Azoles resistance reversal by oridonin in Candida albicans CURRENT STATUS: POSTED

Candida albicans ( C. albicans ) is a yeast causing hazardous fungal infections with high mortality, especially accompanied by resistance to azole drugs (fluconazole, itraconazole and voriconazole). To overcome the azoles resistance of C. albicans , we explored the Oridonin (ORI) with three azole drugs mainly focused on the synergistic activity. In this study, C. albicans strains were obtained from cancer patients, and the reversal of drug resistance of azole-resistant C. albicans was further studied. The synergistic antifungal activities of ORI and azoles were measured by checkerboard microdilution and time-kill assays. The resistance reversal mechanisms, inhibition of drug efflux and induction of apoptosis, were investigated by flow cytometry after Annexin V-FITC/PI co-staining. The expression levels of efflux pump related genes CDR1 and CDR2 were quantitatively detected by qRT-PCR. The azole-resistant isolates identified by checkerboard microdilution method and time-kill curves. The efflux pump inhibition assay with ORI showed that the MIC of fluconazole (128-fold), itraconazole (64-fold) and voriconazole (250-fold) decreased significantly. The upregulation of genes coding for CDR1 and CDR2 were confirmed by qPCR with respect to the housekeeping gene ACT1 in the resistant strain. The sensitizing effect of ORI on fluconazole in the of C. albicans also We demonstrated that the combination of azoles with ORI exerted potent synergism and further that ORI could promote the sensitization to azoles for azoles-resistant C. albicans . µg/ml, µg/ml and 2 to 128 µg/ml, respectively. The Candida cells in exponential phase of harvested and in buffer saline (PBS; The concentrations of the Candida suspensions were measured by a hemocytometer (Shanghai followed by serial dilutions. A volume of 100 µl of the inoculum was added to the polystyrene plates and the final size of the inoculum was 2 × 10 3 CFU/ml for all stains. The plates were incubated at 35 °C. After 24 h and 48 h, 100 µl of the reagent containing 0.5 mg/ml XTT and 10 µmol/L menadione was added, followed by 2 h incubation in the dark at 35 °C. Then the colorimetric changes were measured at 492 nm with a microtiter plate reader (Bio-Rad, USA). The minimum inhibition concentration (MIC) is defined as the lowest drug concentration that caused an 80% reduction in optical density compared with that of drug-free control well. All the experiments were repeated three times.


Background
Fungal invasive infections of humans are now referred to as "hidden killers" [1]. Candidemia was cited as the fourth most prevalent nosocomial bloodstream infection in the United States and has a 3 highly attributable mortality rate, causes prolonged hospitalization and rising health care costs [2,3].
Due to low immunity, tumor patients have an increased risk of nosocomial infection. Infection may affect anti-tumor therapies and even lead to death. Invasive candidiasis may be a presenting symptom of cancer and a predictor of increased cancer risk in later years [4]. More attention should be paid to fungal infection in tumor patients [5].
Azole has been widely applied clinically in the treatment for fungal infection and is still the first line treatment for Candida infection. However, the resistance to azoles in C. albicans is constantly emerging [6]. This makes infections caused by azole-resistant C. albicans often recalcitrant to conventional antifungal therapy. C. albicans can become resistant to azoles by increasing the number of efflux pumps in the cell, as described above [7,8]. Efflux pumps are membrane-associated transporters that work by preventing the intracellular accumulation of drug, thereby avoiding toxic levels that would kill the cell [7,8]. Due to this overexpression of efflux pumps, cross-resistance between azoles is often seen in C. albicans, both in vitro and clinically [9]. Up-regulation of CDR1 and CDR2 was mainly responsible for the resistance of CA-R [10].
Although amphotericin B and echinocandins are two antifungal compounds which only display effective antifungal activity against azole-resistant C. albicans, their high cost and toxicity limit their clinical application. Therefore, it is imperative to find novel treatment strategies to overcome the problem. Interest is increasing in the study of the antifungal effects of azoles combined with one nonantifungal drug [11][12][13]. Using traditional Chinese medicines or their extracts to reverse the resistance of C. albicans to azoles has emerged to be one of the most promising means. Oridonin (ORI) has a molecular formula of C 20 H 28 O 6 ( Fig. 1), an ent-kaurane diterpenoid identified from the Chinese medicinal herb Rabdosia rubescens. It was reported that ORI has multiple health-promoting effects, including antioxidant, anti-inflammatory, and antitumor effects [14][15][16]. There is little evidence showing that ORI can effectively treat infections caused by azole-resistant C. albicans, and the relevant mechanisms need to be further clarified. In this study, we investigated the effects of ORI combinations of azoles against azoles-resistant C. albicans, and characterized the resistance reversal mechanisms in vitro. 4 Methods Strains and chemicals.
Candidemia was defined by a Candida species-positive culture of blood from a patient with cancer (e.g., leukemia, lymphoma, multiple myeloma, or solid tumor) who presented with fever. According to the routine method, C. albicans were identified and screened by Candida CHROMagar medium after cultured according to the routine method. The C. albicans strains used in this work (CA2489, CA3208, CA10 and CA136) were clinical isolates from Shandong Tumor Hospital and Shandong Provincial Qianfoshan Hospital. C. albicans strains were grown routinely on yeast-peptone-dextrose (YPD) agar medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose and 2% (w/v) agar at 35℃. RPMI 1640 medium (pH 7.0) with L-glutamine and without sodium bicarbonate was purchased from Gibco and buffered with MOPS (Sigma). Their susceptibilities were determined according to CLSI (Clinical and Laboratory Standards Institute, formerly NCCLS) M27-A3 document with C. albicans ATCC 10231 as reference strain [17]. The break points at 24 [18]. FLC, ITR and VOR were kindly provided by Cheng Chuang Pharmaceutical Co., Ltd., China; ORI (chemical structure shown in Fig. 1) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Stock solution of FLC was prepared in sterile distilled water. ITR, VOR and ORI were dissolved in dimethyl sulfoxide (DMSO) to make a stock solution. DMSO concentration was kept below 0.01% in all the cell cultures, and did not exert any detectable effect on cell growth or cell death.
In order to determine possible synergistic interactions between the azoles and ORI against all Candida strains, checkerboard analysis was used and fractional inhibitory concentration index (FICI) values were calculated. The FICI was calculated by the formula FICI = FICI A + FICI B, where FICI A is calculated as MIC A alone/MIC A combination and FICI B is calculated as MIC B alone/MIC B combination. The interpretation of the FICI was as Odds suggested: the value of FICI ≤ 0.5 should be considered synergy, 0.5 < FICI ≤ 4 should be considered no interaction, and FICI > 4 antagonism [19]. Serial 2-fold dilutions were performed in RPMI 1640 medium and 50 µl of each drug dilution was added to each well of a round-bottomed 96-well plate. The final concentration of FLC, ITR, VOR and ORI ranged from 1 to 512 µg/ml, 0.016 to 8 µg/ml, 0.016 to 8 µg/ml and 2 to 128 µg/ml, respectively. The Candida cells in exponential phase of growth were harvested and suspended in sterilized phosphate buffer saline To investigate the effect of concentration and exposure time on the antifungal activities of the azoles with or without ORI, a time-killing test was performed against azole-resistant C. albicans, at the starting inoculum of 10 4 CFU/ml. Time-killing studies were conducted in eight groups: (1) drug-free control; (2) azole (FLC/ITR/VOR) alone; (3) ORI (8 µg/ml); (4) ORI (16 µg/ml); (5) ORI (32 µg/ml); (6) azole + ORI (8 µg/ml); (7) azole + ORI (16 µg/ml); (8) azole + ORI (32 µg/ml) FLC, ITR and VOR were used at the concentration of 8, 0.125 and 0.125 µg/ml, respectively. At predetermined time points (0, 6, 12, 24, and 48 h after incubation at 35 °C), an aliquot (100 µl) was aspirated from each group and transferred to a well of a 96-well plate. Then 100 µl of the reagent containing 0.5 mg/ml XTT and 10 µmol/L menadione was added, followed by 2 h incubation in the dark at 35 °C. After 2 h, the colorimetric changes were measured at 492 nm with a microtiter plate reader (Bio-Rad, USA). All experiments were conducted in triplicate, and the results were reported as mean values ± standard deviation (SD). The OD value for each incubation time point was plotted as the vertical ordinate.
In yeast, phosphatidylserine is predominantly located on the inner leaflet of the lipid bilayer on the cytoplasmic membrane and is translocated to the outer leaflet during apoptosis [20]. The apoptotic marker, phosphatidylserine externalization, was analysed via staining with FITC-labelled annexin V and PI with the FITC-annexin V apoptosis detection kit (Solarbio Science and Technology Co., Ltd.). C.
albicans cells were cultured overnight in YPD liquid medium and collected after 18 h. The exponentially growing yeasts were adjusted to 5⋅10 6 CFU/ml with PBS, and diluted to 4⋅10 5 CFU/ml in RPMI 1640 medium. FLC, ORI and combination of both were respectively added to cultures of C.
albicans. Cell cultures without drug treatment served as controls. Cells were incubated for 10 h at 35 °C, and afterwards were collected by centrifugation and washed with cold PBS. Then cells in each group were incubated for 15 minutes at room temperature in dark in an annexin-binding buffer containing 5µ l Annexin V-FITC and 5µ l PI, respectively. Samples were then detected with FACS Calibur flow cytometer (Becton Dickinson).
Flow cytometric analysis of the efflux of rhodamine 6G (rh6G).
Rh6G (Sigma) can be absorbed into yeast cells and the efflux of rh6G uses the same membrane transporter as FLC in yeasts [21]. The intracellular rh6G concentration can be used to investigate the drug efflux mechanism in azole-resistant C. albicans [22]. The rh6G efflux was investigated by a flow cytometer (Becton Dickinson FACS Calibur) at 525 nm with the logarithmic-phase C. albicans cells (5 × 10 6 CFU/ml). C. albicans cells were firstly incubated at 35 °C at 120 rpm in glucose-free PBS buffer containing 10 µM rh6G. When rh6G absorbed into the cell reached equilibrium, uptake of rh6G was stopped by cooling the tubes on ice. The reaction mixture was washed three times with cold PBS buffer to remove rh6G, and then the fluorescence of the cells was determined. After removing the excess rh6G, 8 µg/ml ORI was added to detect efflux. The cells were subjected to a second incubation in PBS buffer containing 5% glucose. Cell cultures in the absence of ORI served as controls. At 90 min after the second incubation, the fluorescence of the cells was measured. Ten thousand cells with similar size and complexity were selected for evaluation in every assay. Experiments were replicated three times. Raw data were analyzed and plotted with GraphPad Prism 5.
Determination of possible resistance mechanisms to FLC by qPCR. The MIC distributions of ORI and azoles alone and in combination against C. albicans were shown in Table 1 and Table 2. ORI acted synergistically with azoles against resistant C. albicans at 24 h and 48 h. However, the synergistic effect on sensitive strains (CA2489 and CA3208) was not obvious.
Although ORI alone had very limited antifungal activity, significant decrease in MICs of the three azoles could be observed when resistant strains were treated in combination with various concentrations of ORI. The MICs for FLC, ITR and VOR alone were respectively > 512, > 8 and > 8 µg/ml against C. albicans (CA10 and CA136). The data indicated that there were C. albicans resistance toward azoles. After adding ORI to the culture system, the concentration of FLC, ITR and VOR to azoles resistance C. albicans were decreased dramatically. When the concentration of combined ORI was 8 µg/ml, the MIC of FLC, ITR and VOR decreased by 128 times, 64 times and 250 times respectively at 24 h, and the FICI values were all less than 0.1. In order to visually present the strong synergism between the azoles and ORI against CA10, the results were illustrated in Fig. 2 as three-dimension graphics. Although the FICI values for the susceptible strains were > 0.5, the MIC of each antifungal agent was reduced 2-fold when it was in combination with ORI. The trend of CA136 was consistent with CA10, and the image was not shown. In the experiment, it was found that the drug-resistant strain CA10 had good repeatability and stability in the treatment of fluconazole, and its mechanism was studied later. The MIC of the quality control strain, ATCC 10231, fell within the normal range.  Association between the degree of reversal activity and FLC susceptibility.
Determination of ORI and azoles by checkerboard microdilution method and time-kill curves showed concentration-dependent synergistic effects in all C.albicans. The intensity and nature of interactions between ORI and azoles against CA10 were shown in Fig. 3. The antifungal effect of ORI hardly changed by increasing concentration, and the curves of ORI alone almost stayed close to the curve of drug-free group. When the antifungal agent was used alone, either azole assumed weak antifungal effect against the drug-resistant C.albicans CA10. However, the antifungal effects of azoles against CA10 cells were dramatically enhanced by addition of ORI (4 µg/ml, 8 µg/ml, 16 µg/ml), and the discernible improvement in the extent of fungistatic activity was noted as the amount of ORI in solution was increased. When the concentration of ORI was 16 µg/ml, the sensitization exerted by ORI was the most potent and the curves were almost straight lines, suggesting that the drug combinations composed of 16 µg/ml ORI and 8 µg/ml FLC, or 0.125 µg/ml ITR, or 0.125 µg/ml VOR almost completely inhibited the growth of the CA10 cells. Sensitized concentrations of ORI resulting in inhibitory ratios below 10% ranged from 0 to 1 µg/ml, and the IC 80 was 8 µg/ml (Fig. 2), so this concentration was used in subsequent experiments.
Determination of changes in expression of CDR1 and CDR2 genes by qPCR.
Quantitative detection of the expression of efflux pump related genes CDR1 and CDR2 by qRT-PCR. (p = 0.017) and CDR2 genes (p = 0.455). The expression level of CDR2 was the most upregulated, which was 3.73 ( FLC alone) and 1.06 (with ORI) times higher than that of the control (Fig. 3).
Inhibition of C. albicans drug resistance through the modulation of efflux pumps.
The efflux of rh6G in CA10 cells was evaluated by flow cytometry and the results were shown in Combination of FLC and ORI induced apoptosis in C. albicans.
The apoptotic and living cells are distinguished by double staining with annexin V-FITC and PI. The results were shown in Fig. 6. Apoptosis was barely detectable in untreated control cells (0%, right quadrant in Fig. 6a), ORI-treated cells (0.14%, right quadrant in Fig. 6b) and FLC-treated cells (0.92%, right quadrant in Fig. 6c). However, the number of early and late apoptotic cells increased in cells treated with ORI and FLC (62.3%, right quadrant in Fig. 6d). In detail, C. albicans cells showed an AnnexinV-FITC + /P − phenotype (6.09%) and AnnexinV-FITC + /PI + phenotype (56.21%). These results indicated that the sensitizing effect of ORI on FLC was observed through induction apoptosis of C. albicans.

Discussions
The patients suffering from cancer prone to C. albicans infection are due to decreased immunity caused by radiotherapy, chemotherapy, and long-term use of corticosteroids and broad-spectrum antibiotics [14]. In solid cancer patients, C. albicans candidemia accounted for 32.8% [14]. C. albicans was more frequently associated with solid tumors of the gastrointestinal and genitourinary tracts and breast patients [24]. It is efficacious for azole antifungal agents to prevent and treat the infections caused by C. albicans. However, the ever-increasing azole resistance in C. albicans emerges. The development of strategies to combat azole-resistances in C. albicans can be greatly facilitated by studying drug combinations of azoles and antifungal sensitizers. Combined use of drugs can improve the curative effect and reduce the adverse reactions and economic costs. The focus of research and development of antifungal synergist began to shift to traditional Chinese medicine extracts, which are potential sources for new drugs. In this work, a FLC-resistant clinical isolate of C. albicans was studied. The first goal was to explore a potential mechanism of azole resistance.
In this study, the activities of three azole drugs (fluconazole, itrconazole and voriconazole) were examined against C. albicans strains with different susceptibilities in presence of ORI, a traditional Chinese medicine extract. ORI has potential as anti-tumor, anti-microbial, anti-inflammatory, and antioxidant agent [14,15,25]. confirmed that ORI reduced the efflux of FLC, which may be related to the inhibition of ORI on the efflux pump function on the cell membrane. It is well acknowledged that interruption of membrane integrity is an important reason why apoptosis of late stages occurs in yeast cells [26]. Therefore, inhibition of the efflux of FLC by ORI may bring about the occurrence of cell apoptosis in C. albicans, and these may together contribute the synergy against resistant C. albicans.
It has been reported that some Chinese medicine exert antifungal activity by inducing apoptosis in C.
albicans [27][28][29], and the induction of cell apoptosis has become an important antifungal pathway [30]. However, the reversal of azole-resistance in C. albicans by ORI has never been described.
Apoptosis is a highly regulated cellular suicide programme crucial for metazoan development. As eukaryotic cells, C. albicans have an asymmetric distribution of phospholipids within the cytoplasmic 13 membrane, with 90% of phosphatidylserines oriented toward the cytoplasm [31]. In this study, the reversal mechanism of ORI in FLC-resistant C. albicans was determined by comparing the cells apoptosis. During apoptosis, phosphatidylserine is externalized from the inner to the outer layer [32].
Annexin V is a phospholipid-binding protein with high affinity for phosphatidylserine, and PI is amembrane-impermeant fluorescent dye which stains DNA. Thus, we investigated this specific apoptotic hallmark using AnnexinV/PI double staining. In FLC-resisitant C. albicans, the combination of ORI and FLC induces early (annexinV-FITC + /PI − , 6.09%) and late apoptosis (annexinV-FITC + /PI + , 56.21%). Although there is almost no apoptotic population on ORI/ FLC alone, apoptosis pathway is switched on in the majority of yeast cells when they are used together.

Conclusions
Taken as a whole, the combination of ORI and azoles exerts synergistic effects against resistant C.
albicans. The mechanism of ORI reversing FLC resistance is that it affects the expression level of efflux-related genes, inhibits drug efflux, and induces apoptosis of C. albicans after entering cells.
Nevertheless, the precise synergistic mechanisms require further investigation owing to the unclear resistance mechanism in the resistant C. albicans strain. This study provides new information about the synergistic antifungal effects and mechanism of this drug combination, as well as insight into antifungal agent discovery.

Ethics approval and consent to participate
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as the research in this article related to micro-organisms.

Consent for publication
Not applicable The fold changes of CDR 1 and CDR 2 by qRT-PCR after the treatments of no drugs (control), 8 μg/ml FLC, 8 μg/ml ORI, and 8 μg/ml FLC + 8 μg/ml ORI on CA10. The Tukey HSD analysis was used to calculate the differences between groups. *** p < 0.001 , compared with the control.