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, 24 and 36 h) (P<0.01) (Fig. 1).
C1: Untreated C. albicans SC5314; F1: farnesol-treated C. albicans SC5314; C2: untreated resistant strain; F2: farnesol-treated resistant strain. F1/C1 and F2/C2: Comparison of gene expression between the farnesol-treated and untreated groups of C. albicans biofilms.
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.
Ras1 involved in the resistance of C. albicans to antifungals
For the spot assay, RAS1OE was more resistant to fluconazole (4 µg/ml) and itraconazole (0.5 µg/ml) than the wild-type strain (pCaEXP-CAI4) (Fig. 2). Moreover, ras1Δ/Δ was more susceptible to fluconazole (4 µg/ml), itraconazole (0.5 µg/ml), amphotericin B (2 µg/ml), nystatin (2 µg/ml), caspofungin (0.5 µg/ml), terbinafine (15 µg/ml) and 5-flurocytosine than the wild-type strain (SN152) (Fig. 2).
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 SMIC50 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 SMIC50 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 SMIC50 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 SMIC50 of the antifungals against the biofilms were similar between that formed from the ras1Δ/Δ and the wild strain.
Table 1. The SMIC values of antifungals in C. albicans biofilms of mutant strains with and without farnesol
Farnesol
(µM)
|
SMIC50 of Fluconazole (µg/ml)
|
pCaEXP-CAI4
|
RAS1OE
|
SN152
|
ras1Δ/Δ
|
6 h
|
|
|
|
|
0
|
>1024
|
>1024
|
128
|
4
|
100
|
>1024
|
256
|
128
|
8
|
200
|
>1024
|
128
|
64
|
4
|
300
|
>1024
|
128
|
64
|
4
|
12 h
|
|
|
|
|
0
|
512
|
>1024
|
1024
|
128
|
100
|
512
|
512
|
1024
|
128
|
200
|
512
|
256
|
1024
|
128
|
300
|
512
|
256
|
1024
|
1024
|
24 h
|
|
|
|
|
0
|
1024
|
>1024
|
1024
|
1024
|
100
|
1024
|
1024
|
1024
|
1024
|
200
|
512
|
512
|
1024
|
1024
|
300
|
512
|
512
|
1024
|
1024
|
36 h
|
|
|
|
|
0
|
>1024
|
>1024
|
1024
|
1024
|
100
|
>1024
|
>1024
|
1024
|
1024
|
200
|
>1024
|
>1024
|
1024
|
1024
|
300
|
>1024
|
>1024
|
1024
|
1024
|
Farnesol
(µM)
|
SMIC50 of Itraconazole (µg/ml)
|
pCaEXP-CAI4
|
RAS1OE
|
SN152
|
ras1Δ/Δ
|
6 h
|
|
|
|
|
0
|
>16
|
64
|
16
|
4
|
100
|
>16
|
16
|
16
|
4
|
200
|
>16
|
1
|
8
|
4
|
300
|
>16
|
16
|
4
|
4
|
12 h
|
|
|
|
|
0
|
>16
|
128
|
>256
|
4
|
100
|
>16
|
64
|
256
|
4
|
200
|
>16
|
1
|
>256
|
4
|
300
|
>16
|
64
|
4
|
4
|
24 h
|
|
|
|
|
0
|
>16
|
256
|
>256
|
>64
|
100
|
>16
|
>64
|
>256
|
64
|
200
|
>16
|
16
|
>256
|
16
|
300
|
>16
|
64
|
>256
|
128
|
36 h
|
|
|
|
|
0
|
>16
|
256
|
>256
|
>256
|
100
|
>16
|
>64
|
>256
|
>256
|
200
|
0.125
|
32
|
>256
|
>256
|
300
|
0.5
|
64
|
>256
|
>256
|
Farnesol
(µM)
|
SMIC50 of Amphotericin B (µg/ml)
|
|
pCaEXP-CAI4
|
RAS1OE
|
SN152
|
ras1Δ/Δ
|
6 h
|
|
|
|
|
0
|
1
|
4
|
8
|
8
|
100
|
0.5
|
4
|
8
|
1
|
200
|
1
|
4
|
4
|
4
|
300
|
1
|
2
|
2
|
8
|
12 h
|
|
|
|
|
0
|
0.5
|
64
|
8
|
4
|
100
|
0.5
|
8
|
4
|
4
|
200
|
0.5
|
4
|
4
|
4
|
300
|
0.5
|
16
|
2
|
2
|
24 h
|
|
|
|
|
0
|
1
|
64
|
32
|
16
|
100
|
1
|
8
|
16
|
16
|
200
|
0.5
|
8
|
8
|
8
|
300
|
1
|
16
|
8
|
8
|
36 h
|
|
|
|
|
0
|
0.25
|
64
|
32
|
32
|
100
|
0.25
|
8
|
2
|
2
|
200
|
0.125
|
16
|
2
|
2
|
300
|
0.125
|
16
|
2
|
2
|
Farnesol
(µM)
|
SMIC50 of Caspofungin (µg/ml)
|
|
pCaEXP-CAI4
|
RAS1OE
|
SN152
|
ras1Δ/Δ
|
6 h
|
|
|
|
|
0
|
0.125
|
1
|
16
|
4
|
100
|
0.0625
|
0.0625
|
0.25
|
0.25
|
200
|
0.0625
|
0.25
|
0.25
|
0.25
|
300
|
0.0625
|
0.0625
|
0.25
|
0.25
|
12 h
|
|
|
|
|
0
|
>8
|
32
|
32
|
4
|
100
|
>8
|
0.125
|
0.5
|
0.5
|
200
|
0.25
|
0.25
|
0.5
|
1
|
300
|
0.25
|
0.0625
|
0.25
|
0.5
|
24 h
|
|
|
|
|
0
|
>8
|
32
|
64
|
64
|
100
|
>8
|
16
|
0.5
|
0.5
|
200
|
>8
|
0.25
|
0.25
|
0.5
|
300
|
>8
|
2
|
0.5
|
0.5
|
36 h
|
|
|
|
|
0
|
>8
|
32
|
>64
|
>64
|
100
|
0.25
|
0.125
|
1
|
0.5
|
200
|
0.125
|
0.5
|
0.5
|
0.5
|
300
|
0.0625
|
16
|
0.25
|
0.5
|
Farnesol
(µM)
|
SMIC50 of Terbinafine (µg/ml)
|
|
pCaEXP-CAI4
|
RAS1OE
|
SN152
|
ras1Δ/Δ
|
6 h
|
|
|
|
|
0
|
>256
|
512
|
64
|
16
|
100
|
>256
|
256
|
>512
|
>512
|
200
|
>256
|
256
|
>512
|
>512
|
300
|
>256
|
256
|
>512
|
>512
|
12 h
|
|
|
|
|
0
|
>256
|
>512
|
512
|
64
|
100
|
>256
|
256
|
>512
|
>512
|
200
|
>256
|
256
|
>512
|
>512
|
300
|
>256
|
512
|
>512
|
>512
|
24 h
|
|
|
|
|
0
|
>256
|
>512
|
>512
|
>512
|
100
|
>256
|
512
|
>512
|
>512
|
200
|
>256
|
>512
|
>512
|
>512
|
300
|
>256
|
512
|
>512
|
>512
|
36 h
|
|
|
|
|
0
|
>256
|
>512
|
>512
|
>512
|
100
|
>256
|
64
|
>512
|
>512
|
200
|
>256
|
512
|
>512
|
>512
|
300
|
>256
|
512
|
>512
|
>512
|
Farnesol
(µM)
|
SMIC50 of 5-Flucytosine (µg/ml)
|
|
pCaEXP-CAI4
|
RAS1OE
|
SN152
|
ras1Δ/Δ
|
6 h
|
|
|
|
|
0
|
2
|
512
|
>512
|
256
|
100
|
1
|
4
|
512
|
128
|
200
|
2
|
2
|
512
|
256
|
300
|
16
|
16
|
512
|
128
|
12 h
|
|
|
|
|
0
|
256
|
512
|
>512
|
128
|
100
|
1
|
1
|
512
|
512
|
200
|
4
|
4
|
16
|
64
|
300
|
16
|
0.5
|
512
|
512
|
24 h
|
|
|
|
|
0
|
512
|
512
|
>512
|
>512
|
100
|
2
|
4
|
512
|
512
|
200
|
8
|
2
|
512
|
512
|
300
|
64
|
0.5
|
512
|
512
|
36 h
|
|
|
|
|
0
|
512
|
512
|
>512
|
>512
|
100
|
0.5
|
0.5
|
512
|
512
|
200
|
16
|
2
|
512
|
512
|
300
|
64
|
64
|
512
|
512
|
Farnesol
(µM)
|
SMIC50 of Nystatin (µg/ml)
|
|
pCaEXP-CAI4
|
RAS1OE
|
SN152
|
ras1Δ/Δ
|
6 h
|
|
|
|
|
0
|
2
|
4
|
4
|
2
|
100
|
4
|
4
|
8
|
8
|
200
|
2
|
2
|
4
|
8
|
300
|
4
|
4
|
8
|
16
|
12 h
|
|
|
|
|
0
|
4
|
8
|
4
|
4
|
100
|
4
|
4
|
8
|
8
|
200
|
2
|
2
|
4
|
8
|
300
|
4
|
4
|
16
|
32
|
24 h
|
|
|
|
|
0
|
4
|
8
|
4
|
4
|
100
|
2
|
8
|
4
|
8
|
200
|
2
|
2
|
4
|
8
|
300
|
4
|
4
|
16
|
16
|
36 h
|
|
|
|
|
0
|
8
|
8
|
4
|
4
|
100
|
4
|
8
|
8
|
8
|
200
|
2
|
2
|
4
|
8
|
300
|
0.5
|
1
|
16
|
16
|
Effects of farnesol on the susceptibility of the biofilms formed from the RAS1 mutant strains to antifungals
Significant SMIC reductions were observed between farnesol-treated and untreated control when farnesol was added to the biofilms of the RAS1OE strains. Compared to the control, farnesol decreased the resistance of RAS1OE to fluconazole (at the 6 h, 12 h and 24 h biofilms), amphotericin B (at the 12 h, 24 h and 36 h biofilms), itraconazole, caspofungin, terbinafine, 5-flurocytosine and nystatin (at the 6 h, 12 h, 24 h and 36 h biofilms). Moreover, the inhibitory effects of farnesol on the antifungal resistance (including fluconazole, itraconazole, amphotericin B, nystatin, caspofungin and 5-flucytosine) of RAS1OE were more obvious than that on the wild-type strain (PCaEXP-CAI4) (P<0.05) (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 5-flurocytosine. 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).
A: farnesol-untreated pCaEXP-CAI4; B: farnesol-treated pCaEXP-CAI4; C: farnesol-untreated RAS1OE; D: farnesol-treated RAS1OE. A1, B1, C1 and D1 show 6 h C. albicans biofilms; A2, B2, C2 and D2 show 12 h C. albicans biofilms; A3, B3, C3 and D3 show 24 h C. albicans biofilms; A4, B4, C4 and D4 show 36 h C. albicans biofilms. The biofilms of C1C2 showed fewer hyphae but more pseudohyphae and blastospores than did those of D1D2. The inhibitory effects of C1D1, C2D2 were more obvious than did that of A1B1, A2B2. Magnification: 400×; farnesol: 200 µΜ.
E: farnesol-untreated SN152; F: farnesol-treated SN152; G: farnesol-untreated ras1Δ/Δ; H: farnesol-treated ras1Δ/Δ. E1, F1, G1 and H1 show 6 h C. albicans biofilms; E2, F2, G2 and H2 show 12 h C. albicans biofilms; E3, F3, G3 and H3 show 24 h C. albicans biofilms; E4, F4, G4 and H4 show 36 h C. albicans biofilms. The biofilms of G1G2 showed fewer hyphae but more pseudohyphae and blastospores than did those of H1H2. The inhibitory effects of G1H1, G2H2, G3H3, G4H4 were less obvious than did that of E1F1, E2F2, E3F3, E4F4. Magnification: 400×; farnesol: 200 µΜ
SEM analysis showed that the cells in farnesol-treated biofilms of RAS1OE and ras1Δ/Δ appeared in the short pseudohyphae and had a rough cell surface than did those in the biofilms without farnesol (Fig. 5 C1D1, C2D2, C3D3, C4D4 and Fig. 6 G1H1, G2H2, G3H3, G4H4). In addition, the biofilms formed from RAS1OE had more hyphae than did those formed from the control (pCaEXP-CAI4) (Fig. 5 A1C1, A2C2, A3C3, A4C4), while the biofilms formed from ras1Δ/Δ had fewer hyphae and more pseudohyphae than did those formed from the control (SN152) (Fig. 6 E2G2, E3G3, E4G4). The surface of the cells was similar between the biofilms of RAS1OE and ras1Δ/Δ.
A: farnesol-untreated pCaEXP-CAI4; B: farnesol-treated pCaEXP-CAI4; C: farnesol-untreated RAS1OE; D: farnesol-treated RAS1OE. A1, B1, C1 and D1 show 6 h C. albicans biofilms; A2, B2, C2 and D2 show 12 h C. albicans biofilms; A3, B3, C3 and D3 show 24 h C. albicans biofilms; A4, B4, C4 and D4 show 36 h C. albicans biofilms. The biofilms of C1-4 appeared in the short pseudohyphae and had a rough cell surface, while D1-4 appeared in the long hyphae and had smooth surfaces. The inhibitory effects of C1D1, C2D2 were more obvious than did that of A1B1, A2B2. Magnification: 2000×; farnesol: 200 µΜ
E: Farnesol-untreated SN152; F: farnesol-treated SN152; G: farnesol-untreated ras1Δ/Δ; H: farnesol-treated ras1Δ/Δ. E1, F1, G1 and H1 show 6 h C. albicans biofilms; E2, F2, G2 and H2 show 12 h C. albicans biofilms; E3, F3, G3 and H3 show 24 h C. albicans biofilms; E4, F4, G4 and H4 show 36 h C. albicans biofilms. The biofilms of G1-4 appeared in the short pseudohyphae and had a rough cell surface, while E1-4 appeared in the long hyphae and had smooth surfaces. The inhibitory effects of G1H1, G3H3, G4H4 were less obvious than did that of E1F1, E3F3, E4F4. Magnification: 2000×; farnesol: 200 µΜ
Farnesol decreased the expression of Ras1 in the biofilms formed from the RAS1OE strains
The results of RT-qPCR and western blotting showed that farnesol significantly reduced the expression of RAS1 and the level of Ras1 in the biofilms formed by RAS1OE compared with those of the untreated controls at all studied phases (P<0.05) (Figs. 7 and 8). Farnesol also significantly reduced the expression of RAS1 and the level of Ras1 in the biofilms formed by the wild-type strains compared with those of the untreated controls at all studied phases (P<0.05) (Figs. 7 and 8). Moreover, farnesol reduced the level of Ras1 and the expression of RAS1 more obviously in the biofilms of the RAS1OE than that of the wild strains (P<0.05). Meanwhile, there was no expression of RAS1 and Ras1 in the biofilms formed by ras1Δ/Δ, with or without farnesol.