A semisynthetic borrelidin analogue BN-3b exerts potent antifungal activity against Candida albicans through ROS-mediated oxidative damage

In the process of investigating the antifungal structure-activity relationships (SAR) of borrelidin and discovering antifungal leads, a semisynthetic borrelidin analogue, BN-3b with antifungal activity against Candida albicans, was achieved. In this study, we found that oxidative damage induced by endogenous reactive oxygen species (ROS) plays an important role in the antifungal activity of BN-3b. Further investigation indicated that BN-3b stimulated ROS accumulation, increased malondialdehyde (MDA) levels, and decreased reduced/oxidized glutathione (GSH/GSSG) ratio. Moreover, BN-3b decreased mitochondrial membrane potential (MMP) and ATP generation. Ultrastructure analysis revealed that BN-3b severely damaged the cell membrane of C. albicans. Quantitative PCR (RT-qPCR) analysis revealed that virulence factors of C. albicans SAPs, PLB1, PLB2, HWP1, ALSs, and LIPs were all down-regulated after BN-3b exposure. We also found that BN-3b markedly inhibited the hyphal formation of C. albicans. In addition, in vivo studies revealed that BN-3b significantly prolonged survival and decreased fungal burden in mouse model of disseminated candidiasis.

Effect of BN-3b on ultrastructure of C. albicans. We next utilized transmission electron microscopy to reveal ultrastructure of C. albicans SC5314 cells after treated with BN-3b. As shown in Fig. 2, BN-3b treated cells exhibited obvious alteration in the morphology compared to vehicle. C. albicans SC5314 cells showed normal cellular morphology with a distinct cell wall and an intact cell membrane in vehicle treated group ( Fig. 2A). In contrast, the cell membrane of C. albicans was seriously destroyed after treated with BN-3b ( Fig. 2D-F). The results indicated that BN-3b killed fungi through destroying the cell membrane.

BN-3b inhibited C. albicans in vivo.
To evaluate the antifungal effect of BN-3b against C. albicans in vivo, we used two wild-type strains of C. albicans (C. albicans SC5314 and CGMCC 2.2086) in a model of disseminated candidiasis in mice. Percent survival over time for BN-3b, AMB, and FLC are shown in Fig. 3. All animals in the vehicle treatment group died by day 11 after infection with C. albicans SC5314, or day 13 with strain CGMCC 2.2086. Survival times for all groups of treated mice were significantly prolonged compared to the vehicle group. BN-3b or FLC treatments at dose of 2.0 mg/kg survived through days 15 and 19 for C. albicans SC5314-infected mice, respectively; days 17 and 20 for strain CGMCC 2.2086, respectively. The percent survival at day 21 after challenge of mice with 4.0 mg/kg BN-3b or 2.0 mg/kg AMB treatment were 28.6% and 64.3% for strain SC5314, respectively; 35.7% and 71.4% for strain CGMCC 2.2086, respectively (Fig. 3). In order to evaluate the impact of drugs on the body weight of mice, we also monitored the body weight of mice treated with BN-3b, AMB, FLC, or vehicle. The mean weight of mice prior to C. albicans SC5314 and CGMCC 2.2086 inoculations were 22.10 ± 0.30 g and 22.36 ± 0.31 g, respectively. After inoculation with C. albicans, a dramatic decrease in the body weights of vehicle-treated mice (Fig. 3). In contrast, body weights for all groups of treated mice were significantly higher compared to the vehicle treatment groups (P < 0.05). Figure 4 presents the fungal burdens in livers, kidneys, spleens, and lungs of mice treated with vehicle, BN-3b (2.0 mg/kg or 4.0 mg/kg), AMB (2.0 mg/kg), and FLC (2.0 mg/kg) by intraperitoneal injection. The BN-3b significantly reduced the number of CFU/g of liver tissues, kidney tissues, spleen tissues, and lung tissues in mice infected by C. albicans compared with vehicle treatment, and it was in a dose-dependent manner (Fig. 4). The results indicated that BN-3b significantly prolonged survival and decreased the fungal burdens of livers, kidneys, spleens and lungs in mouse model of disseminated candidiasis. www.nature.com/scientificreports www.nature.com/scientificreports/ BN-3b enhanced the ROS production. Intracellular ROS production was detected by using the oxidant-sensitive probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) 9 . Generation of ROS was monitored by incubation of BN-3b at 25.0, 50.0, and 75.0 μg/mL with the C. albicans SC5314 cells for 8 h, respectively. As shown in Fig. 5A, the ROS production induced by BN-3b increased significantly in a dose-dependent manner (p < 0.01), which was in turn attenuated by the addition of antioxidant N-acetylcysteine (NAC). The data indicated that BN-3b promoted ROS production in C. albicans.
Phospholipid peroxidation of C. albicans induced by BN-3b. Overproduction of ROS lead to phospholipid peroxidation of membrane 9 . MDA was one of the final products of phospholipids peroxidation and could directly reflect the level of cell membrane damage 19 . To further determine the involvement of oxidative damage induced by ROS in BN-3b antifungal activity, we examined phospholipid peroxidation levels of   www.nature.com/scientificreports www.nature.com/scientificreports/ Effect of BN-3b on GSH. GSH plays a vital role in the protection of yeast cells against damage induced by oxidative stress 20 . As overproduction of ROS may consume GSH, we therefore examined the GSH levels of C. albicans SC5314 cells after treated with BN-3b. As shown in Fig. 5C, the content of GSH were significantly reduced in a dose-dependent manner after treated with BN-3b compared with the control (p < 0.01). Moreover, we also www.nature.com/scientificreports www.nature.com/scientificreports/ found that the ratio of GSH/GSSG were significantly decreased in the BN-3b treatment groups (Fig. 5D). These results further confirmed that oxidative damage induced by ROS was involved in the antifungal activity of BN-3b.
Effect of BN-3b on MMP and ATP synthesis. In general, excessive ROS production triggered the mitochondria dysfunction 21 . To investigate whether BN-3b affected the function of mitochondria, we determined the intracellular MMP level and ATP production of C. albicans SC5314 treated with or without BN-3b. The MMP was measured using a JC-1 fluorescent probe, and the ratio of red/green fluorescence intensity represents MMP 22 . Our results showed that the level of MMP was significantly decreased in a dose-dependent in the BN-3b treated groups (Fig. 5E). The JC-1 red/green fluorescence intensity ratio decreased to 91.0 ± 3.6% (p < 0.05), 57.3 ± 2.1% (p < 0.01), and 45.3 ± 2.5% (p < 0.01), when the C. albicans SC5314 cells were treated with 25.0, 50.0, and 75.0 μg/ mL BN-3b, respectively. www.nature.com/scientificreports www.nature.com/scientificreports/ In addition, ATP content is one of the important indexes of mitochondrial activity 21 . As shown in Fig. 5F, the content of intracellular ATP decreased significantly in a dose-dependent manner after treatment with BN-3b. The content of intracellular ATP decreased to 86.7 ± 4.2% (p < 0.05), 59.3 ± 2.5% (p < 0.01), and 48.7 ± 3.1% (p < 0.01), respectively, when the C. albicans SC5314 cells were exposed to 25.0, 50.0, and 75.0 μg/mL BN-3b. At the same time, the decrease of intracellular ATP content caused by BN-3b could be in turn attenuated by the addition of antioxidant NAC. The above data suggested that the mitochondria function was impaired in BN-3b treated cells.
Effect of BN-3b on the hyphal formation in C. albicans. The ability to switch from yeast to hypha was important for virulence of C. albicans 23 . C. albicans SC5314 cells incubated with vehicle or different concentrations of BN-3b (25.0, 50.0, and 75.0 μg/mL) for 8 h, and then observed by microscopy. In the vehicle treated group, large numbers of hyphae were observed in C. albicans. In contrast, BN-3b markedly inhibited the hyphal formation of C. albicans in a dose-dependent manner (Fig. 6). Especially, BN-3b completely inhibited the hyphal formation of C. albicans at the concentration of 75.0 μg/mL (Fig. 6F).
Effect of BN-3b on expression of virulence-related genes. The effect of BN-3b on the virulence-related genes (Supplementary Table S1) was examined in C. albicans SC5314 using RT-qPCR. RT-qPCR analyses revealed that the expressions of virulence factors of C. albicans SC5314 were significantly down-regulated in a dose-dependent manner after treated with BN-3b compared with the control (p < 0.01) (Fig. 7). These data indicated that BN-3b could exert additional anticandidal activity by inhibiting the expression of virulence factors (SAPs, PLB1, PLB2, HWP1, ALSs, and LIPs) in C. albicans.

Discussion
The kill curves study indicated that BN-3b significantly reduced the cell viability of C. albicans in a dose-dependent manner (Fig. 1B). Following, we evaluated the in vivo antifungal activity of BN-3b by establishing the systemic mouse model of disseminated candidiasis. The results revealed that BN-3b significantly prolonged survival and decreased fungal burdens in mouse model of disseminated candidiasis. Based on the good antifungal activity of BN-3b in vivo and in vitro, we further discussed its action mechanism.
Increasing evidence demonstrated that oxidative damage induced by endogenous ROS was involved in the antifungal activity of antifungal agents [7][8][9][10][11][12]24 . Kobayashi et al. reported that the ROS production of C. albicans were significantly increased in a dose-dependent manner after treated with 0.125 (MIC), 1.25, and 12.5 μg/ mL miconazole, respectively. FLC treatment also enhanced ROS production, especially in the 5 and 50 μg/mL groups 25 . In another study, Li et al. showed that treatment with 8 μg/mL of AMB significantly increased the ROS production in C. albicans 26 . According to the literatures and our experiment results, we used 25 μg/mL FLC or (and) 2 μg/mL AMB as positive controls for the antifungal mechanism research in the current study.
Excessive ROS may lead to oxidative damage of nucleic acids, proteins, and lipids, and ultimately lead to cell death 19 . In this study, we found that BN-3b stimulated ROS production in C. albicans cells (Fig. 5A). To further confirm the ROS production, we examined the intracellular MDA production and GSH concentration. The excess ROS produced will react with cell membranes, produce lipid peroxide radical, and eventually form MDA 27 . Our www.nature.com/scientificreports www.nature.com/scientificreports/ current study showed that the production of MDA were significantly increased in a dose-dependent manner in the BN-3b treated cells (Fig. 5B). GSH protects the cells from oxidative damage by scavenging ROS 27 . Thus, the generation of excess ROS may consume GSH. In fact, our results indicated that the content of GSH were significantly reduced in a dose-dependent manner in the BN-3b treated cells (Fig. 5C). Besides, BN-3b treatment could markedly decrease the ratio of GSH/GSSG (Fig. 5D). Conversely, antioxidant NAC could significantly attenuate BN-3b induced oxidative stress. These results indicated that BN-3b treatment impaired the balance of antioxidant system in C. albicans cells. In general, the overproduction of ROS might damage the membranes. Consistent with this, the cell membrane of C. albicans appeared obvious shrinkage and breakage (Fig. 2D-F) after exposure to BN-3b. Collectively, these data strongly suggested that BN-3b induced the endogenous ROS-mediated oxidative damage and destroyed the cell membrane, ultimately resulted in cell death.
The above results confirmed that BN-3b stimulated ROS accumulation in C. albicans. The excessive ROS production can lead to mitochondrial dysfunction, mainly characterized by the loss of MMP and decrease of ATP generation 28 . Our current study showed that the MMP levels of C. albicans cells were significantly reduced in a dose-dependent manner after treated with BN-3b. Furthermore, the results also showed that the intracellular ATP content was significantly decreased after treated with BN-3b compared with the control. These results indicated that the mitochondria function was impaired in BN-3b treated cells.
BN-3b belongs to the BN ester analogue and was synthesized by our research group. In comparison with BN, the cytotoxicity of BN-3b was significantly decreased. More significantly, BN-3b displayed potent antifungal activity against C. albicans, while BN was inactive 5 . It is generally accepted that the multiple bioactivities of BN were associated with the inhibition of threonyl-tRNA synthetase (ThrRS) 5,6 . The molecular docking study indicated that the side chain of BN-3b was projected deeper into the bottom of binding pocket in ThrRS than BN, which indicated the action mechanisms of BN-3b was different from BN 5 . In the present study, we found that oxidative damage induced by endogenous ROS plays an important role in the antifungal activity of BN-3b. Besides, we speculated that the unique interaction of BN-3b with ThrRS might also play a vital role in its antifungal activity, but it needs to be further verified.
Many virulence factors of C. albicans are involved in the infective process, such as extracellular hydrolases production, adhesion to host tissue, and hyphal formation 13,14 . In this study, we also examined the mRNA level of the virulence-related genes after exposure to BN-3b using RT-qPCR in C. albicans. The three most significant extracellular hydrolases produced by C. albicans are the SAPs, LIPs, and phospholipase B (PLBs) 29 . C. albicans SAPs, encoded by a multigene family 15 (SAP1 to SAP10), contribute to pathogenesis by digestion of host cell membranes and molecules of the host immune system 16 . C. albicans LIPs, encoded by at least ten members (LIP1 to LIP10), contribute to the provision of nutrients and promote fungal penetration of host barriers 17 . Phospholipase B encoded by at least two genes (PLB1 and PLB2) also contributes to the pathogenicity of C. albicans by abetting the fungus in damaging and traversing host cell membranes 15,29 . The results of our study showed that all of these genes were significantly down-regulated after BN-3b treatment (p < 0.01). The pathogenic potential of C. albicans was positively correlated with its adhesive capacity of the organism 30 . Currently, most studies focus on two well-characterized adhesins, Hwp1 and the ALS family 29 . The C. albicans ALS family has eight members (ALS1 to ALS7, and ALS9), each encodes a large glycoprotein whose function is adhesion to host 31 . Furthermore, it has been suggested that some ALS proteins were involved in growth-related functions 17 . Our current study showed that these genes were significantly down-regulated after BN-3b treatment (p < 0.01). Therefore, down-regulation of these genes not only affect the infection ability of C. albicans but also affect the cell proliferation of C. albicans. HWP1, a hypha-specific gene, which encodes a cell-surface adhesin that promotes interactions between C. albicans and host cells 17,29 . It is interesting to note that the mRNA level of HWP1 decreased significantly after treatment with BN-3b. Hyphae formation plays a key role in C. albicans pathogenicity 13,32 . In this study, we found that BN-3b strongly inhibited the hyphal formation of C. albicans. Thus we suggested that BN-3b could inhibit the yeast-to-hypha transition and down-regulate the expressions of virulence factors to weaken the pathogenicity of C. albicans.
Our results indicated that BN-3b exerts antifungal effect through increasing the generation of ROS, decreasing the MMP, reducing the intracellular ATP level, and destroying the cell membrane. In addition, BN-3b exerts added anticandidal activity by inhibiting the yeast-to-hypha transition and down-regulating the expressions of virulence factors. These findings suggested that BN-3b may be a promising lead for the development of antifungal agent. In vitro assay for antifungal activities. The MICs of BN-3b were determined using the method described by CLSI guidelines 34 . FLC and AMB were used as positive controls. The tested compounds were dissolved in DMSO and 2-fold serially diluted to eight different concentrations 5 (10-0.078 mg/mL for BN-3b, 0.8-0.00625 mg/ mL for FLC and AMB). The above samples (1 μL) and 100 μL of prepared fungal suspensions (in RPMI-1640 medium) containing 2 × 10 3 cfu/mL of fungus were added to each well of 96-well microtiter plates 5 . The vehicle treated wells were used as control. The plates were incubated for 48 h at 28 °C, and the absorbance was recorded spectrophotometrically at 620 nm using a microplate reader (BioTek Synergy H1, BioTek Instruments, Inc., Vermont, USA). The MICs of the BN-3b and AMB were defined as the lowest concentrations that completely inhibited visual growth of an organism 35 . The concentration of FLC which caused a 80% reduction in the absorbance compared to the control was considered as the MIC 36 . the viability assay of C. albicans. Fungal suspensions at 6 × 10 5 CFU/mL in YPD liquid medium were In order to quickly establish mouse model of disseminated candidiasis, all mice used in this study were received CY at 100 mg/kg body weight administered intraperitoneally 3 days before and 1 day after infection 38 .
Fungal suspension (C. albicans: 2 × 10 5 CFU/mouse in volume of 0.1 mL) was inoculated into the lateral tail vein of mice, 3 days (72 h) after the intraperitoneal injection of CY. Mice were randomly separated into five groups (n = 14 per group). BN-3b therapy with a dose of 2.0 mg/kg or 4.0 mg/kg of body weight daily, AMB or FLC therapy with a dose of 2.0 mg/kg of body weight daily by intraperitoneal injection were initiated at 24 h after infection, and continuous administration for 5 days. Control group were treated with vehicle (60% 1,2-propanediol) in the same way. Twenty-four hours after the last dose of antifungal agent, four mice of each group were sacrificed to determine the fungal burden in the liver, kidney, spleen, and lung. The organs were excised by a sterile technique, weighed, and homogenized in 5 mL of sterile saline 9 . The homogenates were serially 10-fold diluted in sterile saline, and 100 μL was plated on SDA 39 . Plates were incubated for 48 h at 35 °C and the number of CFU/g of tissue was calculated 9 . In the study of the survival rate and body weight in mice, fourteen mice in each group were monitored daily until 15 days after the end of therapy (21 days after infection).
Measurement of ROS production. Followed the methods as previously described, C. albicans SC5314 (10 7 CFU/mL) incubated with 40 μM DCFH-DA at 37 °C for 60 min in the dark 9 , the cells were collected, washed twice and then diluted to 6 × 10 5 CFU/mL with YPD 12 . After that, a series of BN-3b were added and incubated at 37 °C for 8 h. And then washed and re-suspended in 100 μL of PBS. The fluorescence intensity of a cell suspension (100 μL) containing 10 7 cells was measured using a microplate reader with excitation at 480 nm and emission at 530 nm 9 28 . Green (excitation/emission wavelength: 514/529 nm) and red (excitation/emission wavelength: 585/590 nm) fluorescence were detected on a microplate reader 28 . The ratio of red/green fluorescence intensity represents MMP 22 .
Synthesis of ATP assay. The cellular ATP level was detected using an ATP Bioluminescence Assay Kit (Beyotime Biotechnology Co., Shanghai, China) 28 . C. albicans SC5314 (6 × 10 5 CFU/mL) incubated with vehicle or different concentrations of BN-3b (25.0, 50.0, and 75.0 µg/mL) for 8 h. After that, cells (5 × 10 6 ) from each culture were lysed and centrifuged, and then 100 μL of ATP detection working solution as well as 50 μL supernatant were added to 96-well plate, and then luminescence was measured on a microplate reader 28 .
Hyphal formation assay. The hyphal formation of C. albicans induced by YCB/FBS medium 40,41 . C. albicans SC5314 (6 × 10 5 CFU/mL) incubated with different concentrations of BN-3b for 8 h at 37 °C. The hyphal formation of C. albicans was recorded with a microscope with the magnification of 400×. The treatment of AMB (2.0 μg/mL) and FLC (25.0 μg/mL) severed as positive controls.
RNA extraction and RT-qPCR. C. albicans SC5314 cells were treated with vehicle and BN-3b (25.0 or 50.0 μg/mL) in YCB/FBS medium as the same method above. Total RNA was extracted with AxyPrep Multisource Total RNA Miniprep Kit (Axygen, China) and reverse transcribed with GoScript TM reverse transcription system (Promega, USA) by following the manufacturer's instructions. RT-qPCR was conducted according to our previous report 33 . RT-qPCR was performed with the primer sets listed in Supplementary Table S1. ACT1 gene was used as the internal control. Fold changes were calculated using the 2 −△△Ct method 33 .
Statistical analysis. All data were represented as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was determined by using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, p ≤ 0.05. The SPSS 17.0 statistical software package was used for data analysis.

Data availability
The datasets that were generated and/or analysed during the current study are freely available from the corresponding author on a request.