Anandamide prevents the adhesion of filamentous Candida albicans to cervical epithelial cells

Candidiasis is a fungal infection caused by Candida species that have formed a biofilm on epithelial linings of the body. The most frequently affected areas include the vagina, oral cavity and the intestine. In severe cases, the fungi penetrate the epithelium and cause systemic infections. One approach to combat candidiasis is to prevent the adhesion of the fungal hyphae to the epithelium. Here we demonstrate that the endocannabinoid anandamide (AEA) and the endocannabinoid-like N-arachidonoyl serine (AraS) strongly prevent the adherence of C. albicans hyphae to cervical epithelial cells, while the endocannabinoid 2-arachidonoylglycerol (2-AG) has only a minor inhibitory effect. In addition, we observed that both AEA and AraS prevent the yeast-hypha transition and perturb hyphal growth. Real-time PCR analysis showed that AEA represses the expression of the HWP1 and ALS3 adhesins involved in Candida adhesion to epithelial cells and the HGC1, RAS1, EFG1 and ZAP1 regulators of hyphal morphogenesis and cell adherence. On the other hand, AEA increased the expression of NRG1, a transcriptional repressor of filamentous growth. Altogether, our data show that AEA and AraS have potential anti-fungal activities by inhibiting hyphal growth and preventing hyphal adherence to epithelial cells.


Results
Anandamide and N-arachidonoyl serine prevent the yeast-hypha transition of Candida albicans. It is well known that the virulence of Candida depends on its transition from the yeast form to filamentous hyphae 2 . 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, Scientific RepoRtS | (2020) 10:13728 | https://doi.org/10.1038/s41598-020-70650-6 www.nature.com/scientificreports/ 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. 1ad). 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).
Anandamide impairs the further growth of preformed hyphae. 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).
Anandamide (AeA) prevents the adherence of Candida albicans to cervical epithelial cells. 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 3fivefold 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). N-Arachidonoyl serine (AraS), but not 2-arachidonoylglycerol (2-AG), prevents the adherence of Candida albicans to cervical epithelial cells. 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). www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ AEA altered the expression of genes involved in adhesion and hyphal morphogenesis. 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 www.nature.com/scientificreports/ 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.

Discussion
Candidiasis is a major health problem where Candida species forms biofilm on endothelial and epithelial cells. In immunosuppressed people it can lead to systemic infection and even death 6,22 . The oral cavity, the genitourinary tract and the intestine are the most frequent infection sites. It is important to find treatments that can interfere with the early adhesion of the fungi to the host cells. Here we have shown that treatment of C. albicans with either AEA or AraS strongly reduced their adherence to cervical epithelial cells, making them potential drugs in the co-treatment of this infectious disease. Not only do these compounds affect the hyphal attachment to epithelial cells, but they also lead to a strong reduction in the hyphal length in comparison to control. The appearance of shorter hyphae in the AEA and AraS-treated samples is a direct result of their inhibitory effect on hyphal growth. These compounds were also shown to prevent the yeast-hypha transition. Since the hyphae are associated with higher infectivity than the yeast form 6,22 , the perturbation of hyphal growth by AEA and AraS might be beneficial in reducing the virulence of C. albicans. Of note, AEA didn't affect the biofilm formation on polystyrene plastic surface, suggesting different requirements for the two modes of adhesion. In order to gain better insight into the action mechanism of AEA, we undertook a gene expression study focusing on genes relevant to adhesion, biofilm formation and hyphal morphogenesis. We found genes that were upregulated by AEA, others that were down-regulated and even others that were not significantly affected. Of the genes whose expression was altered by AEA, the upregulation of ALS1, TEC1 and NRG1 and the downregulation of HWP1, ALS3, HGC1, RAS1, ZAP1, CSH1, ECE1 and PLD1 were the most outstanding. Als1, Als3 and Hwp1 are adhesins that are involved in the attachment of C. albicans to epithelial cells 7,12,[23][24][25][26] . Eap1 and Als1 are important for the initial attachment to a surface, while Hpw1 and Als3 are important for the stable attachment www.nature.com/scientificreports/ to epithelial cells 2,6 . EAP1 expression was unaffected by AEA, while ALS1 was only upregulated at the lower AEA concentrations (10-50 μg/ml). The upregulation of ALS1 showed a similar pattern to that of the transcription factor TEC1, which is known to regulate ALS1 expression 6 . In contrast, HPW1 and ALS3 were downregulated at the higher AEA concentrations (50-250 μg/ml), an effect that seems to outweigh the upregulation of ALS1. This conclusion is based on the observation that C. albicans strains lacking HWP1 are unable to form stable attachments to human buccal epithelial cells 23 , and specific antibodies to Als3 blocks C. albicans adhesion to vascular endothelial cells and buccal epithelial cells 24 . The downregulation of HWP1 and ALS3 together with the simultaneous downregulation of ECE1, which is also known to support adhesion 26 , might explain, at least partly, the reduced adhesion of AEA-treated C. albicans to epithelial cells.
Interesting is the AEA-mediated downregulation of RAS1, an upstream regulator of the Cdc35/cAMP/ PKA/Efg1 and the Cdc24/Cst20/Hst7/Cph1 MAPK signal transduction pathways that regulate hyphal morphogenesis 6,22 . Ras1 is considered a master hyphal regulator and mutant RAS1 strains show severe defects in hyphal growth 27 and reduced adherence to epithelial cells 12 . Alteration in RAS1 levels by AEA has thereby direct influence on hyphae formation and adhesion to epithelial cells. In addition, AEA reduced the gene expression of hyphal-promoting transcription factor EFG1 whose activity is affected by the Ras1/Cdc35/cAMP/PKA pathway, while it had no significant effect on the expression of the hyphal-promoting transcription factor CPH1 that is affected by the Ras1/Cdc24/Cst20/Hst7 MAPK pathway. Efg1 has been shown to be required for the adhesion of fungi to both reconstituted human epidermis and reconstituted intestinal epithelium 28 and the cAMP/PKA/Efg1 www.nature.com/scientificreports/ signal transduction pathway has been demonstrated to be necessary for all stages of oral C. albicans infection 12 . Efg1 is an upstream regulator of the adhesins ALS1, ALS3, ECE1 and HWP1 29 . The gene expression of the intermediate mediators CDC35, CST20, HST7 were, however, unaffected by AEA. Since the signals transmitted by Ras1 are reduced by AEA, the activity of these intermediate mediators as well as the activity of the transcription factors Efg1 and Chp1 will consequently be dampened, resulting in retarded hyphal morphogenesis. The retardation of hyphae growth might further be effectuated by the prominent downregulation of the hypha-specific G1 cyclin-related protein 1 (HGC1) that regulates the Cdc28 kinase during hyphal growth 30 . On top of these effects, the AEA-mediated upregulation of NRG1, a transcriptional repressor of filamentous growth 31 , may further contribute to the observed reduction in hyphae length and size. Nrg3 has been shown to repress the expression of ALS3 29 , ECE1 31 and HPW1 31 . Thus the upregulation of NRG1 together with the downregulation of EFG1 may fortify the repression of the adhesin genes. Moreover, the AEA-mediated downregulation of the zinc-responsive transcription factor ZAP1 that is known to be required for efficient hyphae formation 32,33 , might have an additional impact. Altogether, the combined alterations in gene expression caused by AEA might explain both the AEA-induced inhibition of hyphal growth and the reduced adherence of AEA-treated hyphae to epithelial cells. AEA and 2-AG were originally discovered to be endocannabinoids that bind to the cannabinoid receptor CB1 34 . It later emerged that AEA has anti-anxiety activities by regulating the neurotransmitter system by being a retrograde synaptic messenger 35,36 . AEA binds also to other membrane molecules in mammalian besides CB1 including CB2, GRP55 (CB3) and TRPV1 36 . AEA usually does not bind to the extracellular part of the receptors, but rather, its binding domain is frequently located deep in the plasma membrane 37 . The interactions of AEA with membrane-bound cholesterol and ceramides facilitate its transport to the receptor binding domains 37 . The binding of AEA to TRPV1 leads to transient calcium influx in neurons 38 . It remains to be determined whether AEA also affects calcium influx in fungi.
In conclusion, AEA and AraS prevent yeast-hyphae transition, inhibit hyphal growth and reduce the ability of C. albicans hyphae to adhere to epithelial cells. This is, among others, achieved by altered expression of genes involved in cell-cell interaction and of genes regulating hyphal morphogenesis.

Material and methods
Chemicals. Anandamide (AEA), N-arachidonoyl serine (AraS) and 2-arachidonoylglycerol (2-AG) were synthesized as described [39][40][41]  Fungal strain and growth conditions. C. albicans SC5314 that has the GFP gene integrated within the ENO1 genomic locus 42 , 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  TCT TCT TGA TTT TGT GGG TGG  TCG ATA GTC CCT CTA AGA AGTG   ACT1  AAG AAT TGA TTT GGC TGG TAG AGA  TGG CAG AAG ATT GAG AAG AAG TTT   ALS1  TTG GGT TGG TCC TTA GAT GG  ATG ATT TCA AAG CGT CGT TC   ALS3  TAA TGC TGC TAC GTA TAA TT  CCT GAA ATT GAC ATG TAG CA   CDC35  TTC ATC AGG GGT TAT TTC AC  CTC TAT CAA CCC GCC ATT TC   CDR1  GTA CTA TCC ATC AAC CAT CAG CAC TT GCC GTT CTT CCA CCT TTT TGTA   CDR2  TGC TGA  www.nature.com/scientificreports/ 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.
Statistical analysis. 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.