Farnesol promotes epithelial cell defense against Candida albicans through Toll-like receptor 2 expression,interleukin-6 and human β-defensin 2 production

Farnesol, a quorum-sensing molecule, regulates virulence and morphogenesis in Candida albicans and is involved in various human pathologies including oral candidiasis. Oral epithelial cells are involved in innate immunity against Candida infections via Toll-like receptors (TLRs) and inflammatory mediators. We investigated the effects of farnesol on host cells and its possible synergistic interaction with gingival epithelial cells against C. albicans infection by studying the expression of TLR2, 4 and 6. The production of IL-6, IL-8, and human β-defensins 1 and 2 was also examined using engineered human oral mucosa tissue put in contact with various concentrations of farnesol with and without C. albicans. Our findings indicate that 24 h after contact with C. albicans, epithelial cells expressed more TLR2 than did non-infected cells. The addition of exogenous farnesol upregulated the TLR2 expression by the gingival epithelial cells in the presence or absence of C. albicans. In contrast, TLR4 was down regulated when farnesol was added to the tissue with or without C. albicans. Finally, farnesol alone was shown to have no effect on TLR6, yet in the presence of both C. albicans and farnesol, TLR6 expression was down regulated. Farnesol modulated TLR2 expression by the epithelial cells following tissue contact with C. albicans. This effect was paralleled by IL-6 but not IL-8 secretion. Farnesol’s effect on innate immunity was strengthened by its capacity to increase human β-defensin 2 production, and by the efficacy of β-defensin against C. albicans growth. Overall results showed that exogenous farnesol promoted epithelial cell defense against C. albicans infection through the involvement of TLR2, IL-6, and human β-defensin 2.     

Production of tyrosol by Candida albicans biofilms and its role in quorum sensing and biofilm development

Tyrosol and farnesol are quorum-sensing molecules produced by Candida albicans which accelerate and block, respectively, the morphological transition from yeasts to hyphae. In this study, we have investigated the secretion of tyrosol by C. albicans and explored its likely role in biofilm development. Both planktonic (suspended) cells and biofilms of four C. albicans strains, including three mutants with defined defects in the Efg 1 and Cph 1 morphogenetic signaling pathways, synthesized extracellular tyrosol during growth at 37°C. There was a correlation between tyrosol production and biomass for both cell types. However, biofilm cells secreted at least 50% more tyrosol than did planktonic cells when tyrosol production was related to cell dry weight. The addition of exogenous farnesol to a wild-type strain inhibited biofilm formation by up to 33% after 48 h. Exogenous tyrosol appeared to have no effect, but scanning electron microscopy revealed that tyrosol stimulated hypha production during the early stages (1 to 6 h) of biofilm development. Experiments involving the simultaneous addition of tyrosol and farnesol at different concentrations suggested that the action of farnesol was dominant, and 48-h biofilms formed in the presence of both compounds consisted almost entirely of yeast cells. When biofilm supernatants were tested for their abilities to inhibit or enhance germ tube formation by planktonic cells, the results indicated that tyrosol activity exceeds that of farnesol after 14 h, but not after 24 h, and that farnesol activity increases significantly during the later stages (48 to 72 h) of biofilm development. Overall, our results support the conclusion that tyrosol acts as a quorum-sensing molecule for biofilms as well as for planktonic cells and that its action is most significant during the early and intermediate stages of biofilm formation.     

Effect of Farnesol on Planktonic and Biofilm Cells of <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis>

Staphylococcus epidermidis is now amongst the most important pathogenic agents responsible for bloodstream nosocomial infections and for biofilm formation on indwelling medical devices. Its increasing resistance to common antibiotics is a challenge for the development of new antimicrobial agents. Accordingly, the goal of this study was to evaluate the effect of farnesol, a natural sesquiterpenoid, on Staphylococcus epidermidis planktonic and biofilm cells. Farnesol displayed a significant inhibitory effect on planktonic cells. Small concentrations (100 μM) were sufficient to exhibit antibacterial effect on these cells. In biofilm cells the effect of farnesol was not so pronounced and it seems to be strongly dependent on the cells metabolic activity and amount of matrix. Interestingly, the effect of farnesol at 200 μM was similar to the effect of vancomycin at peak serum concentration either in planktonic or biofilm cells. Overall, the results indicate a potential antibacterial effect of farnesol against S. epidermidis, and therefore the possible action of this molecule on the prevention of S. epidermidis related infections.     

Farnesol-induced cell death and stimulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in tobacco cv bright yellow-2 cells

Growth inhibition of tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells by mevinolin, a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) could be partially overcome by the addition of farnesol. However, farnesol alone inhibited cell division and growth as measured by determination of fresh weight increase. When 7-d-old tobacco cv Bright Yellow-2 cells were diluted 40-fold into fresh culture, the cells exhibited a dose-dependent sensitivity to farnesol, with 25 microM sufficient to cause 100% cell death, as measured by different staining techniques, cytometry, and monitoring of fragmentation of genomic DNA. Cells were less sensitive to the effects of farnesol when diluted only 4-fold. Farnesol was absorbed by the cells, as examined by [1-(3)H]farnesol uptake, with a greater relative enrichment by the more diluted cells. Both mevinolin and farnesol treatments stimulated apparent HMGR activity. The stimulation by farnesol was also reflected in corresponding changes in the steady-state levels of HMGR mRNA and enzyme protein with respect to HMGR gene expression and enzyme protein accumulation.     

The mevalonate/isoprenoid pathway inhibitor apomine (SR-45023A) is antiproliferative and induces apoptosis similar to farnesol

Apomine (SR-45023A) is a new antineoplastic compound which is currently in clinical trials and representative of the family of cholesterol synthesis inhibitors 1,1-bisphosphonate esters. Apomine inhibits growth of a wide variety of tumor cell lines with IC(50) values ranging from 5 to 14 microM. The antiproliferative activity of apomine was studied in comparison with that of other inhibitors of the mevalonate/isoprenoid pathway of cholesterol synthesis, simvastatin, farnesol, and 25-hydroxycholesterol. All these compounds inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. Apomine (IC(50) = 14 microM), simvastatin (IC(50) = 3 microM), farnesol (IC(50) = 60 microM), and 25-hydroxycholesterol (IC(50) = 2 microM) inhibited HL60 cell growth. Growth inhibition due to simvastatin was reverted by mevalonate, whereas the antiproliferative activity of apomine, farnesol, and 25-hydroxycholesterol was not. Apomine triggered apoptosis in HL60 cells in less than 2 h. Apomine and farnesol induced caspase-3 activity at concentrations similar to their IC(50) values for cell proliferation, whereas a 10-fold excess of simvastatin was necessary to trigger apoptosis compared to its potency on proliferation. Caspase-3 activity was not induced by 25-hydroxycholesterol. The overall similar profile on mevalonate synthesis inhibition, cell growth inhibition, and apoptosis suggests that apomine acts as a synthetic mimetic of farnesol.     

Virulence Factors in <Emphasis Type="Italic">Candida albicans</Emphasis> and <Emphasis Type="Italic">Streptococcus mutans</Emphasis> Biofilms Mediated by Farnesol

The aim of this study was to evaluate the effect of farnesol on the production of acids and hydrolytic enzymes by biofilms of Streptococcus mutans and Candida albicans. The present study also evaluated the time-kill curve and the effect of farnesol on matrix composition and structure of single-species and dual-species biofilms. Farnesol, at subinhibitory concentrations, showed a significant reduction in S. mutans biofilm acid production, but did not alter C. albicans hydrolytic enzyme production. The number of cultivable cells of both microorganisms was significantly reduced after 8 h of contact with farnesol. Extracellular matrix protein content was reduced for biofilms formed in the presence of farnesol. In addition, confocal laser scanning and scanning electron microscopy displayed structural alterations in all biofilms treated with farnesol, which included reduction in viable cells and extracellular matrix. In conclusion, farnesol showed favorable properties controlling some virulence factors of S. mutans and C. albicans biofilms. These findings should stimulate further studies using this quorum-sensing molecule, combined with other drugs, to prevent or treat biofilm-associated oral diseases.     

Suppression of anti-Candida activity of macrophages by a quorum-sensing molecule, farnesol, through induction of oxidative stress

Farnesol is well known as a quorum-sensing molecule of Candida albicans . To assess the pathological function of farnesol, its effects on macrophage viability and functions including growth inhibitory activities against C. albicans were examined in vitro . Murine macrophages, when cultured in the presence of 56–112 μM of farnesol for 1–2 hr, decreased their activity inhibiting the mycelial growth of C. albicans and lost their viability. This suppression of macrophage function by farnesol was neutralized by the coexistence of the anti-oxidants probucol and trolox. Macrophages cultured in the presence of farnesol for 2 hr displayed morphological change of nuclei and DNA fragmentation, which suggested apoptosis of the cells. Intracellular production of ROS in the farnesol-treated macrophages was shown by fluorescence of DCFH-DA and increase of peroxidized materials. These effects of farnesol were blocked by probucol or trolox. These results indicate that farnesol lowered viability of the murine macrophages and suppressed their anti- Candida activity, perhaps through induction of ROS.     

Farnesol-induced apoptosis in Aspergillus nidulans reveals a possible mechanism for antagonistic interactions between fungi

The dimorphic fungus Candida albicans secretes farnesol, which acts as a quorum-sensing molecule and prevents the yeast to mycelium conversion. In this study we examined the effect of farnesol in the filamentous fungus Aspergillus nidulans. We show that externally added farnesol has no effect on hyphal morphogenesis; instead, it triggers morphological features characteristic of apoptosis. Additional experiments suggest that mitochondria and reactive oxygen species (ROS) participate in farnesol-induced apoptosis. Moreover, the effects of farnesol appear to be mediated by the FadA heterotrimeric G protein complex. Because A. nidulans does not secrete detectable amounts of farnesol, we propose that it responds to farnesol produced by other fungi. In agreement with this notion, growth and development were impaired in a farnesol-dependent manner when A. nidulans was co-cultivated with C. albicans. Taken together, our data suggest that farnesol, in addition to its quorum-sensing function that regulates morphogenesis, is also employed by C. albicans to reduce competition from other microbes.     

Effect of farnesol on Candida dubliniensis morphogenesis

AIMS: Cell-cell signalling in Candida albicans is a known phenomenon and farnesol was identified as a quorum sensing molecule determining the yeast morphology. The aim of this work was to verify if farnesol had a similar effect on Candida dubliniensis, highlighting the effect of farnesol on Candida spp. morphogenesis. METHODS AND RESULTS: Two different strains of C. dubliniensis and one of C. albicans were grown both in RPMI 1640 and in serum in the presence of absence of farnesol. At 150 micromol l(-1) farnesol the growth rate of both Candida species was not affected. On the contrary, farnesol inhibited hyphae and pseudohyphae formation in C. dubliniensis. CONCLUSION: Farnesol seems to mediate cell morphology in both Candida species. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of farnesol on C. dubliniensis morphology was not reported previously.     

Protective effects of farnesol against oral candidiasis in mice

Farnesol is known as a quorum-sensing molecule for Candida albicans and is recognized to play pathogenic roles in Candida infection. To assess the possible role of farnesol in mucosal C. albicans infection, the effects of farnesol treatment against experimental oral candidiasis in mice were examined. Prednisolone-pretreated ICR mice were orally infected with C. albicans and 3, 24 and 30 hr later the animals were orally given farnesol. Forty-eight hr later they were killed for observation. Farnesol treatment in a dose ranging between 1.125 and 9 micromol/mouse showed a protective effect against oral candidiasis in a dose-dependent manner, at least as estimated by symptom scores of tongues. At 9 micromol/mouse it decreased bodyweight loss. Histological studies of 2.25 micromol/mouse farnesol-treated animals indicated that farnesol suppressed mycelial growth of C. albicans on the surface of tongues, but microbiological study did not prevent the change of CFU of C. albicans cells not only on tongues but also in feces, kidneys and livers. These results suggest that farnesol has very characteristic roles in protection against mucosal candidiasis.     

Release from quorum-sensing molecules triggers hyphal formation during Candida albicans resumption of growth

Candida albicans is a pathogenic fungus able to change morphology in response to variations in its growth environment. Simple inoculation of stationary cells into fresh medium at 37 degrees C, without any other manipulations, appears to be a powerful but transient inducer of hyphal formation; this process also plays a significant role in classical serum induction of hyphal formation. The mechanism appears to involve the release of hyphal repression caused by quorum-sensing molecules in the growth medium of stationary-phase cells, and farnesol has a strong but incomplete role in this process. We used DNA microarray technology to study both the resumption of growth of Candida albicans cells and molecular regulation involving farnesol. Maintaining farnesol in the culture medium during the resumption of growth both delays and reduces the induction of hypha-related genes yet triggers expression of genes encoding drug efflux components. The persistence of farnesol also prevents the repression of histone genes during hyphal growth and affects the expression of putative or demonstrated morphogenesis-regulating cyclin genes, such as HGC1, CLN3, and PCL2. The results suggest a model explaining the triggering of hyphae in the host based on quorum-sensing molecules.     

Inhibition of Candida albicans biofilm formation by farnesol,a quorum-sensing molecule

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 micro M) and incubated at 37 degrees C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 micro M farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.     

Farnesol kinase is involved in farnesol metabolism, ABA signaling and flower development in Arabidopsis

Farnesol, which is toxic to plant cells at high concentrations, is sequentially phosphorylated to farnesyl phosphate and farnesyl diphosphate. However, the genes responsible for the sequential phosphorylation of farnesol have not been identified and the physiological role of farnesol phosphorylation has not been fully elucidated. To address these questions, we confirmed the presence of farnesol kinase activity in Arabidopsis (Arabidopsis thaliana) membranes and identified the corresponding gene (At5g58560, FOLK). Heterologous expression in recombinant yeast cells established farnesol as the preferred substrate of the FOLK-encoded kinase. Moreover, loss-of-function mutations in the FOLK gene abolished farnesol kinase activity, caused an abscisic acid-hypersensitive phenotype and promoted the development of supernumerary carpels under water-stress conditions. In wild-type plants, exogenous abscisic acid repressed FOLK gene expression. These observations demonstrate a role for farnesol kinase in negative regulation of abscisic acid signaling, and provide molecular evidence for a link between farnesol metabolism, abiotic stress signaling and flower development.     

Roles of Ras1 membrane localization during Candida albicans hyphal growth and farnesol response

Many Ras GTPases localize to membranes via C-terminal farnesylation and palmitoylation, and localization regulates function. In Candida albicans, a fungal pathogen of humans, Ras1 links environmental cues to morphogenesis. Here, we report the localization and membrane dynamics of Ras1, and we characterize the roles of conserved C-terminal cysteine residues, C287 and C288, which are predicted sites of palmitoylation and farnesylation, respectively. GFP-Ras1 is localized uniformly to plasma membranes in both yeast and hyphae, yet Ras1 plasma membrane mobility was reduced in hyphae compared to that in yeast. Ras1-C288S was mislocalized to the cytoplasm and could not support hyphal development. Ras1-C287S was present primarily on endomembranes, and strains expressing ras1-C287S were delayed or defective in hyphal induction depending on the medium used. Cells bearing constitutively activated Ras1-C287S or Ras1-C288S, due to a G13V substitution, showed increased filamentation, suggesting that lipid modifications are differentially important for Ras1 activation and effector interactions. The C. albicans autoregulatory molecule, farnesol, inhibits Ras1 signaling through adenylate cyclase and bears structural similarities to the farnesyl molecule that modifies Ras1. At lower concentrations of farnesol, hyphal growth was inhibited but Ras1 plasma membrane association was not altered; higher concentrations of farnesol led to mislocalization of Ras1 and another G protein, Rac1. Furthermore, farnesol inhibited hyphal growth mediated by cytosolic Ras1-C288SG13V, suggesting that farnesol does not act through mechanisms that depend on Ras1 farnesylation. Our findings imply that Ras1 is farnesylated and palmitoylated, and that the Ras1 stimulation of adenylate cyclase-dependent phenotypes can occur in the absence of these lipid modifications.     

Biofilm formation by Candida albicans and Streptococcus mutans in the presence of farnesol: a quantitative evaluation

The aim of this study was to evaluate the effect of the QS molecule farnesol on single and mixed species biofilms formed by Candida albicans and Streptococcus mutans. The anti-biofilm effect of farnesol was assessed through total biomass quantification, counting of colony forming units (CFUs) and evaluation of metabolic activity. Biofilms were also analyzed by scanning electron microscopy (SEM). It was observed that farnesol reduced the formation of single and mixed biofilms, with significant reductions of 37% to 90% and 64% to 96%, respectively, for total biomass and metabolic activity. Regarding cell viability, farnesol treatment promoted significant log reductions in the number of CFUs, ie 1.3–4.2 log₁₀ and 0.67–5.32 log₁₀, respectively, for single and mixed species biofilms. SEM images confirmed these results, showing decreases in the number of cells in all biofilms. In conclusion, these findings highlight the role of farnesol as an alternative agent with the potential to reduce the formation of pathogenic biofilms.     

Study of trans–trans farnesol effect on hyphae formation by Yarrowia lipolytica

Dimorphism is an ability of certain fungi related to its adaptation to the environment and provides a selective advantage under stress conditions and is associated to the development of human diseases. Hyphae inducing- and inhibitory-effect of farnesol on hyphae formation by the dimorphic yeast Yarrowia lipolytica was evaluated through digital image analysis. The agitation speed of the culture was the most effective hyphae inducer in comparison to bovine calf serum and N-acetylglucosamine. In low agitation system, bovine calf serum was more effective for hyphae formation inducing 57 % of hyphae transition. Farnesol inhibited hyphae formation even in low concentration (300 μM) and this effect increased with increasing concentrations. In the presence of N-acetylglucosamine, this effect was more evident in comparison to the presence of bovine calf serum, which might have protected the cells from farnesol. Digital image analysis was an important tool to evaluate this phenomenon.     

The effect of farnesol on amino acid incorporation by a wild-type and cell-wall variant strain of Candida albicans

Farnesol, a quorum sensing (QS) signal, is produced by Candida albicans during high density growth and has been found to inhibit morphogenesis. This QS auto-inducing signal was discovered to increase amino acid incorporation by C. albicans when concentrations of farnesol increased to 10 microg/mL in yeast nitrogen broth. Farnesol concentrations greater than 10 microg/mL abolished the enhanced incorporation, and the magnitude of the increased incorporation was dependent on cell-surface hydrophobicity.