Ras1 involved in the antifungal resistance of Candida albicans and the inhibition of farnesol on the resistance of biofilms

Background: Farnesol enhances the susceptibility of Candida albicans biofilms to antifungals, while the molecular mechanisms of this behavior are poorly understood. RAS1 regulates the hyphal growth of C. albicans, and farnesol inhibited hyphal growth by RAS1 regulation, while the role of RAS1 in the resistance of C. albicans biofilms and the molecular mechanism of the RAS1 in the farnesol-relevant antifungal capacity to C. albicans biofilms is still unknown. The study hypothesized that Ras1 involved in the antifungal resistance of C. albicans and the inhibition of farnesol on the resistance of biofilms. Results: The susceptibility assays showed that RAS1 over-expressing strain (RAS1OE) increased the resistance of C.albicans in both planktonic and biofilm form to antifungals, while RAS1 deletion strain (ras1Δ/Δ) reduced that to antifungals. The SMIC50 of the antifungals were increased with the mature of the biofilms formed from the mutant and the wild strains. Exogenous farnesol decreased the resistance of RAS1OE to antifungals, including fluconazole, amphotericin B, itraconazole, caspofungin, terbinafine, 5-flurocytosine and nystatin. The inhibitory effects of farnesol on the antifungal resistance of the biofilms from the RAS1OE were in accordance to almost all of the growth phases. Moreover, exogenous farnesol decreased the resistance of biofilms from RAS1OE more obviously than that from the wild strains (P<0.05). In addition, Morphological observation showed that that RAS1OE increased hyphal growth the biofilms, while ras1Δ/Δ reduced that of the biofilms. Compare to the wild-type strain, the inhibitory effects of farnesol on hyphal growth were more obvious to the RAS1OE, while less obvious to the ras1Δ/Δ. Furthermore, farnesol reduced the level of Ras1 and the expression of RAS1 of the biofilms formed from the RAS1OE


Abstract
Background: Farnesol enhances the susceptibility of Candida albicans biofilms to antifungals, while the molecular mechanisms of this behavior are poorly understood. RAS1 regulates the hyphal growth of C. albicans, and farnesol inhibited hyphal growth by RAS1 regulation, while the role of RAS1 in the resistance of C. albicans biofilms and the molecular mechanism of the RAS1 in the farnesol-relevant antifungal capacity to C. albicans biofilms is still unknown. The study hypothesized that Ras1 involved in the antifungal resistance of C. albicans and the inhibition of farnesol on the resistance of biofilms.
Results: The susceptibility assays showed that RAS1 over-expressing strain (RAS1OE) increased the resistance of C.albicans in both planktonic and biofilm form to antifungals, while RAS1 deletion strain (ras1Δ/Δ) reduced that to antifungals. The SMIC50 of the antifungals were increased with the mature of the biofilms formed from the mutant and the wild strains. Exogenous farnesol decreased the resistance of RAS1OE to antifungals, including fluconazole, amphotericin B, itraconazole, caspofungin, terbinafine, 5-flurocytosine and nystatin. The inhibitory effects of farnesol on the antifungal resistance of the biofilms from the RAS1OE were in accordance to almost all of the growth phases. Moreover, exogenous farnesol decreased the resistance of biofilms from RAS1OE more obviously than that from the wild strains (P<0.05). In addition, Morphological observation showed that that RAS1OE increased hyphal growth the biofilms, while ras1Δ/Δ reduced that of the biofilms. Compare to the wild-type strain, the inhibitory effects of farnesol on hyphal growth were more obvious to the RAS1OE, while less obvious to the ras1Δ/Δ. Furthermore, farnesol reduced the level of Ras1 and the expression of RAS1 of the biofilms formed from the RAS1OE strain compared with those of the untreated controls at all studied phases. Moreover, farnesol reduced the level of Ras1 and the expression of RAS1 of the biofilms formed from RAS1OE more obviously than that from the wild strains. Conclusions: Ras1 involved in the antifungal resistance of Candida albicans, and the inhibition of farnesol on the resistance of biofilm.

Background
The fungus Candida albicans, an opportunistic human pathogen, is commonly found in the biofilms of the oral cavity and gastrointestinal tract. The biofilms of C. albicans are significantly less susceptible to antimicrobial agents than that of the planktonic cells, which seriously weakens the effectiveness of antifungals [1]. Indeed, 65~80% of C. albicans infections were associated with biofilms [2,3]. The extensive use of antifungals has led to the increased resistance of the biofilms to the few existing antifungals. Thus, it is crucial to explore the mechanisms of therapeutic or preventive strategies targeting biofilm-related infections.
Previous studies have demonstrated that farnesol prevented the germination of yeast cells into mycelia, repressing hyphal growth and biofilm formation [4,5]. Further studies showed that farnesol enhanced the microbial susceptibility to antibiotics of Staphylococcus aureus [6], Paracoccidioides brasiliensis [7], Streptococcus mutans [8], and Fusarium graminearum [9]. Another in vitro study showed that farnesol inhibited the development of C. albicans biofilms formed from the resistant strains, and farnesol in combination with fluconazole, itraconazole and 5-flurocytosine had synergistic effects against C. albicans biofilms [10]. Farnesol possibly via regulation of the ergosterol biosynthesis pathway [11] to enhance the susceptibility of planktonic C. dubliniensis and C. albicans to fluconazole [6]. However, the exact mechanism underlying the farnesol-mediated increase in susceptibility to various antifungals is poorly understood in C. albicans biofilms.
C. albicans RAS proteins are small GTPases that can trigger the MAPK and cAMP signaling pathway, and involved in morphogenesis [4,12], stress resistance [13] and the maintenance of the hyphal state [14]. Ras1 protein are highly conserved signaling proteins that play central roles in key physiological processes, in this process RAS1 regulates a diverse array of phenotypes that are critical for both commensal and pathogenic lifestyles within the host [15]. Recent data suggest that MAPK and cAMP signaling pathways not only regulate the morphogenesis of C. albicans but also participate in the drug resistance of C. albicans biofilms [16]. However, the role of RAS1 in the resistance of C.
albicans biofilms was not discussed in these previous studies, which need to be further studied.
The molecular mechanism of the RAS1 in regulating the farnesol-relevant antifungal capacity to C. albicans biofilms is still unknown. In the present study, we hypothesized that RAS1 is associated to the antifungal resistance of C. albicans biofilm, and also involved in the regulation of farnesol on the resistance of C. albicans biofilm. To test this hypothesis, the antifungal resistance of C. albicans biofilms formed by RAS1 mutant strains (including RAS1 overexpressing strain (RAS1OE) and deletion strain (ras1Δ/Δ) and the wild strains was examined in the presence or absence of farnesol. Meanwhile, the morphological changes were examined using confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM). In addition, the effects of farnesol on the expression of RAS1 and Ras1 in C. albicans biofilms formed from the studied strains were analyzed using RT-qPCR and western blot, respectively.

Ras1 of the C. albicans biofilms is regulated by farnesol
The results showed that RAS1 expression and Ras1 level were down-regulated in the biofilms of C. albicans SC5314 and the resistant strain exposed to farnesol at four different growth phases (6, 12,  RAS1 and Ras1 were down-regulated in the biofilms of C. albicans SC5314 and the resistant strain exposed to farnesol at four different growth phases. Farnesol: 200 µM; *: P<0.05; **: P<0.01.
In the spot assay, antifungals were added to the medium at a concentration of 4 μg/ml for fluconazole, 2 μg/ml for amphotericin B, 0.5 μg/ml for caspofungin, 15 μg/ml for terbinafine, 0.5 μg/ml for itraconazole, 2 μg/ml for nystatin and 8 μg/ml for 5-fluorocytosine. Then, 5µl of tenfold serial dilutions of the suspensions were spotted onto YPD plates in the presence or absence of antifungals with serial concentration gradients.
For the XTT-reduction assay, biofilms formed from the RAS1OE strain showed higher SMIC 50 values for fluconazole (at 12 h and 24 h biofilms), 5-flurocytosine (at 6 h and 12 h biofilms), nystatin (at 6 h, 12 h and 24 h biofilms), itraconazole, amphotericin B, caspofungin and terbinafine (at 6 h, 12 h, 24 h and 36 h biofilms) than did those biofilms formed from the wild-type strain (Table 1). Furthermore, biofilms formed from ras1Δ/Δ showed lower SMIC 50 values for itraconazole (at 6 h, 12 h and 24 h biofilms), amphotericin B (at 12 h and 24 h biofilms), nystatin (at 6 h biofilms), fluconazole, caspofungin, terbinafine and 5-flurocytosine (at the 6 h and 12 h biofilms), than did those bifilms formed from the wild-type strain (SN152) ( Table 1). On the other hand, the SMIC 50 of the antifungals were increased with the mature of the biofilms formed from all the studied strains. As the biofilms grown for 36 h, the SMIC 50 of the antifungals against the biofilms were similar between that formed from the ras1Δ/Δ and the wild strain.  Table 1). In addition, the inhibitory effects of farnesol on the antifungal resistance of the biofilms from the RAS1OE strain were in accordance to almost all of the growth phases.
In addition, compared to the control, farnesol decreased the resistance of the biofilms formed from ras1Δ/Δ to amphotericin B and caspofungin (at 6, 12, 24 and 36 h biofilms). However, farnesol did not decrease the resistance of the biofilms formed from ras1Δ/Δ to fluconazole, itraconazole and 5flurocytosine. On the other hand, fanrnesol increased the resistance of the biofilms formed from ras1Δ/Δ to the nystatin (at the 6h, 12, 24 and 36 h biofilms) and terbinafine (at the 6 and 12 h biofilms) (Table1). The inhibitory effects of farnesol on the resistance of C. albicans biofilm from the RAS1-deletion strain are not accordance at all the growth phase.

The morphologic changes of biofilms
Both CLSM and SEM observations showed that the inhibitory effects of farnesol on the hyphal growth of RAS1OE were more obvious than that on the wild-type strain ( Fig.3 and Fig.5), while the inhibitory effects of farnesol on hyphal growth of the ras1Δ/Δ were less obvious than those on the wild-type strain ( Fig. 4 and Fig. 6). The CLSM showed that the biofilms of RAS1OE and ras1Δ/Δ exposed to farnesol showed fewer hyphae but more pseudohyphae and blastospores than did those control without farnesol (Fig.3 C1D1, C2D2 and Fig. 4 G1H1, G2H2), respectively. In addition, the biofilms formed from RAS1OE had more extensively grown hyphae and pseudohyphae with few blastospores than did those formed from the wild strain (pCaEXP-CAI4) ( Fig. 3 A1C1, A2C2, A3C3, A4C4). Moreover, the biofilms formed from ras1Δ/Δ had fewer hyphae but more pseudohyphae and blastospores than did those formed from the wild strain (SN152) (Fig. 4 E2G2, E3G3).

Discussion
Previous investigations had clearly showed that RAS1 is required for the induction of hyphal growth and the maintenance of the hyphal state [12,14], and also has a close relationship with pathogenic processes [17], such as cell adhesion and biofilm formation. While no study reported the role of Ras1 in the antifungal resistance of C. albicans and the relevant farnesol regulation mechanism of the biofilms, which were discussed in this study.
Our study found that the RAS1 involved in the antifungal resistance of C. albicans. For the C. albicans in planktonic form, RAS1-overexpressing strain increased resistance of planktonic C. albicans to fluconazole and itraconazole, and the RAS1 deletion strain reduced resistance to all the studied antifungals. For the C. albicans in biofilm form, the RAS1 overexpression increased the resistance of the biofilms to the fluconazole, itraconazole, amphotericin, nystatin, caspofungin, terbinafine, and 5flurocytosine, and RAS1 deletion reduced the resistance of those above antifungals. The findings indicated that RAS1 is associated with the antifungal resistance of C. albicans in planktonic and biofilms forms.
Currently, azoles, polyenes, echinocandins and miscellaneous antifungals are the main antifungal families used in clinical practices. In this study, the antifungal susceptibilities of the studied strains were analyzed for the main antifungals including fluconazole, itraconazole, amphotericin B, nystatin, caspofungin, terbinafine and 5-flurocytosine. Fluconazole/itraconazole impairs ergosterol synthesis and leads to a cascade of membrane abnormalities in the fungus [18]. Amphotericin B/nystatin acts through pore formation at the cell membrane and induces the ergosterol sequestration after binding to ergosterol [19]. Caspofungin blocks the synthesis of β-(1,3)-D-glucan, an essential component of the fungal cell wall [20]. Terbinafine inhibits the ergosterol synthesis and preventing the conversion of squalene to lanosterol by inhibiting squalene epoxidase [21]. 5-flurocytosine works by being converted into 5-fluorouracil inside the fungal, and blocks its ability to make protein [22]. These antifungal drugs have different mechanisms of action, then the effects of antifungals to the mutant strains might be complex. The results that RAS1 overexpression increased its susceptibility, and the RAS1 deletion decreased its susceptibility, to multiple antifungals with different mechanisms, implies that RAS1 might has a non-specific mechanism in regulating the resistance action of C. albicans against those antifungals.
In this study, farnesol decreased the expression of RAS1 gene and the level of Ras1 protein in C.
albicans biofilm formed from the RAS1-overexpressing strain and the wild strain. Meanwhile, the inhibitory effects of farnesol on the resistance of C. albicans biofilm from the RAS1-overexpressing strain were more obvious than that formed from the wild-type strain (PCaEXP-CAI4) and the RAS1- Morphological observations in this study showed that the RAS1-overexpression increased the hyphal growth and the RAS1-deletion decreased the hyphal growth of the C. albicans biofilms. Previous studies showed that RAS1 deletion mutants are impaired in hyphal development under many different induction conditions [23]. The morphological observations of the deletion strain in the present study were in accordance with the previous study. Furthermore, both CLSM and SEM observations showed that the inhibitory effects of farnesol on RAS1-overexpression were more obvious than that on the wild-type strain, and the inhibitory effects of farnesol on RAS1-deletion strain were less obvious than those on the wild-type strain. Combined with the results that farnesol increased the susceptibility of those biofilms to most antifungals, the morphological results suggested that farnesol decrease the hyphae growth through regulating the RAS1 expression of the biofilms, and involve in the antifungal resistance of C. albicans biofilms subsequently.
The formation of mature biofilms was composed of a series developed steps. The formation of C.
albicans biofilms started with the initial adherence of yeast cells (0-2 h), followed by germination and micro-colony formation (2-4 h), filamentation (4-6 h), monolayer development (6-8 h), proliferation (8-24 h), and maturation (24-48 h) [24]. In this study the main stages of biofilm were investigated to clarify the effects of farnesol on RAS1 in C. albicans biofilms. A phase-specific mechanism might be involved in biofilm resistance, which appeared of the increased resistance of C. albicans with the mature biofilms (24h and 36 h). Because of this phase-specific increased resistance, the inhibitory effects of farnesol were reduced with the mature of biofilms. In addition, the study showed that the inhibitory effects of farnesol on Ras1 level and the antifungal resistance of the biofilms from the RAS1-overexpressing strain were in accordance to almost all of the growth phases, which indicated that RAS1 involved in the antifungal resistance caused by farnesol.
On the other hand, the effects of farnesol on the resistance of C. albicans biofilm from the RAS1deletion strain are not accordance at the different growth phase. The possible reason of this might be the strain deleted RAS1 caused the resistance change of itself, a phase-specific mechanism involved in the resistance of biofilm, and other complicated and multiple factors affected the resistance of the biofilms of the RAS1-deletion strain, which need to be further discussed.
In conclusion, our data indicate that RAS1 involved in the resistance of C. albicans biofilms, and also involved in the inhibitory effects of farnesol on the resistance of C. albicans biofilms. To the best of our knowledge, no previous studies reported that RAS1 is associated with the resistance of C. albicans biofilms, and also no study of the RAS1 responding to farnesol associated with its antifungal effectiveness or capacity. In addition, the observations that RAS1 deletion or over-expression, with or without farnesol, affected susceptibility to multiple antifungals with different mechanisms of action implies a non-specific effect. Additional research involving the specific molecular mechanisms should be further pursued to elucidate the antifungal resistance mechanisms of C. albicans biofilms.

Conclusion
The

Strains, plasmids and media
The strains and plasmids used in the study are listed in Table 2 plasmids. The wild-type strain PCaEXP-CAI4 was generated from CAI4 using the plasmid pCaEXP (BioVector NTCC Inc., China). RAS1 deletion strain (ras1Δ/Δ) was generated from SN152 (School of Pharmacy, The Second Military Medical University). Fusion PCR was performed to create disruption fragments of C. albicans HIS1 and LEU2 coding sequences, and the HIS1 and LEU2 disruption fragments were transformed into SN152 to obtain ras1Δ/Δ [25].
Freshly grown yeast cells from Sabourd's Dextrose Agar (SDA) plates were incubated in yeast peptone dextrose (YPD) medium and grown overnight in an orbital shaker at 30°C. The cells were collected and resuspended in RPMI 1640 supplemented with L-glutamine and buffered with morpholine propane sulfonic acid (Gibco Ltd., Paisley, U.K.). The solution was adjusted to a cell density of 1×10 6 cells/ml for the experiments. All experiments were performed in triplicate on three separate occasions. were dissolved in 100% (vol/vol) methanol and frozen at -70°C until use. The effect of methanol on the growth of C. albicans was evaluated by testing different methanol concentrations without antifungals, ranging from 0.05-1% (v/v) [26]. The highest methanol concentration used in the microdilution plates was 0.1% (v/v), which presented no antifungal activity.

Susceptibility tests
The The susceptibility of RAS1 mutant strains in planktonic form to antifungals was determined using a spot assay [27]. The mutant and the wild-type strains were incubated in YPD medium and grown for Growth differences were measured after incubation at 30°C for 48 h.
The susceptibility of RAS1 mutant strains in biofilm to antifungals was determined using the XTT [2,3bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide]-menadione (0.5 mg/ml XTT, 1 μM menadione) reduction assay (Sigma-Aldrich, St Louis, MO, USA) [28]. Colorimetric changes were analyzed in a microtiter plate reader (BioTek Instruments, Inc., VT, USA) at a wavelength of 490 nm, which measured the changes in the metabolic activity of the biofilms [29]. The biofilms were incubated as described above. A series of antifungal agent-free wells and biofilm-free wells were also included to serve as the positive and negative controls. Drugs were prepared in serial 2-fold dilutions, and their final concentrations ranged from 4 to 1024 µg/ml for fluconazole, 1 to 256 µg/ml for itraconazole, 0.25 to 64 µg/ml for nystatin, amphotericin B and caspofungin, and 2 to 512 µg/ml for terbinafine and 5-flucytosine. Antifungals were added to the biofilms and incubated for an additional 24 h at 37°C. The value of the background OD measurement, obtained from biofilm-free wells processed in the same manner as the inoculated wells, was subtracted from the OD measurement of each well. After this subtraction, the percentage of growth in each well was calculated as the OD of each well/OD of the drug-free well. The lowest drug concentrations that inhibited biofilm growth by 50% were considered the sessile MIC 50 (SMIC 50 ) of this antifungal.

The morphological observations using CLSM and SEM
Biofilms formed from the mutant strains, the wild-type strains, and the farnesol-treated strains were formed on the glass bottom of cell culture dishes for morphological observation. For CLSM observation, the formed biofilms were fixed with 4% paraformaldehyde overnight, washed with PBS and stained with 500 μl calcofluor white stain (0.0025 g/ml; Sigma Chemical Co., St. Louis, MO) [30] for 30 min at 37°C in the dark. The biofilms were observed using a Zeiss LSM700 microscope with a video capture system, automatic camera, image analysis hardware and software (Carl Zeiss, Inc., Oberkochen, Germany), and a 488 nm argon ion laser. For SEM observation, the formed biofilms were fixed with 2.5% glutaraldehyde solution overnight. The biofilms were subsequently washed twice with distilled water, dehydrated in an ethanol series (70% for 10 min, 95% for 10 min and 100% for 20 min) and air-dried overnight in a desiccator. Then, the biofilms were metalized by gold sputtering for 45 s in a High Vacuum Evaporator, followed by bonding to carbon double-side tape and processing for SEM (FEI Inc., Hillsboro, USA).

Ras1 of the biofilms regulated by farnesol via RT-qPCR and confirmed by Western blot
analysis RNA samples of the farnesol-treated and untreated control biofilms were purified using a modified hot phenol method [31]. The cDNA was synthesized using the reverse transcription system (TaKaRa, Bio analysis was performed to ensure the absence of primer dimers. The expression of genes was calculated using the 2 -ΔΔCt method [11]. ACT1 was used as a reference gene. Primers were all designed by Shanghai Generay Bio-Tech Co., Ltd. (Table 3).
Total protein extracts were prepared as described for C. albicans biofilms using an immunoprecipitation protocol [32]. Protein concentrations were determined using a BCA Protein Assay Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA) and exposed to Kodak X-ray films. GAPDH was used as an internal control in the experiments.

Statistical analysis
All quantitative experiments were performed in triplicate for statistical analyses. Data were analyzed using SPSS19.0 software (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) were used to analyze the differences between the groups, while a paired t-test was performed for intragroup comparisons. The rank sum test was used to analyze ranked data. Comparisons resulting in P values of less than 0.05 were considered statistically significant.

Ethics approval and consent to participate
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Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.  Susceptibility tests of the RAS1 mutant strain of planktonic C. albicans   The morphological changes in 6 h, 12 h, 24 h and 36 h C. albicans biofilms of RAS1OE shown by SEM. Figure 6 The morphological changes in 6 h, 12 h, 24 h and 36 h C. albicans biofilms of ras1Δ/Δ shown by SEM. Figure 7 The expression of the RAS1 gene in farnesol-treated RAS1OE Figure 8 The expression of the Ras1 protein in RAS1OE and pCaEXP-CAI4