In vitro and in vivo antifungal activities and mechanism of heteropolytungstates against Candida species

The antifungal activities of heteropolytungstates, α-1,2,3-K6H[SiW9V3O40] (SiW-3), K13[Ce(SiW11O39)2]·17H2O (SiW-5), K13[Eu(SiW11O39)2]·25H2O (SiW-10), K6PV3W9O40 (PW-6), α-K4PVW11O40 (PW-8), were screened in 29 Candida albicans, 8 Candida glabrata, 3 Candida krusei, 2 Candida parapsilosis, 1 Candida tropicalis, and 1 Cryptococcus neoformans strains using the CLSI M27-A3 method. SiW-5 had the highest efficacy with a minimum inhibitory concentration (MIC) values of <0.2–10.2 μM in vitro. The antifungal mechanism, acute toxicity and in vivo antifungal activity of SiW-5 were then evaluated in C. albicans. The results showed that SiW-5 damaged the fungal cell membrane, reduce the ergosterol content and its main mode of action was through inhibition of ergosterol biosynthesis. Real-time PCR showed that ERG1, ERG7, ERG11 and ERG28 were all significantly upregulated by SiW-5. An acute toxicity study showed the 50% lethal dose (LD50) of SiW-5 for ICR mice was 1651.5 mg/kg. And in vivo antifungal studies demonstrated that SiW-5 reduced both the morbidity and fungal burden of mice infected with C. albicans. This study demonstrates that SiW-5 is a potential antifungal candidate against the Candida species.

Following the ever increasing application of antibiotics, immunosuppressive agents, and invasive medical devices, as well as increasing numbers of immunocompromised patients, fungal infections have dramatically increased worldwide 1,2 . Among fungal infections, the Candida species is one of the dominant fungal pathogens, associated with high rates of morbidity and mortality 2,3 . However, the development of antifungal drugs has always lagged behind fungal infections incidence, and most of antifungals have limited potential as systemic agents due to issues of toxicity, adverse effects or restricted bioavailability. Moreover, their historic long-term application has resulted in many drug resistance 4,5 . Therefore, there is an increasing need for novel, more efficient and safer antifungals.
Polyoxometalates (POMs) are early transitional metal oxygen anion clusters and have garnered worldwide attention due to the versatility of their chemical structures. They have numerous applications in chemistry, materials science, catalysis, redox, magnetism, medicine, and have potential to be especially promising as antibacterial, antiviral and antitumor agents [6][7][8] . To the best of our knowledge, there are only a small number of reports on their antibacterial activities [9][10][11] , and no reports on their antifungal activities.
Herein, a series of antifungal susceptibility tests were carried out for heteropolytungstates, α-1,2,3-K 6 H[Si-W 9 V 3 O 40 ] (SiW-3), K 13   . To investigat the antifungal mechanism of heteropolytungstates, the ultrastructure of C. albicans was visualized by transmission electron microscopy (TEM), the ergosterol contents were measured by high performance liquid chromatography (HPLC), and real-time PCR was carried out to evaluate the inhibition of heteropolytungstates on the ergosterol biosynthesis at the molecular level. In addition, the in vivo efficacy of heteropolytungstate in a C. albicans systemic infection murine model was evaluated.

MIC determination.
Time kill kinetics. The antifungal activity of SiW-5 was confirmed by the time kill test. As shown in the Fig. 1, no appreciable antifungal activity of SiW-5 at 0.5MIC was observed. But the groups at MIC and 2MIC showed fungicidal effect and led to a decrease of 3.93 and 6.63-log 10 CFU/mL at 24 h. The inhibition of the SiW-5 continuously increased with the enhancement of the concentration and time, indicating that the antifungal effect of SiW-5 is time-and dosed dependent.
Transmission electron microscopy (TEM). TEM was used to study the ultrastructure changes of the C. albicans HL27 treated by SiW-5. As shown in Fig. 2A, the C. albicans HL27 cells in control groups showed uniform density enveloped with an intact and regular cell wall and membrane. While treated with 0.6 μM of SiW-5 for 24 h, the cells were smaller and irregular. The cell wall and membrane were rough and the intracellular content leakage. Moreover, structural disorganization in the cytoplasm were clearly observed, shown in Fig. 2B.

Assessment of ergosterol content.
Ergosterol is an important constituent of fungal membrane for maintaining cell integrity, membrane fluidity and the cell metabolism. It is the target of most existing antifungals. Therefore, the effect of SiW-5 on the ergosterol content of C. albicans was conducted by HPLC. The HPLC results showed that the retention time of ergosterol was about 14.798 min. The standard curve was linear and R 2 = 0.9999 (Fig. S4). The ergosterol inhibition ratios of MIC of FLC and 0.5MIC, MIC and 2MIC of SiW-5 were 61.29%, 19.35%, 51.61%, and 80.65%, respectively (Fig. S5, Fig. 3). The inhibition ratio of SiW-5 on ergosterol synthesis continuously increased with the enhancement of concentration, indicating that the antifungal effect of SiW-5 was dependent on its concentrations and its main mode of action was through inhibition of ergosterol biosynthesis.

Real-Time PCR.
To study the interference of ergosterol biosynthesis caused by SiW-5 on a molecular level, real-time PCR was performed. The C. albicans HL27 cells were exposed to SiW-5 (MIC values) for 24 h, their total RNA was extracted, and cDNA synthesized. This cDNA was then used as a template for a series of RT-PCRs. The ERG1, ERG7, ERG11 and ERG28 genes were found significantly upregulated with the fold change relative to control in gene expression of 5.95 ± 1.30, 4.90 ± 0.24, 7.40 ± 2.56 and 6.29 ± 0.71, respectively. No obvious change was found in the expression of ERG27 with the relative fold change of 1.47 ± 0.46. Acute toxicity. In vivo toxicity of SiW-5 was evaluated in healthy ICR mice. The LD 50 in ICR mice was determined to be 1651.5 mg/kg by the Bliss method, and the confidence level of 95% was 1539.6 mg/kg to 1926.5 mg/ kg, which was classified as low toxicity. There were no adverse effects or clinical signs of toxicity for the surviving mice, and no significant influence of SiW-5 on body weight during the observation period.
In vivo antifungal studies. The in vivo efficacy of SiW-5 or FLC was determined against a systemic infection by C. albicans HL27 in mice with CY-induced immunosuppression. The survival ratios over time of mice treated with SiW-5 and FLC were shown in Fig. 4. The water-sham group all died within 6 days, and the median survival time was 5 days. The groups treated with 10 mg/kg FLC and 100 mg/kg SiW-5 showed 70% and 60% survival after 15 days. The median survival time of the groups treated with 50 mg/kg and 25 mg/kg SiW-5 were 7 and 6 days. PAS-stained histological examinations of kidneys from infected mice treated with sterile water showed obvious tissue damage and a large amount of yeasts and hyphae, which slightly and moderately decreased in the group treated with 25 mg/kg and 50 mg/kg SiW-5. While in the groups treated with 100 mg/kg SiW-5 and 10 mg/kg FLC, the fungal cells were markedly reduced and showed a yeast form (Fig. 5). Treatments with 100, 50 and 25 mg/kg SiW-5 and 10 mg/kg FLC caused significant reduction in CFU counts per gram of kidney compared to the water sham group (Table 2).

Discussion
Candida species are the main fungal pathogen that causes infections in humans, ranging from superficial mucosal infection to systemic mycoses 17 . Candida infections are recurrent diseases, which have increased due to the rise in the number of immunocompromised host populations 18 .
Sun et al. reported the antifungal activity of organothiophosphoryl polyoxotungstates against Fusarium graminearum in 2002 19 . Until now, antifungal activities of polyoxometalates against human fungal pathogens are seldom reported. In present study, the antifungal activities of a series of heteropolytungstates were evaluated through MIC determination compared with FLC. The test compounds exhibited potent antifungal activities against fungal strains. Among them, SiW-5 has the highest efficacy, and higher than that of the positive control group FLC. The structure of the polyoxotungstates seems to be important for the antifungal activity. The lacunary-Keggin sandwiched polyoxotungstates and Keggin-structural polyoxotungstates are known to exhibit various biological activities such as the anti-human immunodeficiency virus. Herein, the antifungal activity of lacunary-Keggin sandwiched polyoxotungstates is higher than the Keggin-structural polyoxotungstates. Maybe the stability of the lacunary-Keggin sandwiched polyoxotungstates is higher than the others at pH 7.5. The charge number of the polyoxotungstates is also an important factor. The most potent antifungal polyoxotungstates, SiW-5 and SiW-10, show highly negative charges of 13, while the others showing weak antifungal activities have lower negative charges. The polyoxotungstates, especially highly negative-charged lacunary-Keggin sandwiched polyoxotungstates, exhibited potent antifungal activities. Based on the previous studies, the antiviral property of heteropolytungstate Cs 2 K 4 Na[SiW 9 Nb 3 O 40 ]·H 2 O is mainly due to its localization on the host cell surface, inhibiting viral adsorption. In addition, polyoxotungstates with antibacterial activity were preferentially located on the membrane of bacteria with intact composition 20 . And the antibacterial polyoxotungstates uptaken in the cell were preferentially located on the membrane with intact composition 21 . Therefore, the fungal membrane maybe the target of SiW-5 22 . 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. Ergosterol biosynthesis pathway is thus a significant target of most existing antifungals, for instance, fluconazole, itraconazole, amphotericin B, terbinafine, etc 23 . In present study, the ergosterol contents of the cells treated with 0.5MIC, MIC and 2MIC of SiW-5 resulted in a dose-dependent reduction of 19.35%, 51.61% and 80.65% versus negative control group (Fig. S5, Fig. 3). Furthermore, TEM results evidenced that the membrane of C. albicans treated with SiW-5 was indeed damaged, leading to the morphological change from oval form to smaller irregular form, the intracellular content leakage and structural disorganization in the cytoplasm (Fig. 2).
To study the effect of heteropolytungstates on ergosterol biosynthesis, real-time PCR using five of the essential genes involving in ergosterol biosynthesis was conducted. The results revealed a nearly global upregulation including gene ERG1 (5.95 ± 1.30), ERG7 (4.90 ± 0.24), ERG11 (7.40 ± 2.56) and ERG28 (6.29 ± 0.71), which is consistent with previous reports 24 . When sterol levels are reduced, the expression of ergosterol biosynthesis (ERG) genes are substantially increased. This induction can be mediated by Upc2p, which mainly controls the genes for  ergosterol synthesis in yeasts. Upc2p can activate the expression of ERG genes in response to sterol depletion. The pathway is subject to negative feedback regulation 25,26 .
In the in vivo antifungal study, immunosuppressed mice were inoculated with C. albicans via tail vein, leading to systemic infections, affecting kidneys, heart, liver, spleen, lung and brain. Because the kidney was the main target organ, histopathological examinations and fungal burdens of kidneys were thus used to evaluate the in vivo antifungal efficacy of SiW-5. The groups treated with 10 mg/kg FLC and 100 mg/kg SiW-5 showed 70% and 60% survival (Fig. 4), and obviously reduced the fungal burden ( Fig. 5 and Table 2).
In conclusion, a heteropolytungstate (SiW-5) shows potent antifungal activity in vitro and in vivo.

Methods
Materials. RPMI   each patient once the purpose and nature of the study had been fully explained. We confirm that all methods were performed in accordance with the relevant guidelines and regulations. The Candida species were preliminarily identified according to the colored colony morphology on CHROMagar Candida medium (CHROMagar Co., France) and Vitek 2 automated system (bioMérieux, Marcy l'Etoile, France). All the strains were preserved in the Department of Health Laboratory, School of Public Health, Jilin University, China.

Drugs Dose (mg/kg) log CFU/g in kidney a
Water sham -6.63 ± 0.06  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 SiW-5. Total RNA was extracted from C. albicans HL27 incubated with or without MIC of SiW-5 for 24 h using the hot phenol method as previously described 30 . Reverse transcription was conducted in a total volume of 20 μL with Primescript RT reagent kit (TaKaRa, China). Real-time PCR reactions were performed with SYBR Green I (Roche, China), using qTOWER 2.0 PCR system (Analytic Jena AG, Germany) 31 . The pimer sequences used in Real-Time PCR were listed in Table S2, using 18 S rRNA as the internal control. The expression level of each gene in the SiW-5 treated sample relative to that of untreated sample was calculated using 2 −ΔΔCt method.
Acute toxicity. All animal experiments including acute toxicity and in vivo antifungal studies were approved by the Animal Care and Use Committee at Jilin University. All animal procedures were conducted in compliance with the guideline of the China Association of Laboratory Animal Care. The mice were housed at the Laboratory Animal Center, School of Public Health, Jilin University, China. A total of 70 ICR mice (Changchun, China; 20-22 g) were randomly divided into 7 groups, with equal numbers of female and male in each group. SiW-5 was dissolved in sterile water and intragastrically administered to the mice at a single dose of 1300, 1400, 1500, 1600, 1700 and 1800mg/kg body weight, respectively; the control group received sterile water. Then the mortalities were recorded within 14 days. The values of 50% lethal dose (LD 50 ) and 95% confidence were calculated by Bliss method.
In vivo antifungal studies. In vivo antifungal studies were performed as described previously 32,33 , with some modifications. The C. albicans HL27 was used to infect BALB/c mice (Changchun, China; 18-22 g). Immunosuppression was induced in the mice by intraperitoneal injections of cyclophosphamide (CY, 150 mg/ kg of body weight) on days-3 and -1, and prolonged by CY injections (150 mg/kg) on days 3, 6, and 9. The immunosuppressed mice were inoculated with 2 × 10 5 CFU of C. albicans in 0.1 mL sterile saline via tail vein on day 0. Treatments consisted of SiW-5 administered at 100, 50 or 25 mg/kg intraperitoneally once a day or FLC administered at 10 mg/kg intraperitoneally once a day 24 h postinfection for 7 consecutive days, containing 14 mice each with equal numbers of female and male. Infection control group was administered sterile distilled water intraperitoneally, containing 10 mice with equal numbers of female and male. Mice were monitored for 15 days after inoculation. Survival rates, periodic acid-Schiff (PAS) staining histopathological examinations and tissue fungal burdens (the number of CFU per gram of kidney) were used to evaluate the in vivo antifungal efficacy of SiW-5.
SCIenTIFIC REPORTS | 7: 16942 | DOI:10.1038/s41598-017-17239-8 Statistical analysis. Statistical analysis was conducted using SPSS 13.0. All experiments were performed in triplicate. Data were represented as mean ± SD. Student's t-test was used to test the significance between two groups. One-way analysis of variance (ANOVA) was used to test the significance among different groups. P < 0.05 was considered to indicate statistically significant differences.