In Vitro Antifungal Activity and Mechanism of Ag3PW12O40 Composites against Candida Species

Fungal infections pose a serious threat to human health. Polyoxometalates (POMs) are metal–oxygen clusters with potential application in the control of microbial infections. Herein, the Ag3PW12O40 composites have been synthesized and verified by Fourier transform infrared (FT-IR) spectrum, transmission electron microscopy (TEM), scanning electron microscope (SEM), elemental analysis, and X-ray diffraction (XRD). The antifungal activities of Ag3PW12O40 were screened in 19 Candida species strains through the determination of minimum inhibitory concentration (MIC) by the microdilution checkerboard technique. The minimum inhibitory concentration (MIC50) values of Ag3PW12O40 are 2~32 μg/mL to the Candida species. The MIC80 value of Ag3PW12O40 to resistant clinical isolates C. albicans HL963 is 8 μg/mL, which is lower than the positive control, fluconazole (FLC). The mechanism against C. albicans HL963 results show that Ag3PW12O40 can decrease the ergosterol content. The expressions of ERG1, ERG7, and ERG11, which impact on the synthesis of ergosterol, are all prominently upregulated by Ag3PW12O40. It indicates that Ag3PW12O40 is a candidate in the development of new antifungal agents.


Introduction
Fungal infections pose a serious threat to human life and health, especially among immunocompromised patients, such as organ transplant patients, implanted medical devices, and those with indwelling catheters [1][2][3]. The Candida species such as C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei are the main pathogens that may cause severe mucosal and systemic infections [2]. Effective antifungal drugs are the key to the treatment of Candida infections [4]. Azoles, such as fluconazole, itraconazole, posaconazole, and voriconazole are considered as the first-line treatment of patients with Candida infections [5,6]. However, the broad utilization of azoles has led to drug resistance, which is an increasing problem in clinic isolates [7,8]. Therefore, it is important to develop novel antifungal agents.
Composite antimicrobial materials are a new class of functional materials developed on the basis of composites, which usually consist of antimicrobial metals and inorganic or organic carriers. Antimicrobial metals are the key compositions in composite antimicrobial materials, and their ability varies greatly. Some studies in the literature report that inorganic nanoparticles, such as silver and ZnO, can be used as antifungal agents [9][10][11][12]. At present, silver antimicrobial materials are hot spot researched and widely used in medical catheters, bone adhesives, and other medical devices [13]. The FT-IR spectrum of Ag 3 PW 12 O 40 has the characteristic asymmetric stretching vibration peaks at 1002, 952, 883, 771, 586, and 516 cm −1 , which were attributed to ν(W-O terminal ) and ν(W-O bridge -W) of polyoxoanion, as shown in Figure 1g. The characteristic peaks of Ag 3 PW 12 O 40 were nearly consistent with the reported H 3 PW 12 O 40 date in the literature. The other strong features at 3448 cm −1 were assigned to the water molecules.

Antifungal Susceptibility Testing
The Ag 3 PW 12 O 40 composites were assessed in suspension against the standard and clinical isolated strains of Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Cryptococcus tropicalis with fluconazole (FLC) as positive control, as shown in Table  The effect of Ag 3 PW 12 O 40 and FLC against C. albicans HL 963 in different concentrations was further proved by the MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) method. As shown in Figure 2, after treatment by Ag 3 PW 12 O 40 , the viability of C. albicans HL 963 cells have a significant reduction than in the negative control group (p < 0.05). The inhibition ratio of C. albicans HL 963 treated by Ag 3

Growth Inhibition Curves
The growth inhibition effect on the C. albicans HL 963 with various concentrations of the Ag 3 PW 12 O 40 , negative control (culture medium without durgs), and FLC at different times is shown in Figure 3. The number of fungi began to proliferate rapidly from the 10th h in the control group; in contrast, the fungi treated with Ag 3 PW 12 O 40 and FLC was significantly delayed and began to grow 24 h later. After treatment for 24 h, the inhibit delay curve quickly increased up in FLC groups, especially in the low-dose group (8 µg/mL). Meanwhile, the growth of fungi treated with the same dose of Ag 3 PW 12 O 40 was significantly inhibited, and no reduction of inhibition was observed with the extension of time. All three concentrations of Ag 3 PW 12 O 40 could inhibit the proliferation of C. albicans HL 963.

LIVE/DEAD Assays
The cells were stained by acridine orange (AO) and ethidium bromide (EB) double-fluorescent staining. AO can penetrate the intact cell membrane, embed the nuclear DNA, and make it emit bright green fluorescence. However, EB can only penetrate the damaged cell membrane, embed nuclear DNA, and fluoresce orange-red. After culturing 24 h, the fungus was treated with 64 µg/mL Ag 3 PW 12 O 40 and FLC for 24 h. AO and EB were used to double stain C. albicans, and the cell morphology was observed under a fluorescence microscope. The results are shown in Figure 4; after incubation for 24 h, the fungal grew well in the RPMI 1640 group, while cell death (dead cells were stained red) occurred in the Ag 3 PW 12 O 40 and FLC groups. The massive dead cells were found in the Ag 3 PW 12 O 40 group, meaning that Ag 3 PW 12 O 40 is more effective at killing fungi. The result is consistent with the result of the MTS method. In the bright-field images, we observed that C. albicans HL 963 formed a very dense cell layer and grew well in the untreated group. Meanwhile, in Ag 3 PW 12 O 40 and FLC groups at the same dose, the FLC group still had a dense cell layer and compact arrangement. However, in the Ag 3 PW 12 O 40 group, the number of cells was significantly reduced, and the cells became dispersed. The results showed that Ag 3 PW 12 O 40 has better antifungal properties than FLC.

Assessment of Ergosterol Content
The HPLC results showed that the retention time of ergosterol was about 12.60 min ( Figure 5). Ergosterol contents were extracted from control, FLC, and the Ag 3 PW 12 O 40 composites after 24 h treatment. The ergosterol contents were 2.20 ± 0.15, 0.10 ± 0.10, and 0.79 ± 0.12 mg/mL, respectively (p < 0.05) ( Table 2).The standard curve was linear (R 2 = 0.9945). As shown in Figure 6, the Ag 3 PW 12 O 40 and FLC treatment for C.albicans HL 963 resulted in the ergosterol content reduction of 59.08 ± 7.23% and 95.43 ± 0.19% (p < 0.05). The results showed that one of the inhibitory effects of Ag 3 PW 12 O 40 was through the reduction of ergosterol content on the membrane of C. albicans.

The Level of Ergosterol Biosynthesis-Related Genes
To further investigate the mechanism of Ag 3 PW 12 O 40 reduction of ergosterol biosynthesis, real-time PCR was used to evaluate the expression of five important genes involved in ergosterol biosynthesis. The C. albicans HL 963 cells were treated with 8 µg/mL of Ag 3 PW 12 O 40 for 24 h; then, total RNA was extracted, and cDNA was synthesized by reverse transcription. This cDNA was used as a template for a series of real-time PCRs. The melt curves of reference gene and target genes in C. albicans HL 963 treated with RPMI1640 or Ag 3 PW 12 O 40 are shown in Figure 7. As shown in Figure 8, the expression of ERG1, ERG7, and ERG11 was significantly upregulated with the fold change relative to control of 1.91 ± 0.19, 2.09 ± 0.29, and 1.45 ± 0.17, respectively.

Discussion
Invasive fungal diseases have been increasing in recent years. Candida spp. is the main pathogen causing infection, ranging from superficial mucous membranes to the whole body.
Although several potent antifungal agents are available to treat Candida spp., the drug-resistant newstrains frequently emerge.
Polyoxometalates (POMs) are a kind of metal-oxygen clusters, which are formed by the self-assembly of a transition metal and an oxygen atom and form an inorganic compound having a spatial network structure with many different biological activities than mononuclear complexes. As a new candidate drug, these compounds have the advantages of simple synthesis, low cost, flexible design and modification of molecules, low side effects, and excellent physical and chemical properties (surface charge distribution, molecular shape, pH, solubility, etc.). There are few research studies on the antimicrobial activity of POMs. Yamase group conducted relevant studies and found that some polyoxometalates have good antibacterial activity. For example, decavadate can selectively inhibit Streptococcus pneumoniae; Keggin, Keggin deficiency, Double Keggin and Keggin sandwich polytungstate showed a strong synergistic effect with β-lactam antibiotics and synergistic resistance against methicillin-resistant Staphylococcus aureus (MRSA), which can improve the antibacterial activity of β-lactam antibiotics against MRSA [20,25,26]. The antifungal effects of polyoxometalates are less studied. In 2002, Sun et al. reported that a series of polymetallic tungstate had antibacterial activity against Fusarium graminae, which mainly caused field gramineous crop diseases [27]. In 2011, Chen et al. reported the antimicrobial activity of polyoxometalates on agricultural food fungi [28]. Our group is mainly focused on the design and synthesis of polyoxometalates and their biological activities research in interdisciplinary fields. According to our reports previously, more than 10 polyoxometalates were screened for antifungal susceptibility on 44 strains of human pathogenic fungi, and five POMs were found to have potential antifungal activities [22]. In this study, the antifungal activity of Ag 3 PW 12 O 40 composites has been evaluated. The results showed that Ag 3 PW 12 O 40 showed high activity against fungal strains. In particular, it showed an inhibitory effect on FLC-resistant fungus, such as HL 963 (Table 1). An MTS method was further used to evaluate the antifungal activity of Ag 3 PW 12 O 40 against HL 963. The result is similar to that from the broth microdilution method. The antifungal activity of Ag 3 PW 12 O 40 is dose-dependent. In comparison with the growth inhibition curves (Figure 3), the Ag 3 PW 12 O 40 groups had significantly higher inhibition rates than those in the FLC groups (p < 0.05). Meanwhile, the AO and EB double fluorescent staining proved that the Ag 3 PW 12 O 40 group had more dead cells than that in the FLC group ( Figure 4).
Moreover, the structure of the Ag 3 PW 12 O 40 seems to be important for the antifungal activity. POMs have various types of structures, such as Keggin, Wells-Dawson, Anderson, Lindqvist, sandwich Keggin, Preyssler, Strandberg, and Krebs et al. The Keggin-structural polyoxotungstates are known to exhibit various biological activities such as the anti-human immunodeficiency virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV-1, HSV-2), and human cytomegalovirus (HCMV) [29][30][31]. Herein, the PW 12 O 40 3− anions in Ag 3 PW 12 O 40 also owns a Keggin structure, which may deduce the high antifungal activities. In addition, our previous work show that the cytotoxicity of Ag 3 PW 12 O 40 against L-02 (human normal liver) cells was low (the 50% inhibition concentration value of 274.8 µg/mL). The cytotoxicity value is 8.6-137.4 times higher than the MIC 50 values (2-32 µg/mL) on the fungi cells in this work [23].
Ergosterol is an important component throughout the fungal cell membranes, which distinguishes fungi from bacteria, plant, and animal cells. It plays a vital role in many biological functions such as maintaining cell integrity, regulating membrane fluidity and the cell cycle. Thus, the ergosterol biosynthesis pathway is a significant target of most existing antifungal agents; for instance, fluconazole, itraconazole, amphotericin B, terbinafine, etc. [32]. In this work, the effect of Ag 3 PW 12 O 40 on the ergosterol content was investigated. Similar to fluconazole, Ag 3 PW 12 O 40 had also an inhibitory effect on the ergosterol content ( Figure 6). Five of the essential genes involved in ergosterol biosynthesis were conducted by real-time PCR. Genes ERG1 (1.91 ± 0.19), ERG7 (2.09 ± 0.29), and ERG11 (1.45 ± 0.17) were unregulated (Figure 8) by Ag 3 PW 12 O 40 . The content of ergosterol decreased with the upregulation of three genes. These results are consistent with previous reports in which C. albicans was treated with azole [33,34]. A compensatory response to re-establish the plasma membrane ergosterol levels may account for the observed upregulation of the ERG genes in the ergosterol pathway [2]. When sterol levels are reduced, the expression of ergosterol biosynthesis (ERG) genes are substantially increased. So, the results shown that Ag 3 PW 12 O 40 may interfere with the expression of ergosterol biosynthesis related genes, decreasing ergosterol content and destroying Candida albicans cells.
In conclusion, Ag 3 PW 12 O 40 exhibited a potent antifungal effect through mechanisms associated with membrane disruption. Therefore, Ag 3 PW 12 O 40 is a potential candidate for the development of new antimicrobial agents.

Materials
All the chemicals were analytical grade reagents and used without further purification. The RPMI-1640 with l-glutamine, without sodium bicarbonate (Sigma, Mendota Heights, MN, USA), buffered to pH 7.0 with 165 mM morpholine propanesulfonic acid (MOPS) (Sigma) was used for MIC determination and liquid culture of fungal strains. FLC was obtained from The TCI Company. Ergosterol standard was obtained from Dr. Ehrenstorfer Company (Augsburg, Germany). Prime script RT reagent kit (TaKaRa, Shiga, Japan) was used for reverse transcription. SYBR Green I (Roche, Basel, Switzerland) was used for real-time PCR reactions.

Synthesis of Ag 3 PW 12 O 40 Composites
Ag 3 PW 12 O 40 was synthesized and purified by modifying the published methods [35]. The synthesis process was as follow: 3 mL aqueous solution of AgNO 3 (0.33 M) was added dropwise at a rate of 1 mL/min to 3.

Physical Characterization of Ag 3 PW 12 O 40 Composites
The IR spectrum in the range 400-4000 cm −1 was recorded on an Alpha Centaurt FT/IR Spectrophotometer using KBr pellets (Shimadzu, Japan). Ag, P, and W were determined by an inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 7000 DV, Perkin Elmer). The morphology and size of the Ag 3 PW 12 O 40 composites were acquired by transmission electron microscopy (TEM, JEM-1011, Tokyo, Japan) with a working voltage at 100 kV and a scanning electron microscope (SEM, HITACHI S4800, Tokyo, Japan) was used to characterize the morphology of the samples. Powdered materials were deposited on adhesive tape fixed to specimen tabs and ion-sputter-coated with gold using a BAL-TEC SCD 005 Sputter Coater prior to SEM measurements. The analyses were done under an acceleration voltage of 30 kV, a beam current of 20 nA and a spot size of 1 µm. The X-ray powder diffraction method was carried out in a D/max-α power diffractometer (Rigaku, Tokyo, Japan) using Cu-Kα monochromatic radiation (λ = 1.5418 Å). Confocal laser scanning microscope (CLSM) images were obtained from an Olympus FV1000 equipped with 488, 559 nm one-photon laser. Particles' surface charge (ζ-potential, mV) and diameter were characterized using a Zetasizer Nano Z dynamic light scattering detector (Malvern Instruments Ltd., Worcestershire, UK). Prior to the measurements, composites were diluted to 1 mg/mL with deionized water.

Isolation and Culture Conditions of Fungi
The strains used in this study included 15 (China). The Candida species were preliminarily identified according to the colored colony morphology on CHRO Magar Candida medium (CHRO Magar Co., Paris, France) which was used to confirm the Candida species. The reference strains, Candida tropicalis ATCC 750, C. parapsilosis ATCC 22,019, and C.albicans ATCC 90,028 were purchased from American Type Culture Collection. All isolates were incubated at 35 • C and subcultured onto Sabouraud dextrose agar (SDA, Conda) at 4 • C in School of Public Health, Jilin University, China.

Determination of MIC of Ag 3 PW 12 O 40 Composites
ATCC 22,019 is the quality control strain, which was used in each susceptibility test to ensure quality control. The minimal inhibitory concentration (MIC) values of Ag 3 PW 12 O 40 and FLC were determined for all the Candida strains using the broth microdilution protocol of the Clinical and Laboratory Standards Institute (CLSI) methods (M38-A) [36]. Briefly, the fungal strains were added to sabouraud dextrose broth (SDB) and cultured with shaking at 35 • C and 180 rpm for 18 h and after diluted to 0.4-5 × 10 4 cells/mL by RPMI 1640. The 96-well plates were prepared by dispensing into each well 100 µL of RPMI-1640 broth. A 100 µL of drugs initially prepared at the concentration of 512 µg/mL was added into each of the first wells, followed by 2-fold serial dilution to obtain a concentration range of 0.25~256 µg/mL. To this was separately added 100 µL of 0.4~5 × 10 4 cells/mL fungal cell suspensions. The 11th well containing 100 µL medium without drugs and fungal cell was used as the empty control. The last well containing 100 µL fungal cell suspensions without drugs was used as the negative control. The final volume in each well was 200 µL. After 15 s of oscillation, the plates were incubated at 35 • C for 48 h. The absorbance was measured at 600 nm by using a microplate reader (Biotek Co., Winooski, VT, USA). MIC 80 or MIC 50 were determined as the lowest concentration of the drugs that inhibited growth by 80% or 50% compared with drug free wells. The inhibitory rate = (1 − OD drug − OD empty control /OD negative control − OD empty control ) × 100%.

MTS-Reduction Assay
The antifungal activity of Ag 3 PW 12 O 40 on the C. albicans HL 963 determined by the MTS assay as described in the literature. Briefly, C. albicans HL 963 plated on 96-well plates at a density of 1.0 × 10 6 cells/mL. After 24 h, the dilutions of Ag 3 PW 12 O 40 and FLC at different doses (64, 128, 256, and 512 µg/mL) were added and incubated for 48 h. The C. albicans HL 963 cells in the negative control group were treated with the same volume of medium. To evaluated cell viability, an MTS assay was performed according to the manufacturer's instructions (Promega, Madison, WI, USA). The cells were incubated in the dark for another 25 min at 37 • C. Then, using a multichannel pipette, we removed 80 µL of the resulting colored supernatant from each well and transfered it into the corresponding wells of a new microtiter plate. Microplate absorbance was measured at a wavelength of 490 nm on a microplate reader (Biotek Co., Winooski, VT, USA).

Growth Inhibition Curves
The Candida albicans strain was cultured in SDB liquid medium at 35 • C overnight. C.albican HL 963 was diluted at the starting inoculum of 1 × 10 6 cells/mL in glass tubes. Different concentrations of the Ag 3 PW 12 O 40 (8, 16, 32 µg/mL) and FLC (8, 16, 32 µg/mL) were added into tubes. At scheduled time points (0, 2, 4, 8, 12, 24, 36 and 48 h) after incubation in an orbital shaker (about 180 rpm) at 35 • C, a 100 µL aliquot was removed from every solution and at 600 nm with a microtiter plate reader (BioTek, Winooski, VT, USA), and background optical densities were subtracted from that of each well.

LIVE/DEAD Assay
Fungal suspension was prepared from Candida albicans after 2 times of activation and diluted to 1-5 × 10 6 CFU/mL. The sterilized coverslip was put into 6-well plate, and 2 mL fungal solution was added to each well. After incubation for 24 h, the cells were washed 3 times with PBS. The Ag 3 PW 12 O 40 and FLC with a final concentration of 64 g/mL were added as the experimental group, and the RPMI 1640 was added as the control group. The prepared 6-well plates were incubated at 35 • C for 24 h, and the coverslip was cleaned with PBS 3 times. AO (Acridine Orange) (5 µL, 100 mg/L) and EB (ethidium bromide) (5 µL, 100 mg/L) were mixed with lightless condition; then, we added it to the coverslip, straining in darkness for 30 s. A fluorescence inverted microscope was used to observe and photograph. AO and EB were prepared before the experiment.

Assessment of Ergosterol Content
C. albicans HL 963 were treated with Ag 3 PW 12 O 40 at 35 • C for 24 h. The cells were centrifuged and washed with PBS. A 0.5 g wet weight of cell mixed with PBS and fresh saponifier was saponified at 80 • C for 1 h and extracted by petroleum ether. Then, the extract was volatilized to dryness at 60 • C. The dry residues were dissolved by 0.5 mL methanol and filtered through 0.45 µm micro membrane. Then, we determined the quantification of ergosterol in samples with or without the drugs by comparing peak areas of samples to a standard curve generated from HPLC-grade ergosterol. A standard curve of HPLC-grade ergosterol (Dr. Ehrenstorfer Co., Augsburg, Germany) consists of 0.001, 0.004, 0.015, 0.0625, 0.25, and 1 mg/mL. Ergosterol contents were analyzed using LC-20AB prominence Liquid Chromatography (Shimadzu Co., Kyoto, Japan) including a Shimadzu C 18 column (250 mm × 4.6 mm, 5 µm). Eluent was methanol/water (97/3, 100% HPLC grade). The flow rate was 1 mL/min. Temperature was 35 • C. SPD-20AV prominence UV/VIS Detector (Shimadzu, Kyoto, Japan) was used to detect UV at 282 nm [37]. The ergosterol inhibition ratio = (1 − ergosterol content of treated cells/ergosterol content of untreated cells) × 100%.

Real-Time PCR
Real-time PCR was used to measure the transcriptional expressions of the genes involved in ergosterol biosynthesis of C. albicans treated with Ag 3 PW 12 O 40 . Total RNA was extracted using the hot phenol method as previously described [38]. Reverse transcription was conducted in a total volume of 20 µL with Primescript RT reagent kit (TaKaRa, Shiga, China). Real-time PCR reactions were performed with SYBR Green I (Roche, city, China), using qTOWER 2.0 PCR system (Analytic Jena AG, Jena, Germany). The primer sequences used in real-time PCR were listed in Table 3, using 18S rRNA as the internal control. The expression level of each gene in the Ag 3 PW 12 O 40 -treated sample relative to that of the untreated sample was calculated using the 2 − Ct method.

Statistical Analysis
Data were expressed as mean ± SD (standard deviation) and analyzed by SPSS 16.0 statistical software (IBMCorp, Armonk, NY, USA) (significance was established at p < 0.05). All experiments were performed in triplicate. Statistical significance was evaluated by the one-way analysis of variance (ANOVA) and Student's t-test. LSD post hoc analyses of multiple comparisons were statistically analyzed.

Conclusions
In summary, the Ag 3 PW 12 O 40 composite is a potential candidate for the development of a new broad spectrum antifungal agent. Future research may focus on trying to explore the antimicrobial mechanism of Ag 3 PW 12 O 40 and the synthesis of new POM-based compounds.