Fungal Quorum-Sensing Molecules: A Review of Their Antifungal Effect against Candida Biofilms

The number of effective therapeutic strategies against biofilms is limited; development of novel therapies is urgently needed to treat a variety of biofilm-associated infections. Quorum sensing is a special form of microbial cell-to-cell communication that is responsible for the release of numerous extracellular molecules, whose concentration is proportional with cell density. Candida-secreted quorum-sensing molecules (i.e., farnesol and tyrosol) have a pivotal role in morphogenesis, biofilm formation, and virulence. Farnesol can mediate the hyphae-to-yeast transition, while tyrosol has the opposite effect of inducing transition from the yeast to hyphal form. A number of questions regarding Candida quorum sensing remain to be addressed; nevertheless, the literature shows that farnesol and tyrosol possess remarkable antifungal and anti-biofilm effect at supraphysiological concentration. Furthermore, previous in vitro and in vivo data suggest that they may have a potent adjuvant effect in combination with certain traditional antifungal agents. This review discusses the most promising farnesol- and tyrosol-based in vitro and in vivo results, which may be a foundation for future development of novel therapeutic strategies to combat Candida biofilms.


Introduction
It has been estimated that there are 2.2 to 3.8 million fungal species worldwide; however, approximately 300 species have been described to cause human disease [1]. Candida species are among the most common human fungal pathogens. The annual incidence rate of Candida-associated bloodstream infections ranged from 9.5 to 14.4 per 100,000 in the United States of America [2]. This value ranged from 1.4 to 5.7 per 100,000 in Europe, depending on the country [3]. In the last two decades, the prevalence of resistant fungal infections has been steadily increasing due to the widespread use of antifungals in agriculture and veterinary and human medicine [4,5]. Global warming and anthropogenic effects have resulted in the emergence of previously little-known, potentially multi-resistant fungal pathogens in clinical practice, such as Candida auris, azole-resistant Aspergillus spp., or Lomentospora prolificans. These emerging pathogens have caused further challenges for therapy [6,7].
Several fungal species can switch their morphology from yeast to hyphal or pseudohyphal forms, which is coupled with biofilm formation and plays a pivotal role both in fungal virulence and in resistance to antifungals [8][9][10]. The increased number of biofilm-associated infections is exacerbated by a paucity of antifungal agents or therapeutic strategies in development that have unique mechanisms of action or possess alternative approaches, respectively [11]. Currently, the most promising antifungal agents are already in Phase 3 including ibrexafungerp [12], rezafungin [13], super bioavailable itraconazole [14], and VT-1161 [15]. Recently investigated alternative therapeutic approaches involve high-dose therapy with available antifungal agents [16][17][18], antifungal lock therapy [19], and combination-based therapies [20,21].
Despite their importance, Candida biofilms remain a relatively underappreciated and understudied area. Therefore, effective therapeutic strategies against these sessile communities remain scarce. Biofilms are usually found in medical devices such as joint prostheses, pacemakers, urinary and central venous catheters, dentures, and mechanical heart valves, hindering the eradication of Candida infections [10]. In addition, several chronic Candida-related diseases are also associated with biofilm development [29]. Biofilm formation on the vaginal mucosa has been observed in in vivo models of vulvovaginal candidiasis [30]. Oral-and oesophageal mucosae-associated biofilms are a very important contributor to oral diseases caused by Candida species; gastrointestinal and urogenital tracts are also common sites of Candida-associated opportunistic infections [31]. Candida is one of the most commonly identified fungal genera in wounds whose environment can also promote the formation of biofilms [32]. A series of recent studies has indicated that strains defective in hyphal formation display significantly milder symptoms, highlighting the role of biofilm formation in pathogenesis of these chronic or recurrent infections [30,33].
These sessile communities exhibit five-to eightfold higher resistance to all licenced antifungal drugs when compared to their planktonic counterparts [10]. This high rate of resistance can be explained by the increased metabolic activity of cells in the early development phase of biofilm formation [10]. On the other hand, dormant, non-proliferating persister cells have also been observed, especially in mature biofilms, that have demonstrated high tolerance to antifungals [34]. Furthermore, the various Candida species can produce dense extracellular polymeric substances which serve as a solid barrier to prevent the diffusion of antifungal drugs and account for almost 90% of the biofilm dry mass [10]. As has been previously reported in the literature, sessile Candida communities exhibit an altered gene expression profile, including the upregulation of CDR and MDR genes which encode azole resistance transporter proteins, and pose further challenges for treatment [35].
To date, there is no definitive therapy against Candida biofilms; nevertheless, there are several promising in vitro, in vivo and clinical results. The increasing number of resistant Candida species and isolates highlight the need for new molecules with new targets. Alternative therapeutic approaches against multidrug-resistant fungal biofilms may be the result of a combination of traditional antifungal agents with quorum-sensing molecules [36].

Fungal Quorum Sensing
A major mechanism of microbial communication is a population density-dependent stimulus-response system called quorum sensing. This process occurs by the continuous release and monitoring of low molecular weight hormone-like secreted molecules (quorum-sensing molecules), which are not elementary in the central metabolism but have a variety of biological activities. The concentration of these quorum-sensing molecules is proportional with the size of population; after reaching a critical threshold, a response is triggered leading to the coordinated expression or repression of quorum sensing-related target genes [37].
In the fungal kingdom, quorum sensing was a relatively unknown phenomenon until Hornby et al. (2001) described the effect of the isoprenoid farnesol on Candida albicans morphogenesis; this opened a new branch of science focusing on fungal quorum sensing [38]. At the same time, quorum sensing has been already reported in Aspergillus spp. [39] and Penicillium spp. [40]. To date, four main quorum-sensing molecules were described including farnesol, tyrosol, phenylethanol, and tryptophol, which have a remarkable effect on the regulation of morphogenesis (yeast to hyphae transition and vice versa), initiation of fungal apoptosis, and virulence [41].
Recently, several authors reported that certain quorum-sensing molecules may generate oxidative stress, especially at supraphysiological concentrations, which may have an antifungal effect [42][43][44][45]. The majority of data concerning fungal quorum sensing molecule-related therapeutic potential derived from C. albicans experiments, and these results cannot be always directly extrapolated to non-albicans species. Recently, the number of studies dealing with the effect of quorum-sensing molecules on non-albicans species has steadily increased, supporting the comprehensive understanding of the in vitro and in vivo antifungal effects exerted by these molecules.

Physiological Effect of Farnesol in Candida Species
Farnesol (3,7,11-trimethyl-2,6,10-dodecatriene-1-ol) was the first described Candida-derived quorum sensing molecule; it is released in C. albicans as a side product of the sterol synthetic pathway by dephosphorylation of farnesol pyrophosphate [38,46]. It is an acyclic sesquiterpene heat-stable molecule, which is produced primarily under aerobic conditions and it is unaffected by extreme pH and the type of carbon or nitrogen source [38,47]. Generally, the farnesol concentration is proportional to the colony-forming unit number [38]. Under physiological conditions, C. albicans isolates secrete a farnesol concentration with a mean of 35.6 µM (range: 13.7 to 58.5 µM) [48]. This concentration was 35 times higher than that secreted by non-albicans species, with the exception of Candida dubliniensis, which has demonstrated a concentration of 8.3 µM (range: 6.0 to 17.5 µM). All other non-albicans species excreted significantly lower farnesol concentrations, ranging from 0.4 to 1 µM [48]. These differences in excretion may be explained by the species-specific characteristics in sterol synthesis [49].
Based on a cDNA microarray analysis, a total of 274 genes were identified as responsive in C. albicans, with 104 genes up-regulated and 170 genes down-regulated [50]. Farnesol has an ability to influence Candida morphology, biofilm formation, drug efflux pump expression, apoptosis regulation, phagocytic response, surface hydrophobicity, iron metabolism, and heat-shock-related pathways [50][51][52][53][54]. One of the most prominent farnesol-associated effects is the induction of hypha-to-yeast transition and the inhibition of biofilm formation in various Candida species. It should be emphasized that 150-fold more farnesol is needed to block germ-tube formation in the presence of 10% serum, showing that it can bind to serum proteins at a high rate [55,56].
In view of this diverse role, it is not surprising that this compound influences several central signalling pathways in different Candida species. One of the best-studied farnesol-related pathways is the Ras1-cAMP-PKA cascade, where farnesol binds to the cyclase domain of the adenylyl cyclase Cyr1, influencing the level of intracellular cAMP [57]. Moreover, farnesol induces the cleavage of the small GTPase Ras1, resulting in a soluble Ras1; soluble Ras1 is a weak activator of Cyr1 and supports the formation of yeast cells [58]. Furthermore, farnesol can directly inhibit the cAMP signalling pathway, supporting the hypha-to-yeast transition [59]. It is noteworthy that farnesol exposure stabilizes the Nrg1 protein, which is the negative regulator of filamentation [60]. While farnesol was described first in C. albicans, it can inhibit filamentation and growth in other fungal species [27,61], including Saccharomyces cerevisiae [62], Aspergillus niger [63], Aspergillus flavus [64], Aspergillus nidulans [65], Penicillium expansum [66], Fusarium graminearum [67], and Paracoccidioides brasiliensis [68].
Regarding reactive oxygen species production, the supraphysiological farnesol concentrations (200-300 µM) are stressful for most fungi, while the physiological concentrations (30-40 µM) protect them from stress [57]. In addition to the farnesol-related effect on growth in the case of different microbes, the molecule also has a relevant immunomodulator effect [57,69]. Farnesol can stimulate both macrophage chemokine synthesis or macrophage recruitment, and trigger activation of neutrophil granulocytes and monocytes. Farnesol exposure also influences the differentiation of monocytes into dendritic cells [57,69].
Farnesol has been reported to induce cell growth inhibition and/or apoptosis in tumor cells where the observed IC 50 values varied widely for different tumor types and different cell lines [70]. Farnesol caused 100% cell death at >120 µM in A549 and H460 lung cancer cells [71].

Antimicrobial Activity of Farnesol
At physiological concentrations, farnesol has no significant effect on Candida cells that have already begun hyphae development or biofilm formation [25,38]. However, prior results suggest that farnesol can cause biofilm degradation at supraphysiological concentrations, suggesting the potential use of this compound in biofilm-associated infections [36]. In addition, several authors have published studies demonstrating contribution of farnesol to reduced azole resistance of Candida cells, including in biofilms [72]. This phenomenon can be explained by the modulation of Cdr1 efflux pumps, reactive oxygen species production, or changes in glutathione homeostasis [38,61,72]. Furthermore, farnesol has an effect on genes connected to ergosterol synthesis [46]. Dižová et al. (2018) observed that the presence of 200 µM farnesol down-regulated the ERG20, ERG11 and ERG9 genes. However, this farnesol concentration supplemented with 0.5 mg/L fluconazole restored the original expression level of ERG20 and ERG11. Interestingly, the physiological farnesol concentration (~30 µM) only slightly influences the expression of these genes in 48 h-old biofilms [73]. Chen et al. (2018) reported that CYR1 and PDE2 regulate resistance mechanisms against various antifungals in C. albicans biofilms. However, farnesol can diminish the resistance of C. albicans biofilms by regulating the expression of the gene CYR1 and PDE2 [74]. Yu et al. (2012) observed that the sterol biosynthetic pathway may contribute to the inhibitory effects of farnesol, as the transcription levels of the ERG11, ERG25, ERG6, ERG3, and ERG1 genes decreased following farnesol exposure [75]. Jabra-Rizk et al. (2006) showed that farnesol concentrations of 30-50 mM decrease the fluconazole MICs for C. albicans and C. dubliniensis from resistant values to a susceptible dose-dependent range, while concentrations of 100-300 mM resulted in fluconazole susceptibility [76].
One of the first major breakthroughs in combination-based experiments with farnesol and antifungals was published by Katragkou et al. (2015), who found a significant synergy against C. albicans 48 h-old biofilms between fluconazole, amphotericin B, and micafungin in the presence of farnesol [26]. The highest synergistic effect was observed in the case of micafungin combined with farnesol using fractional inhibitory concentration index determination and Bliss independence analysis. Based on the Bliss model, the observed effects were 39-52% higher compared to the expected efficacy if the drugs had been acting independently [26]. It should be noted that synergism was observed only in the case of farnesol/micafungin and farnesol/fluconazole based on calculated fractional inhibitory concentration indices, suggesting the usage of multiple analytical approaches for investigation of drug-drug interaction [26].
Regarding non-albicans species, Kovács et al. (2016) showed that farnesol consistently enhanced the activity of caspofungin and micafungin, as concordantly shown in two independent experimental settings (chequerboard dilution and time-kill experiments) [27]. Fernández-Rivero et al. (2017) reported that a supraphysiological farnesol concentration (300 µM) improved the activity of amphotericin B against Candida tropicalis biofilms but did not affect anidulafungin [77]. Two recent studies by Nagy et al. concluded that farnesol significantly enhanced the activity of echinocandins and triazoles against one-day-old C. auris biofilms in vitro, suggesting an alternative approach to overcome the previously well-documented azole and echinocandin resistance of C. auris biofilms [45,78].
Animal experiments with farnesol raised several questions in terms of in vivo applicability of this compound. In one of the first in vivo studies, Navarathna et al. (2007) concluded that the physiological farnesol production may play a pivotal role as a virulence factor in fungal pathogenesis; furthermore, exogenous oral and intraperitoneal farnesol administration (20 mM) enhances the mortality of mice in their systemic mouse model [79]. Contrary to these results, Hisajima et al. (2008) observed a protective effect against C. albicans in their oral candidiasis mouse model [80]. It should be noted that there was a 1000-fold difference between the administered farnesol dosages (9 µM/mouse) in the experiments of Hisajima et al. (2008) [80]  immunocompromised systemic mouse model, especially when inocula was pre-exposed to farnesol [45].
The farnesol-exerted antifungal activity can be explained by the higher level of reactive oxygen species, especially in the case of non-albicans species [43,45]. Furthermore, farnesol has an amphiphilic property which allows for its integration into cell membranes, influencing membrane fluidity and integrity. In the case of Candida parapsilosis and C. dubliniensis, farnesol affected the cellular polarization and membrane permeability [61,76,85]. These observations can help further elucidate the antifungal effect.  [88]. A clear synergistic interaction was observed between farnesol and nafcillin or vancomycin against S. epidermidis sessile cells [88]. Additionally, it potentiates the activity of beta-lactam antibiotics against antibiotic-resistant bacterium species [89]. Castelo-Branco et al. (2012) showed a potent antimicrobial effect exerted by exogenous farnesol exposure against mature Burkholderia pseudomallei biofilms [90]. Additionally, it increased the activity of amoxicillin, ceftazidime, doxycycline, and sulfamethoxazole-trimethoprim, which are routinely administered for the treatment of melioidoses [91]. Farnesol also had a synergizing effect against ciprofloxacin-resistant Pseudomonas aeruginosa biofilms when used in combination with ciprofloxacin [92]. In vivo data also supports the antibacterial efficacy of farnesol. It has been observed that 6.7 mM farnesol treatment significantly decreased the S. epidermidis associated catheter infection and systemic dissemination [88].
Based on several studies, farnesol has a remarkable effect in Candida-bacterium mixed biofilms. C. albicans-derived farnesol has also been shown to have an effect on the response of S. aureus to antibiotics in mixed species biofilms. Farnesol exposure results in a significant decrease in staphyloxantin, which is an important virulence factor of this bacterium [42]. Černáková et al. (2018) showed that 200 µM farnesol has an inhibitory effect on C. albicans growth in mixed-species biofilms with Streptococcus mutans [93]. Cugini et al. (2010) examined the C. albicans-P. aeruginosa mixed species biofilms, where the C. albicans-derived farnesol enhanced P. aeruginosa quinolone signal production in a LasR-defective strain [94].
Regarding non-albicans species, tyrosol has been recognized as inducing the biofilm-forming ability of C. auris to grow as yeast or pseudohyphae [96]. Based on RNA-Seq analysis, tyrosol treatment resulted in 261 and 181 differentially expressed genes with at least a 1.5-fold increase or decrease in expression in C. parapsilosis, respectively; however, the initial adherence was not affected by the presence of tyrosol [43]. Interestingly, the ortholog of the C. albicans CZF1 gene, which is a key transcription factor of biofilm development in C. parapsilosis, was upregulated following tyrosol exposure [43,100]. Nevertheless, Jakab et al. (2019) did not observe higher rates of biofilm formation in the presence of tyrosol [43]. In C. parapsilosis, tyrosol exposure overexpressed the active efflux pumps and caused an enhanced oxidative stress response, while inhibiting growth, ribosome biogenesis, and virulence. Surprisingly, its metabolism was modulated toward glycolysis and ethanol fermentation [43]. Monteiro et al. (2015) reported that tyrosol exposure did not induce increased adhesion in C. glabrata [101].

Antimicrobial Activity of Tyrosol
Tyrosol is a relatively understudied molecule compared to farnesol in terms of potential antifungal or anti-biofilm activity; despite this, a few studies have examined the potential use of tyrosol in monotherapy or in combination with traditional antifungal agents against Candida species [36,72]. Arias et al. (2016) showed that tyrosol treatment at concentrations ranging from 100 to 200 mM exerted a significant reduction in metabolic activity against C. albicans and C. glabrata two-day-old oral biofilms, which was proportional to a reduction in cell number [103]. Do Vale et al. (2017) showed that tyrosol alone at concentrations of 50 and 90 mM demonstrated inhibition of the planktonic growth of C. albicans and C. glabrata cells, respectively [104]. However, tyrosol does not significantly reduce metabolic activity or the number of cells for one-day-old oral biofilms; in addition, the nature of interaction of tyrosol with chlorohexidine gluconate was indifferent. Nevertheless, 1.25 mM tyrosol with 0.00725 mM chlorhexidine gluconate showed a synergistic interaction in reducing the number of hyphae formed [104]. A combination of tyrosol and farnesol has been explored for oral Candida isolates for both planktonic and sessile growth. This combination showed synergy against C. glabrata, indicating that this combination may contribute to the development of oral care products against Candida species [105].
In another study, tyrosol showed anti-biofilm activity against denture-derived C. albicans isolates. However, it has been shown that the single use of tyrosol cannot decrease hydrolytic enzymes on oral C. albicans [106]. Shanmughapriya et al. (2014) observed that tyrosol treatment caused a 25% and a 50% reduction in intrauterine device-derived Candida krusei and C. tropicalis biofilm production at concentrations of 40 µM and 80 µM, respectively [107]. In addition, amphotericin B combined with tyrosol showed a remarkable inhibitory effect against these non-albicans biofilms. A concentration of 4 mg/L amphotericin B in the presence of 80 µM tyrosol exerted approximately 90% inhibition in biofilm formation [107]. Cordeiro et al. (2015) showed that the addition of tyrosol significantly reduced the MICs for amphotericin B, fluconazole, and itraconazole against planktonic C. albicans and C. tropicalis [108]. Furthermore, exogenous tyrosol alone was able to significantly reduce the biofilm formation of these species at concentrations ranging from 125 to 250 mM. At these concentrations, tyrosol decreased the metabolic activity of growing biofilms by approximately 24 and 30% for C. albicans and C. tropicalis, respectively. Reduction of metabolic activity was more pronounced when tyrosol was combined with traditional antifungal drugs including amphotericin B, fluconazole, and itraconazole. It should be noted that application of amphotericin B with tyrosol markedly decreased the metabolic activity of mature biofilms (35%) [108]. Kovács et al. (2017) reported that tyrosol may be used as an adjuvant agent with caspofungin or micafungin in alternative treatment strategies [109]. Regarding the in vivo antifungal effect of tyrosol, Jakab et al. (2019) reported that daily treatment with 15 mM tyrosol decreased the fungal tissue burden in their immunocompromised mouse model [43]. In this study, the expression of ALS6, which has a pivotal role in adhesion, was significantly reduced by tyrosol treatment. Furthermore, downregulation of the expression of FAD2 and FAD3 may also contribute to decreased virulence and kidney fungal burden. The well-documented antifungal effects exerted by tyrosol may be explained by the enhanced oxidative stress and the inhibition of virulence-related genes, growth, and ribosome biogenesis. In addition, tyrosol can alter the metabolism of Candida cells toward fermentation [43].
Data on the potential antibacterial effects of tyrosol remain scarce. Arias et al. (2016) found a potential anti-biofilm activity of tyrosol against S. mutans in single and mixed species biofilms with C. albicans or C. glabrata developed on acrylic resin and hydroxyapatite surfaces [103]. Their results may contribute to the development of innovative topical therapies focusing on biofilm-associated oral diseases. Abdel-Rhman et al. (2016) reported substantial antibacterial activity of tyrosol against S. aureus; moreover, tyrosol increased susceptibility to gentamicin, amikacin, and ciprofloxacin at subinhibitory concentrations ranging from 3.5 to 14.3 mM [110]. Tyrosol treatment can also influence S. aureus virulence, decreasing the production of protease and lipase enzymes and limiting the ability to form biofilms [110]. In the case of P. aeruginosa, tyrosol strongly inhibited haemolysin and protease production [111].

Future Remarks
Paradoxically, medical advancement has resulted in an increasing number of immunocompromised individuals susceptible to Candida infections. The incidence and mortality rate related to systemic Candida infections has remained unchanged, despite the advances in the field of antifungal therapy. Based on recent comprehensive epidemiological studies, the high incidence and mortality may be attributed to sessile Candida populations, namely biofilms, which show high resistance against environmental factors, immune responses, and traditional antifungal therapy. Although there is no definitive solution or highly effective therapeutic recommendation against Candida biofilms, there are many promising therapeutic strategies including antifungal "lock" therapy, photodynamic inactivation, and the use of natural products or synthetic peptides with antifungal activity. A further solution may be the utilization of quorum-sensing molecules alone or in combination with traditional antifungal agents; however, there are numerous open questions as to their exact action or the interaction between quorum-sensing molecules and the host. In addition, the full understanding of quorum sensing in non-albicans species has remained unelucidated. In this review, we provided an overview on the current status of studies focusing on anti-biofilm activity of farnesol and tyrosol. Hopefully, these in vitro and in vivo results can be implemented in therapeutic practice as soon as possible to overcome Candida biofilm-related infections.