicals are rich sources of antifungal and antibiofilm agents against drug-resistant microorganisms.15 Nepodin from Rumex crispus L. (Polygonaceae) has been previously shown to have in vivo antifungal activity against powdery mildew fungi16 and antibacterial activity against Edwardsiella ictaluri and Streptococcus iniae.17 Nepodin was reported to exhibit antidiabetic activity with minimal cytotoxicity.18 Nepodin has also been found to possess antimalarial19 and anti-inflammatory activities.20 However, the antibiofilm activities of nepodin have not been studied in yeast or any bacterial species. We considered that nepodin would suppress C. albicans biofilm formation because the nepodin-like phytochemicals eugenol,21 thymol,22 purpurin,23 quercetin,24 and alizarin25 have been shown to have antibiofilm activities against C. albicans. Hence, in the present study, the antibiofilm activity of nepodin was initially investigated against an antifungal-resistant C. albicans strain. To understand how nepodin and Rumex japonicus extract inhibit biofilm formation, confocal laserscanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to investigate morphological changes, biofilm formation, and hyphal growth of C. albicans. Furthermore, the molecular basis of alterations in C. albicans

physiology upon exposure to nepodin was also investigated using RNA-seq and qRT-PCR. In addition, the antibiofilm activities of several nepodin derivatives were investigated to determine chemical structure−activity relationships, and the antibiofilm activity of nepodin was studied in two dual-species biofilm models: C. albicans with Staphylococcus aureus or Acinetobacter baumannii. Finally, an in vivo Caenorhabditis elegans model was used to confirm the antivirulence efficacy of nepodin and its noncytotoxic nature.

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RESULTS Inhibition of C. albicans Biofilm Formation by Nepodin. The antibiofilm activity of nepodin was initially investigated against fluconazole-resistant C. albicans DAY185, and planktonic cell growth was also measured in the presence of nepodin. Nepodin at concentrations of 2 and 5 μg/mL inhibited C. albicans biofilm formation by 90 and 97%, respectively (Figure 1A). However, nepodin at a concentration of 20 μg/mL slightly reduced the planktonic cell growth of C. albicans (by 1000 μg/mL. These results support the notion that biofilm formation by C. albicans was effectively inhibited by the antibiofilm activity of nepodin and not by its fungicidal activity, and suggested that unlike conventional fungicides, nepodin may be less prone to the development of drug resistance. Biofilm inhibition was analyzed by CSLM. Although C. albicans formed dense biofilms (thickness >40 μm and almost 1178

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results were in line with the observed antibiofilm activity of nepodin. Furthermore, nepodin inhibited biofilm and hypha formation and cell aggregation of another C. albicans strain, ATCC 10231, without affecting its planktonic cell growth (Figure S2), and it also inhibited biofilm formation of two nonC. albicans strains, Candida glabrata and Candida parapsilosis (Figure S3). Taken together, these results show nepodin potently inhibited hypha formation and cell aggregation and reduced biofilm formation by C. albicans. Inhibition of C. albicans Biofilm and Hyphal Formation by R. japonicus Root Extract. Rumex plant species, such as R. japonicus and R. crispus, have been reported to produce nepodin.16,18 Hence, R. japonicus root extract, which contains ∼30% nepodin, was prepared and investigated with respect to biofilm inhibition and hyphal inhibition. As was expected, R. japonicus root extract markedly and dosedependently inhibited C. albicans biofilm formation (Figure 3A). More specifically, R. japonicus root extract at 5 μg/mL inhibited C. albicans biofilm formation by 85%, and at 10 μg/ mL, it almost abolished C. albicans biofilm formation in 96-well polystyrene plates (Figure 3A). As was observed for nepodin, R. japonicus extract at 20 μg/mL slightly reduced (by 20%) the planktonic cell growth of C. albicans in 96-well plates (Figure 3B) with an MIC of >2000 μg/mL against C. albicans DAY185. Also, the R. japonicus extract at 2 or 10 μg/mL markedly reduced biofilm densities and thicknesses (Figure 3C) and reduced hypha formation in PDB medium (Figure 3D). These results suggest that a nepodin-rich plant extract could be directly used to diminish Candida biofilm and hypha formation. Antibiofilm Activities of Nepodin-like Compounds. Because nepodin is a derivative of naphthalene or naphthol, we also investigated the antibiofilm activities of six nepodin-like compounds, namely, 2-isopropylnaphthalene, 1′-hydroxy-2′acetonaphthone, 2′-hydroxy-1′-acetonaphthone, 6-acetyl-2naphthol, 4-acetyl-1-naphthol, and naproxen. Of these six compounds, naproxen and 4-acetyl-1-naphthol at 20 μg/mL inhibited C. albicans biofilm formation by ≥60% versus biofilm formation in the untreated controls, whereas the other four compounds showed no activity at concentrations up to 50 μg/ mL (Figure 4). Previously, other compounds with motifs similar to that of nepodin, such as eugenol,21 thymol,22 purpurin,23 quercetin,24 and alizarin,25 were reported to exhibit antibiofilm activities against C. albicans. On the basis of these structural similarities, it would appear the presence of two hydroxyl groups in nepodin is largely responsible for its antibiofilm properties, because the presence of methyl or methoxy groups had little effect on antibiofilm activities. Antibiofilm Activities of Nepodin against Dual Biofilms of C. albicans and S. aureus or A. baumannii. C. albicans often forms biofilms with bacteria like S. aureus, Pseudomonas aeruginosa, and Enterococcus species on host and environmental surfaces; these biofilms are formed more rapidly and have greater antimicrobial tolerance.28 We investigated the antibiofilm effects of nepodin on single species biofilms and dual-species biofilms. Initially, we investigated the inhibitory effects of nepodin on biofilms formed by S. aureus, P. aeruginosa, and A. baumannii, individually. Biofilm formation by S. aureus and A. baumannii was dose-dependently inhibited by nepodin (Figure 5A,D), whereas biofilm formation by P. aeruginosa was not (data not shown). This is the first report of the antibiofilm activities of nepodin against S. aureus and A. baumannii. Specifically, nepodin at 5 μg/mL inhibited S. aureus

100% surface coverage) in the nontreated controls, nepodin at 2 or 10 μg/mL dramatically reduced biofilm densities and thicknesses (Figure 1D). Biofilm reduction was also confirmed by COMSTAT analysis, which showed that nepodin at 2 and 10 μg/mL significantly reduced biofilm biomass, average thickness, and substrate coverage (Figure 1E). Specifically, biofilm biomass, thickness, and substrate coverage were reduced by 10 μg/mL nepodin by >95% versus those of the untreated controls. Also as expected, the biofilm dry weight was decreased by nepodin treatment (Figure 1F). Moreover, C. albicans biofilm formation was reduced by nepodin in a silicon catheter for 5 days (Figure S1). Inhibition of Hyphal Growth and Cell Aggregation by Nepodin. It is widely held that the yeast-to-hypha transition and cell aggregation are prerequisites of biofilm development by C. albicans.26 To investigate the effect of nepodin on C. albicans morphology, two culture media were used. In PDB medium, nontreated control C. albicans consisted of mixtures of pseudohyphae and hyphae and few yeast cells. However, nepodin treatment at 2 or 10 μg/mL markedly reduced hypha formation (Figure 2A). Cell-aggregation assays were per-

Figure 2. Inhibition of hyphal filamentation and aggregation by nepodin. (A) Inhibition of hyphal filamentation in PDB medium and (B) inhibition of cell aggregation in RPMI medium containing 10% bovine serum. Hyphae were visualized after incubation for 24 h. The scale bar represents 200 μm. “None” indicates the nontreated control. (C) SEM observation of hyphal inhibition in C. albicans biofilms by nepodin in PDB medium. The scale bar represents 30 μm.

formed using RPMI-1640 medium containing 10% bovine serum, which promotes hypha formation.27 After incubation for 24 h, mostly hyphae and large cell aggregates entangled by hyphae were observed in the nontreated controls, whereas nepodin treatment dose-dependently resulted in much smaller cell aggregates (Figure 2B). Also, SEM analysis showed nepodin at 2 or 10 μg/mL in PDB medium substantially suppressed the hyphal transition on nylon membranes (Figure 2C). Untreated-control-biofilm cells were predominantly large hyphal cells, whereas nepodin-treated cells were mainly yeast cells with few hyphae. In addition, hyphal and cell-aggregation 1179

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Figure 3. Inhibitory effects of Rumex extract on C. albicans biofilm and hypha formation. (A) Antibiofilm activity of Rumex japonicus extract (containing ∼30% nepodin) against C. albicans in PDB medium determined after incubation for 24 h. (B) Planktonic cell growth of C. albicans in the presence of R. japonicus extract without shaking in 96-well plates. (C) Biofilm formation by C. albicans on polystyrene plates in the presence of R. japonicus extract as observed by confocal laser microscopy. Scale bars represent 100 μm. (D) Inhibition of hyphal filamentation by R. japonicus extract in PDB medium. Hyphae were visualized after incubation for 24 h. The scale bar represents 200 μm.

Figure 4. Antibiofilm activities of nepodin-like compounds. Inhibitory effects of six nepodin-like compounds on C. albicans biofilm formation assessed in PDB medium after incubation for 24 h: (A) 2-isopropylnaphthalene, (B) 1′-hydroxy-2′-acetonaphthone, (C) 2′-hydroxy-1′acetonaphthone, (D) 6-acetyl-2-naphthol, (E) 4-acetyl-1-naphthol, and (F) naproxen. Chemical structures are shown.

decent growth of planktonic cells (∼0.8 at OD620) and biofilm formation (∼3.0 at OD570) were achieved for C. albicans and S. aureus separately and in coculture in 96-well plates (Figure 5B). Importantly, nepodin treatment at 10 μg/mL reduced dual-biofilm formation by >85%. Also, SEM analysis was used to visualize the dual biofilms. As previously reported,28,29 we observed polymicrobial associations of C. albicans and S. aureus in mixed media in nontreated control samples, on the basis of the observation that C. albicans formed large hyphae, and there were few yeast cells that were much larger than S. aureus cells.

biofilm formation by 75% after incubation for 24 h, nepodin at 10 μg/mL delayed planktonic cell growth of S. aureus (Figure S6), and the MIC of nepodin was ∼20 μg/mL against S. aureus ATCC 6538. These results suggest that the antibiofilm activity of nepodin was partially caused by antibiotic activity toward S. aureus. To form dual biofilms of C. albicans and S. aureus, we used a 50/50 mixture of PDB and LB media containing (23 ± 3) × 105 and (59 ± 4) × 107 CFU, respectively, to induce the growth of C. albicans and S. aureus.28 Under this condition, 1180

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Figure 5. Antibiofilm activities of nepodin in the dual-species biofilm models. (A) Antibiofilm activities of nepodin against S. aureus in LB medium. (B) Inhibitory effect of nepodin on dual C. albicans and S. aureus biofilms, determined in a 50/50 mix of PDB and LB media after incubation for 24 h. (C) SEM observation of dual biofilms of C. albicans and S. aureus. (D) Inhibition of A. baumannii biofilms in TSB medium. (E) Inhibition of dual biofilms of C. albicans and A. baumannii, investigated in a 50/50 mixture of PDB and TSB media after incubation for 24 h. (F) SEM observation of dual biofilms of C. albicans and A. baumannii. In the insets, the large cells are C. albicans and the small cells are (C) S. aureus or (F) A. baumannii (indicated by yellow triangles). The scale bar represents 30 μm.

descriptions,31 and KEGG analysis was used to elucidate the impacts of the biological pathways targeted by nepodin. GO analysis showed that a high percentage of genes were hypothetical genes; genes for integral membrane components; oxidoreductase-activity genes; transport genes; and genes associated with pathogenesis, filamentous growth, and biofilm formation (Figure S4A,B). KEGG analysis also showed that nepodin treatment targeted metabolic processes, such as the biosynthesis of secondary metabolites, the biosynthesis of antibiotics, glycolysis, and glycerophospholipid and carbon metabolism (Figure S4C). To validate the RNA-seq data, we selected 20 genes that were highly differentially expressed or known to be involved in biofilm and hypha formation and performed qRT-PCR experiments. The qRT-PCR assays showed that the differential changes generally concurred with RNA-seq results (Figure 6 and Table S2). For example, among the genes induced by nepodin, the following increases were found by using qRTPCR and RNA-seq, respectively: 14-fold versus 84-fold for

Furthermore, it appeared that S. aureus cells were encased in C. albicans hyphae (Figure 5C). We observed that nepodin obviously reduced hypha formation of C. albicans and widely distributed S. aureus cells. A dual-biofilm system of C. albicans and A. baumannii was also investigated, with a 50/50 mix of PDB and TSB media containing (23 ± 3) × 105 and (87 ± 13) × 107 CFU, respectively. Nepodin was also found to dose-dependently reduce dual-biofilm formation in this system (Figure 5E). SEM analysis showed that the small A. baumannii cells were attached to C. albicans cells, forming dual biofilms (Figure 5F), as previously reported.30 More importantly, nepodin at 20 μg/mL reduced dual-biofilm formation by >75%. This is the first report of the inhibition of the formation of dual biofilms of C. albicans and A. baumannii. However, nepodin could not eradicate preformed biofilms of C. albicans with S. aureus or C. albicans with A. baumannii (data not shown). Future in vivo studies are required to investigate the efficacies of nepodin and Rumex extract against polymicrobial biofilms. Differential Gene Expression Induced by Nepodin in C. albicans. To understand the molecular basis responsible for biofilm inhibition by nepodin, RNA-seq was first used to identify genes differentially expressed by at least 2-fold in nepodin-treated C. albicans cells. A total of 6024 genes were differentially expressed by nepodin at 10 μg/mL (2929 genes were upregulated, and 3095 were downregulated) indicating that nepodin very widely affects gene expression of C. albicans cells. In order to identify genes more affected by nepodin, we applied a 5-fold-differential-expression criterion. The 170 genes so identified were sorted into five functional categories, namely, biofilm-related genes, hypha- and filamentousgrowth-related genes, virulence-related genes, stress-response genes, and other genes or hypothetical genes (Table S1). Notably, the addition of nepodin significantly changed (upand downregulated) the expression of 22 biofilm- and hypharelated genes by more than 5-fold. In addition, gene-ontology (GO) analysis was used improve the accuracy of gene-product

Figure 6. Relative transcriptional profiles of C. albicans cells treated with and without nepodin. C. albicans was incubated with or without nepodin at 10 μg/mL for 4 h with shaking at 250 rpm. Transcriptional profiles were obtained by qRT-PCR. Fold changes represent changes in the transcription of treated versus untreated C. albicans. The experiment was performed in duplicate, and six qRT-PCR reactions were performed per gene. *P < 0.05 vs nontreated controls. “None” indicates the nontreated control. 1181

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Figure 7. Combinatory effects of nepodin and an efflux-pump inhibitor or an antifungal. Antibiofilm activity of nepodin (1 μg/mL) along with (A) efflux-pump inhibitor FK506, (B) antifungal agent amphotericin B, or (C) antifungal agent butoconazole against C. albicans DAY185 in PDB medium, determined after incubation for 24 h. *P < 0.05 vs nontreated controls. “None” indicates the nontreated control.

Figure 8. Chemical structures of phytochemicals that have antibiofilm activity against C. albicans and are also similar on a structural level to nepodin.

CDR4, 23-fold versus 11-fold for CDR11, 131-fold versus 63fold for IFD6, 37-fold versus 49-fold for TPO2, 162-fold versus 34-fold for UCF1, and 54-fold versus 51-fold for YHB1. Among the genes repressed by nepodin, the following decreases were found by using qRT-PCR and RNA-seq, respectively: 5-fold versus 14-fold for ECE1, 5-fold versus 12-fold for HWP1, 4fold versus 2-fold for UME6, and 15-fold versus 31-fold for HGT10, respectively. The expression of other biofilm- and hypha-related genes (ALS1, BCR1, CYR1, EFG1, RAS1, and TUP1) was less affected by nepodin. Taken together, qRTPCR and RNA-seq results showed that nepodin significantly repressed biofilm- and hypha-related genes (ECE1, HGT10, HWP1, and UME6) and induced transporter-related genes (CDR4, CDR11, and TPO2) and biofilm-related genes (IFD6, UCF1, and YHB1). Coadministration of Nepodin and an Efflux-Pump Inhibitor or Antifungal Agent. Because C. albicans cells overexpressed several transporter genes (CDR4, CDR11, and TPO2) in the presence of nepodin, an additional biofilm assay was performed with an efflux-pump inhibitor, FK506,32 along with nepodin. The antibiofilm efficacy of nepodin was clearly enhanced in the presence of FK506 (Figure 7A). Furthermore, antifungal agents amphotericin B and butoconazole33 were administered along with nepodin. Coadministration of antifungal agents and nepodin significantly enhanced the antibiofilm efficacy of nepodin (Figure 7B,C). Reduction of C. albicans Virulence in a Nematode Model by Nepodin with Minimal Cytotoxicity. The

hyphal form of C. albicans is more lethal to C. elegans than its yeast form because the hyphae pierce the nematode’s cuticle and kill the worm.34 Hence, we investigated whether nepodin could reduce C. albicans virulence in a C. elegans nematode model. C. albicans infection caused 85% C. elegans fatality (15% survival) in 4 days, but the presence of nepodin at 5 or 10 μg/ mL rescued C. elegans (50% survival, Figure S5A,B). In addition, the cytotoxicity of nepodin was investigated using noninfected C. elegans (Figure S5C) and mouse B16 melanoma cells (Figure S5D). We found that nepodin was not toxic to C. elegans at concentrations up to 200 μg/mL (Figure S5C). Similarly, MTT assays showed that nepodin at up to 100 μg/ mL had no cytotoxic effects on mouse tumor B16 cells and only a slight effect at 200 μg/mL (Figure S5D). These results show that nepodin effectively reduced C. albicans virulence in the nematode model and that its cytotoxicity was minimal on the nematodes and mouse tumor B16 cells.

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DISCUSSION This study shows for the first time that nepodin from Rumexroot extract suppresses biofilm formation by a drug-resistant C. albicans strain and by polymicrobial biofilms of C. albicans and S. aureus or A. baumannii by inhibiting the hyphal growth of C. albicans without any cytotoxic effects. The switching of yeast cells to hyphal cells is considered as playing an important role in biofilm formation11 and the pathogenesis of fungal infections.35,36 Several compounds, including phytochemicals, have been reported to inhibit C. 1182

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albicans biofilm formation by reducing hypha formation.37−39 Thus, it appears that inhibition of the formation of hyphae is not rare in the plant kingdom, and it represents a practical means of inhibiting biofilm formation by C. albicans. The phytochemicals eugenol,21 thymol,22 purpurin,23 magnolol and honokiol,40 quercetin,24 emodin,41 hinokitiol,42 curcumin,43 and alizarin25 are similar at a structural level as they all possess aromatic structures functionalized with hydroxyl, alkyl, and methoxy groups (Figure 8). In the present study, transcriptomic studies showed that the expression of several hypha-specific and transport-related genes was substantially altered in nepodin-treated C. albicans cells (Figure 6). Specifically, ECE1, HGT10, HWP1, and UME6 were downregulated, and ABC transport genes (CDR4, CDR11, IFD6, and TPO2) were upregulated (Figure 6). HWP1 (also called ECE2) and ECE1 are essential for hyphal development, and their expression has been shown to be correlated with cell elongation, biofilm formation,10,44 and intercellular adhesion.45 UME6 is a filament-specific regulator of the hyphal extension of C. albicans,46 and it enhances biofilm formation.47 HGT10 is involved in glycerol acquisition48 but plays no known role in biofilms.49 Interestingly, the expression of TUP1 (another important regulator for hyphal growth) was not altered by nepodin. Therefore, it seems the suppression of hyphal growth and biofilm formation by nepodin may be partially explained by the repression of these hypha-specific genes (ECE1, HWP1, and UME6). On the other hand, nepodin upregulated the expressions of CDR1, CDR11, TPO2, IFD6, UCF1, and YHB1 by more than 20-fold (Figure 6). The expression of Candida drug resistance genes and genes for multidrug transporters and major ABC transporters (CDRs) have been reported as being positively correlated with increased azole resistance in C. albicans isolates and upregulated during biofilm growth.50,51 Like that of other transporter CDRs, gene expression of polyamine transport protein52 (TPO2) was also induced by nepodin. Although speculative, it may be that C. albicans strives to pump nepodin out of the cell via several transporter proteins. Additionally, coadministration of nepodin and an efflux-pump inhibitor could partially solve this issue (Figure 7A). Also, coadministration of nepodin and antifungal agents could enhance the antibiofilm activity of nepodin (Figure 7B,C). The alcohol dehydrogenase encoded by IFD6 is an inhibitor of biofilmmatrix production.53 Furthermore, a YHB1-deletion mutant was reported as being hyperfilamentous.54 These findings are in line with our observation that the overexpression of IFD6 and YHB1 by nepodin inhibits filamentation and biofilm formation. Although UCF1 is upregulated by cAMP in filamentous growth55 and upregulated by nepodin in the present study (Figure 6), the addition of cAMP to 10 mM does not complement hyphal growth or biofilm formation (data not shown). Also, the gene expression of other hyphal-regulatory genes, ALS1, ALS3, and TUP1, is relatively unaffected by nepodin (Figure 6), indicating that nepodin does not directly control the cAMP pathway and suggesting its action mechanism is complex. Nepodin has been found in the roots of R. japonicas18 and R. crispus,16 which are common perennials in East Asia that are used in herbal medicines to treat hemoptysis, scabies, hematochezia, and neurodermatitis.56 Furthermore, their pharmaceutical abilities to scavenge free radicals, inhibit proliferation, induce the apoptosis of cancer cells, and suppress pathogenic plant fungi have been described recently.56 In

addition, it has also been reported that nepodin exhibits antidiabetic activity and minimal cytotoxicity,18 and its low cytotoxicity has been confirmed in this study (Figure S5C,D).

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CONCLUSION The continuing evolution of drug-resistant microorganisms has driven the development of novel antifungals and antibiotics. This study shows that the antibiofilm and antihyphal activities of nepodin on C. albicans are due to prevention of the yeast− hypha transition and not to the inhibition of fungal growth. Nepodin was also found to inhibit the formation of biofilms composed of C. albicans and S. aureus or A. baumannii and to reduce C. albicans virulence effectively in vivo in a C. elegans model with minimal cytotoxicity. In conclusion, our findings indicate that nepodin and Rumex extract present bases for the design of potent antibiofilm and anti-hypha-formation agents.

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EXPERIMENTAL SECTION Strains, Chemicals, and Culture Materials. For this study, fluconazole-resistant C. albicans strain DAY185 (MIC > 1024 μg/mL), C. albicans ATCC 10231, C. glabrata ATCC 2001, and C. parapsilosis ATCC 22019 were used and cultured in potato dextrose agar (PDA), potato dextrose broth (PDB), or yeast malt (YM) medium supplemented with 2% glucose. S. aureus strain ATCC 6538 and P. aeruginosa PAO157 were cultured in LB medium, and A. baumannii ATCC 17978 was cultured in trypticase soy agar (TSA) or trypticase soy broth (TSB). All experiments were performed at 37 °C. Nepodin (>98%) and naproxen (>98%) were purchased from Santa Cruz Biotech., Inc. 2-Isopropylnaphthalene (>95%), 1′hydroxy-2′-acetonaphthone (>98%), and 2′-hydroxy-1′-acetonaphthone (>98%) were purchased from TCI Company, and 6-acetyl-2-naphthol (>97%) and 4-acetyl-1-naphthol (>97%) were purchased from Sigma-Aldrich. They were dissolved in dimethyl sulfoxide (DMSO), which was used as a negative control in all experiments at a concentration in media of ≤0.1% (v/v); it did not affect cell growth or antibiofilm activity. R. japonicus root extract containing 30% (w/v) nepodin was prepared as follows. The roots of R. japonicus (7.0 kg) were cut into small pieces and extracted with 95% ethanol (14 L) for 9 h. The aqueous ethanol extract was then concentrated under reduced pressure, and water was added. This aqueous mixture was then centrifuged at 7000 rpm for 10 min at room temperature. Two liters of 0.1 M sodium hydroxide solution was added to the obtained solid residue, and the solution was sonicated for 10 min and centrifuged at 7000 rpm for 10 min at room temperature. The obtained supernatant was adjusted to pH 7.0 with acetic acid and centrifuged at 7000 rpm for 10 min. The residue so obtained (10.3 g; nepodin content of 30%, w/w) was stored in a refrigerator at 4 °C. Nepodin was identified as the major component in the residue by mass spectrometry and nuclear magnetic resonance (NMR) by comparing results with published data.17 Planktonic (free-floating) cell growths of bacteria were measured using a spectrophotometer (UV-160, Shimadzu) at 620 nm. The minimum inhibitory concentration (MIC) of nepodin was determined using a microdilution method in 96-well polystyrene plates (SPL Life Sciences) as previously described.58 Overnight cultures of C. albicans were treated with nepodin at various concentrations (0−1000 μg/ mL) and incubated at 37 °C for 24 h. The MIC was defined as 1183

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aggregation assay, C. albicans cells were inoculated into 2 mL of RPMI medium supplemented with 10% bovine serum, which promotes hypha formation,27 at density of 105 CFU/mL in 14 mL test tubes. After incubation for 24 h, aggregated cells were visualized in bright field using an iRiS Digital Cell Imaging System (Logos Bio Systems) at magnifications of 4× and 10×. At least four independent experiments were conducted. Hyphal Analysis by SEM. Scanning electron microscopy was used to observe hypha formation on nylon membranes, as previously described.63 Briefly, a nylon membrane was cut into 0.5 × 0.5 cm pieces and placed in 96-well plates containing C. albicans or mixed species grown with or without nepodin (2 and 10 μg/mL) and incubated for 24 h at 37 °C. PDB medium was used for growth of C. albicans biofilms, a 50/50 mix of PDB and LB media was used for the dual C. albicans and S. aureus biofilms, and a 50/50 mix of PDB and TSB media was used for the dual biofilms of C. albicans and A. baumannii. Cells that adhered to the nylon membrane were fixed with glutaraldehyde (2.5%) and formaldehyde (2%) for 24 h, then postfixed using osmium, and dehydrated using an ethanol series (50, 70, 80, 90, 95, and 100%) and isoamyl acetate. After critical-point drying, cells were examined and imaged using an S-4100 scanning electron microscope (Hitachi) at a voltage of 15 kV. RNA Isolation for Transcriptomic Studies. For transcriptomic analyses, 25 mL of C. albicans at an initial turbidity of 0.1 at OD600 (∼105 CFU/mL) was inoculated into PDB broth in 250 mL Erlenmeyer flasks and incubated for 4 h at 37 °C with agitation (250 rpm) in the presence or absence of nepodin (10 μg/mL). To prevent RNA degradation, RNase inhibitor (RNAlater) was added to the cells. Total RNA was isolated using a hot acidic phenol method,64 and RNA was purified using a Qiagen RNeasy Mini Kit. RNA-Seq Analyses. For RNA-seq analysis, an RNA library was constructed using the SMARTer Stranded RNA-Seq Kit (Clontech Laboratories, Inc.), as previously described.65 Differentially expressed genes were identified on the basis of counts from unique and multiple alignments using Bedtools.66 Read count (RT) data were processed by quantile normalization using Bioconductor.67 RNA-seq data were deposited at NCBI Gene Expression Omnibus and are accessible through accession number GSE119822. Differentially expressed genes were further analyzed using the DEG-analysis method in the Excel-based Differentially Expressed Gene Analysis program (ExDEGA). Gene-ontology analysis was performed at QuickGO (www.ebi.ac.uk/QuickGO/). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the RNA-seq data were performed with KEGG Mapper (http:// www.genome.jp/kegg/tool/map_pathway2.html). qRT-PCR. qRT-PCR was used to determine the expression of hypha-related genes (ALS1, ALS3, BCR1, CDR4, CDR11, CYR1, ECE1, EFG1, EFG1, GST3, HGC1, HGT10, HWP1, IFD6, RAS1, TPO2, TUP1, UCF1, UME6, and YHB1) and to confirm RNA-seq results. The specific primers and housekeeping gene (RDN18) used for qRT-PCR are listed in Table S3. The expression of RDN18 was not affected by nepodin. The qRT-PCR method used was that described by Kim et al.63 and was performed using SYBR Green Master Mix (Applied Biosystems) and an ABI StepOne Real-Time PCR System (Applied Biosystems). At least two independent cultures were used.

the lowest concentration that inhibited yeast growth by 80% as assessed by spectrophotometry (620 nm) and colony counting. Crystal-Violet Biofilm Assay. Biofilms of Candida and other bacteria were produced on 96-well polystyrene plates, as previously described.59 For C. albicans, a 2 day old single colony was inoculated into 25 mL of PDB medium and incubated overnight at 37 °C. Overnight cultures at an initial turbidity of 0.1 OD at 600 nm (∼105 CFU/mL) were then inoculated into PDB (final volume of 300 μL) with or without nepodin or Rumex extract and incubated for 24 h without shaking at 37 °C. S. aureus and P. aeruginosa PAO1 biofilms were produced in LB medium, and A. baumannii biofilms were produced in TSB medium. Biofilm cells that adhered to 96-well plates were stained with 0.1% crystal violet (Sigma-Aldrich) for 20 min, washed repeatedly with sterile distilled water, and resuspended in 95% ethanol. Plates were read at 570 nm, and the results are presented as the means of at least six repetitions. The inhibition percentage represents the relative biofilm formation: 100 × (biofilm formation with chemical/biofilm formation of untreated control). Biofilm Observations by CSLM. C. albicans biofilms or dual biofilms were formed on 96-well polystyrene plates with or without nepodin without shaking for 24 h. Planktonic cells were then removed by washing with distilled water three times, and the single or dual biofilm was stained with carboxyfluorescein diacetate succinimidyl ester (a minimally fluorescent lipophile; Invitrogen, Molecular Probes, Inc.).60 Plate bases were then visualized using a 488 nm Ar laser (emission 500 to 550 nm) under a confocal laser microscope (Nikon Eclipse Ti). To quantify biofilm structures, COMSTAT software61 was used to determine biovolumes (μM3/μM2), mean biofilm thicknesses (μM), and substratum coverages (%). Two independent cultures were performed under each experimental condition and at least 10 random positions were assayed. Determination of Biofilm Dry Weight. The dry weight of biofilm cells was measured as previously described using a filter paper.62 Briefly, C. albicans biofilms were produced in a 96-well plate for 24 h, and suspension cells were removed. Only biofilm cells were removed by the addition of PBS to each well with vigorous pipetting two times. All biofilm cells were collected on a preweighed nylon filter (0.45 μm pore size) and washed with PBS. The filter was dried at 85 °C for 5 h, and the dry weight of the cells was weighed and calculated. Two independent cultures were used. Biofilm Observation on a Silicon Catheter. Biofilms of C. albicans were produced on a silicon catheter, which was purchased from Sewoon Medical Company, Ltd. The cells were inoculated into PDB at an initial turbidity of 0.1 OD at 600 nm (∼105 CFU/mL) for overnight culture in glass-bottom dishes. At the beginning of the incubation, a 3 cm long half-cut catheter was placed in the middle of each dish, and the dishes were incubated with or without nepodin (10 μg/mL) at 37 °C. After 72 h of incubation, the medium was replaced with freshly made PDB and incubated 48 h more before the catheter was observed using CSLM. Observation of C. albicans Hyphae and Cell Aggregation in Liquid Media. For hyphal observations, C. albicans cells were inoculated into 2 mL of PDB medium at a density of 105 CFU/mL in 14 mL test tubes with or without nepodin (2 and 10 μg/mL) and incubated at 37 °C for 24 h with shaking at 250 rpm. Cell cultures (2 mL) were then transferred to glass-bottom dishes and observed. For the cell1184

DOI: 10.1021/acsinfecdis.9b00033 ACS Infect. Dis. 2019, 5, 1177−1187

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Antivirulence Efficacy and Cytotoxicity of Nepodin in the Nematode Model. To investigate the effects of nepodin on the virulence of C. albicans, we used C. elegans strain fer15(b26); fem-1(hc17), as previously described.25 Briefly, synchronized adult nematodes were fed on C. albicans lawns for 4 h at 25 °C and collected after washing three times with M9 buffer. Approximately 30 worms were then added to each well of 96-well plates containing PDB medium (300 μL) with or without nepodin (0, 2, 5, or 10 μg/mL). Plates were then incubated for 4 days at 25 °C without shaking. For cytotoxicity assays, 30 noninfected worms were pipetted into single wells of a 96-well dish containing M9 buffer, and nepodin was added to final concentrations of 0, 10, 20, 50, 100, and 200 μg/mL without C. albicans. Plates were then incubated for 4 days at 25 °C without shaking. Three independent experiments were performed in triplicate. Results are expressed as percentages of live worms (survival), as determined by responses to platinumwire touching after incubation for 4 days. Observations were made using an iRiS Digital Cell Imaging System (Logos Bio Systems). Cytotoxicity of Nepodin on Mouse Tumor Cells. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)2,5diphenyl tetrazolium bromide (MTT) as described previously.68 Mouse B16 melanoma cells (ATCC CRL-6322, 2 × 104 cells/well) were cultured in 96-well plates and allowed to attach for 12 h. Cells were then treated with nepodin at concentrations of 0−200 μg/mL for 24 h. Three independent cultures were used. Statistical Analysis. Replication numbers for assays are provided above and results are expressed as means ± standard deviations. The statistical analysis was performed using oneway ANOVA followed by Dunnett’s test using SPSS version 23 (SPSS Inc.). P values of