It is well known that the virulence of Candida depends on its transition from the yeast form to filamentous hyphae². It was therefore important to study the effect of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and the endocannabinoid-like N-arachidonoyl serine (AraS) on this transition. To this end, we exposed GFP-expressing Candida albicans in the yeast form to various concentrations of AEA, AraS and 2-AG and incubated them for 4 h at 37 °C, a condition resulting in the transition to hyphae. Most of the fungi have transformed to filamentous hyphae in the control and in the fungi exposed to 10 μg/ml AEA (Fig. 1a–d). In samples treated with 50 μg/ml AEA, most of the fungi had transformed to hyphae, but also several fungi in yeast form and fungi with short hyphae were seen (Fig. 1e–f). However, almost no transformation to hyphae was observed in Candida exposed to 125 and 250 μg/ml AEA (Fig. 1g–j), indicating that AEA at these concentrations prevents the yeast-hypha transition. Similarly, AraS strongly inhibited yeast-hypha transition at 125 and 250 μg/ml, with partial inhibitory effect at 50 μg/ml (Suppl. Figure 1). 2-AG had no significant effect on the yeast-hypha transition (data not shown). When looking at the vegetative growth of C. albicans in the yeast form, a delay in the growth was observed in the presence of higher concentrations of AEA, AraS and 2-AG (Suppl. Figure 2).

Since AEA prevented the morphogenetic switch from yeast to hyphae, we wondered whether AEA could affect the hyphae after being formed. For this purpose, we allowed the Candida to form hyphae by an overnight incubation at 37 °C, and then exposed the hyphae to various concentrations of AEA for 1 h. The majority of the hyphae length in the control and 10 μg/ml AEA-treated samples ranged between 25–60 μm (Fig. 2a,b). Most of the C. albicans hyphae treated with 50, 125 and 250 μg/ml AEA for 1 h showed shorter hyphae length ranging from 15–30 μm (Fig. 2c–e). Occasionally, some hyphae with exceptional long length (90–100 μm) appeared in both the control and treated samples (Fig. 2). We next wanted to know whether the shorter hyphae observed in the AEA-treated samples are due to an inhibition of hyphal growth. To study this possibility, C. albicans in its yeast form was first allowed to form hyphae by incubating them 4 h at 37 °C, and then subjected to a 3 h time-lapse microscopy at 37 °C in the absence or presence of 125 μg/ml AEA. As expected, the hyphae continued to grow in the control samples (Fig. 3a and Suppl. Figure 3a—time-lapse video). However, most of the hyphae ceased growing after being exposed to AEA (Fig. 3b and Suppl. Figure 3b—time-lapse video), suggesting that AEA interferes with hyphal morphogenesis. Similar inhibition of hyphal growth was observed when exposing the hyphae to 50 μg/ml AEA (data not shown).

Next we studied the effect of AEA on C. albicans adherence to cervical epithelial cells. GFP-expressing C. albicans were allowed to form hyphae by an overnight incubation at 37 °C. The hyphae were pretreated with various concentrations of AEA for 1 h, and then co-cultured on confluent HeLa cervical epithelial cells for another hour (Fig. 4a–e). In parallel, the same fungi samples were incubated on tissue culture plastic plates as controls that reflect the inputs (Fig. 5a–e). The morphology of the whole fungal population prior to incubation with HeLa or on plastic is shown in Fig. 2. Pretreatment of C. albicans hyphae with 50 μg/ml and 125 μg/ml AEA (Fig. 4c,d) reduced their adherence to the epithelial cells by 40 ± 8% and 62 ± 4%, respectively, with a statistical significance of p < 0.001 compared to control (Fig. 4f). Increasing the AEA concentration to 250 μg/ml (Fig. 4e) did only cause a slightly higher inhibition of 72 ± 2% (Fig. 4f), suggesting that a plateau effect is observed at 125 μg/ml. Of note, AEA-treated C. albicans hyphae that were able to bind to the epithelial cells, showed 3–fivefold shorter hyphae (Fig. 4d,e) compared to control fungi (Fig. 4a) with a p < 0.001 for 50–250 μg/ml AEA (Fig. 4g). A maximal effect on hyphae length was observed at 50 μg/ml (Fig. 4g). At a concentration of 10 μg/ml, AEA had no significant effect on the hyphae length (Fig. 4b). The hyphae that adhered to HeLa cells were also thinner and several fungi appeared without hyphae at all when using 50–250 μg/ml AEA. The appearance of shorter hyphae adherent to HeLa cells following AEA treatment is a direct consequence of the AEA effect on hyphal growth as described above (Figs. 2, 3). Of particular importance is the preferential inhibition of hyphal adhesion to HeLa cells in comparison to plastic as shown by the relative reduction in the ratio of HeLa-adherent versus plastic-adherent hyphae (Suppl. Figure 4). Pretreatment of HeLa cells with AEA prior to addition of C. albicans didn't alter the attachment of the fungi to the cells (data not shown). It should be noted that normal untreated hyphae of different lengths exhibited similar ability to adhere to HeLa cells (Suppl. Figure 5), meaning that the preferential appearance of shorter adherent hyphae after exposure to AEA is a direct consequence of the treatment.

In contrast to the reduced adherence of AEA-treated C. albicans to HeLa cells, the AEA-treated fungi adhered even better to polystyrene tissue culture plates in comparison to untreated fungi (Fig. 5a–e) with statistical significance in the concentration range of 50–250 μg/ml AEA (Fig. 5f; p < 0.05). This accords with the higher amount of hyphae observed in AEA-treated samples in comparison to control samples (Fig. 5). The hyphae length of AEA-treated C. albicans that bound to plastic were about twofold shorter on average in comparison to control (Fig. 5g) with statistical significance in the concentration range of 50–250 μg/ml AEA (p < 0.05). When incubating the C. albicans with AEA for 24 h, there was no significant alteration in the biofilm mass formed on plastic (Suppl. Figure 6). However, both 2-AG and AraS significantly reduced the biofilm mass on plastic in a dose-dependent manner with a p < 0.05 (Suppl. Figure 6).

We next analyzed the effect of AraS and 2-AG on the ability of C. albicans to adhere to cervical epithelial cells and compared their effect with that of AEA (Fig. 6a–h). Pretreatment of C. albicans with 125 μg/ml and 250 μg/ml AraS for 1 h (Fig. 6c,g) reduced adherence to the epithelial cells by 30 ± 5% and 63 ± 5%, respectively, with a statistical significance of p < 0.05 compared to control (Fig. 6i). The hyphae of the fungi that adhered to the epithelial cells following AraS treatment (Fig. 6g) were shorter and thinner in comparison to untreated fungi (Fig. 6a,e). However, treatment of C. albicans with 125 μg/ml and 250 μg/ml 2-AG (Fig. 6b,f) didn’t interfere with their adherence to HeLa cells (Fig. 6i). Both AraS and 2-AG treated C. albicans adhered well to polystyrene plastic within 1 h (Fig. 7). This is in contrast to the reduced biofilm mass formation on plastic after 24 h incubation in the presence of the compounds (Suppl. Figure 6).

In order to understand the anti-adhesive effects of AEA on C. albicans interaction with epithelial cells, we exposed the fungi to various concentrations of AEA for 2 h, followed by gene expression analysis of genes relevant for biofilm formation, adherence and hyphal morphogenesis (Tables 1, 2 and 3). Some genes were upregulated including the ALS1 adhesion molecule, the transcription factor TEC1 involved in hyphal development and the transcriptional repressor NRG1 that prevents filamentous growth (Table 1). ALS1 and TEC1 were mainly upregulated at the lower concentrations (10 and 50 μg/ml AEA), while NRG1 was upregulated at the higher concentrations (50–250 μg/ml AEA). Of note, the three multidrug efflux transporters MDR1, CDR1 and CDR2 were strongly upregulated (Table 1). On the other hand, AEA repressed the expression of the adhesins HWP1 and ALS3, the cell elongation protein ECE1, the signal transduction regulators HGC1 and RAS1 and the transcription regulators EFG1 and ZAP1 (Table 2). Also the cell hydrophobicity-associated protein CSH1 was strongly repressed as well as the virulence factor phospholipase D1 (PLD1) (Table 2). There were also several genes that were not significantly affected by AEA including the cell wall adhesion EAP1 and the anti-adhesive protein YWP1 (Table 3). Altogether, these alterations in gene expression may explain, at least in part, the inhibition of hyphal growth by AEA and the reduced adherence of AEA-treated hyphae to epithelial cells.

Anandamide (AEA), N-arachidonoyl serine (AraS) and 2-arachidonoylglycerol (2-AG) were synthesized as described^39–41 and dissolved in ethanol. We also purchased anandamide from Sigma (St. Louis, MO), and N-arachidonoyl serine (AraS) and 2-arachidonoylglycerol (2-AG) from Cayman Chemical.

HeLa cervical carcinoma cells were cultivated in DMEM (Sigma, St. Louis, MO) supplemented with 8% heat-inactivated fetal calf serum (FCS; Biological Industries, Beth HaEmek, Israel), 2 mM L-glutamine and 1 mM sodium pyruvate, and incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

C. albicans SC5314 that has the GFP gene integrated within the ENO1 genomic locus⁴², was kindly provided by Prof. J. Berman (Tel Aviv University, Israel). The fungi were first seeded on potato-dextrose agar plates (Acumedia, Neogen, Lansing, MI) at room temperature where they grow in the yeast form, and then inoculated in RPMI (Sigma, St. Louis, MO) for a 16–18 h incubation at 37 °C to let them form hyphae. For the yeast-hypha transition assay, colonies of C. albicans in yeast form were inoculated in RPMI at an OD_600nm of 0.5 and incubated with different concentrations of AEA, AraS and 2-AG at 37 °C for 4 h. For time-lapse microscopy, hyphae that has been formed after 4 h incubation of C. albicans in yeast form (OD_600nm of 0.25) at 37 °C, were incubated in 300 μl RPMI in a μ-slide 8 well chambered coverslip (ibidi GmbH, Martinsried, Germany) in the absence or presence of 125 μg/ml AEA. The Okolab incubation chamber was used to maintain the temperature at 37 °C. Images were captured each 5 min for 3 h using the Nikon spinning disk microscope (Yokogawa W1), the × 20 CFI PLAN APO VC objective and the SCMOS ZYLA camera. The images were processed using the NIS-Element AR program.

C. albicans that have been cultivated overnight in RPMI at 37 °C, were resuspended and diluted to an OD_600nm of 0.25 in RPMI and then seeded in 96-flat bottomed microplates (Corning, NY) in 200 μl RPMI with different concentrations of test compounds per well. Following an overnight incubation at 37 °C, the biofilms formed in the wells were washed twice with PBS and stained for 20 min with 1% crystal violet. The stained biofilms were washed twice with DDW and after drying, the stain was dissolved in 200 μl of 33% acetic acid and the absorbance read in a Tecan M200 microplate reader at 595nm²⁰.

The adherence assay was performed by a slight modification of Feldman et al.⁴³. Two hundred and fifty thousand HeLa cells were seeded in 1 ml DMEM supplemented with 8% FCS in 24 well tissue culture plate (Corning, NY) the day before assay. At the following morning, the medium was changed to 1 ml of RPMI supplemented with 1% FCS. C. albicans that have grown for 16–18 h at 37 °C, were resuspended in fresh RPMI to an OD of 1.0, and then exposed to different concentrations of AEA, 2-AG, AraS or corresponding concentrations of ethanol for 1 h serving as controls. Thereafter, 100 μl of the pretreated C. albicans were added to the 1 ml HeLa cell cultures, and the co-culture incubated for 1 h at 37 °C. At the end of incubation, the cells were washed twice with 1 ml PBS and fixed with 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS for 30 min. The co-cultures were visualized using NIKON confocal microscope and the NIS-Element AR software. Quantitative analysis of GFP was done using the ImageJ software. For each sample 8–10 images were taken and each image was analyzed for the amount of GFP. The percentage adherence was calculated according to relative GFP staining. In addition, the length of the hyphae was measured using the Adobe Photoshop software. Between 50 and 100 fungi were measured per sample.

C. albicans that have been grown overnight in RPMI at 37 °C were treated for 2 h with various concentrations of AEA or corresponding concentrations of ethanol. At the end of incubation, the RNA was extracted from the fungi using TRI-Reagent (Sigma, St. Louis, MO)⁴⁴. Cell disruption was done in 1 ml TRI-Reagent in the presence of 200 μl 1 mm acid-washed glass beads (Sigma, St. Louis, MO) with the aid of a FastPrep cell disrupter (BIO 101, Savant Instrument). The purified RNA was reverse transcribed into cDNA using the qScript cDNA synthesis kit (Quantabio, Beverly, MA) and PCR amplification was done in a CFX96 BioRad Connect Real-Time PCR apparatus using Power Sybr Green Master Mix (Applied Biosystems, ThermoFischer Scientific) on 2 ng cDNA in the presence of 300 nM forward/reverse primer sets (Table 4). PCR conditions included an initial heating at 50 °C for 2 min, an activation step at 95 °C for 10 min, followed by 40 cycles of amplification (95 °C for 15 s, 60 °C for 1 min). Calculations were done according to the 2^−ΔΔCt method, where both 18S rRNA and ACT1 were used as reference genes.

Experiments were performed in triplicates and repeated twice. Results are presented as average of data obtained from three experiments ± standard error. Results are considered to be statistically significant when the p value was less than 0.05 using the Student's t-test.