Inhibiting roles of farnesol and HOG in morphological switching of Candida albicans

Abstract

Candida albicans is a major opportunistic fungal pathogen of humans, especially in the oral cavity it involves in precancerous lesions. Numerous transcriptional regulators and hypha-specific genes involved in the morphogenesis mechanisms have been identified. Its virulence is predominantly attributed to the potentiality of morphological switching from yeast and pseudohyphae to hyphal growth. Giving attention in farnesol for prevention or intervention of its virulence sense and possible etiologic role in some uncovered premalignant diseases, in addition, to be a quorum-sensing signal molecule and relationship with HOG pathway, although its morphological switching inhibiting function has attracted high attention and got great progress in being elucidated, their exact mode of action is not completely understood. This report provides a review of characteristic aspects of farnesol signaling and HOG pathway during hyphal development. It also includes other associated pathways, molecules, and novel drug development based on the latest researches over the last decade. Furthermore, farnesol as immunomodulatory to host is an important inferring.

Introduction

Human’s opportunistic pathogen Candida albicans (C. albicans) is involved in superficial and systemic diseases such as mucosal premalignant disorders in some population and life-threatening disseminated invasive infections in susceptible individuals [1]. And its yeast-to-hyphal transition and biofilm formation are the predominant virulence-associated traits. Now due to the contribution of some extensive investigations and detailed demonstrations [2-5] about environmental sensing regulation of morphological switching in C. albicans, as well as the development of molecular genetic technologies and the first genome declaration in the world, people have come to further recognize its special dimorphism virulence, which is proposed as an etiologic and therapeutic target [6]. Also, numerous key transcriptional regulators and hypha-specific genes (HSGs) involved in the morphogenesis mechanisms have been identified. The morphological switching of C. albicans includes hyphal initiation, hyphal maintenance, and hypha-to-yeast transition, and its regulatory mechanisms remain in focus [7]. As the drug resistance of fungal or bacteria increases gradually, the research on these mechanisms is conducive to the development of novel antibacterial agents or antibacterial adjuvants.

The first identified quorum sensing molecule (QSM) farnesol [8] and HOG (high osmolarity and glycerol) pathway [9], as the morphological switching inhibitors, have attracted high attention and achieved great progress [10,11], their exact mode of action is not completely understood. Here we review the characteristical aspects of farnesol signaling and HOG pathway during hyphal development, as well as the recently acknowledged other associated pathways and molecules. Besides, we also reviewed their related applications and the research progresses in antifungal therapy.

Farnesol, a complex role in quorum sensing and switching inhibitor

Quorum sensing molecules allow micro-organisms to monitor their growth and control cell density-dependent phenomena. Studies on the roles of farnesol in C. albicans have made progress. Farnesol is secreted by C. albicans white cells only whereas opaque and anaerobic cells turn off farnesol synthesis [4], as a side product of the ergosterol biosynthesis pathway by dephosphorylation of farnesol pyrophosphate, primarily mediated by the pyrophosphatase Dpp3 and C. albicans produces high amounts of farnesol [10,12]. Farnesol inhibits filamentation [12] including and hyphal initiation [5] and filamentous-growth [3,4] without disrupting growth rate. Farnesol blocks the yeast-to-hypha transition at least for a period of 6-10 h after germ tube formation but does not block preexisting hyphal elongation [13]. Farnesol plays an additional role in protecting fungus against oxidative stress [14] and immune modulation [12]. Furthermore, several signaling pathways and molecules are involved in farnesol-mediated mechanisms (Figure 1).

Figure 1.

Morphological switching inhibitor, farnesol signaling in Candida albicans. Arrows indicate stimulation, T-shaped lines indicate inhibition, and gray lines represent relationships that are inactive.

Inhibiting the Ras1-Cyr1/cAMP-PKA signal pathway

The inhibitory function of farnesol on the Ras1-Cyr1/cAMP-PKA cascade [3,12] is well investigated. Ras1p has an advantage of localizing to the membrane and interact with farnesol [15]. The catalytic domain (leucine-rich repeat, LRR) of adenylate cyclase (Cyr1), which once bounds to peptidoglycans will result in filamentation, makes Cyr1p as the central molecular sensor [16]. Therefore, both Ras1p and Cyr1p can be a sensor of farnesol. Farnesol promotes the cleavage of Ras1, resulting in a soluble Ras1 form with a reduced ability to activate Cyr1 [17], and interacts with Ras1 to form a farnesylated Ras1 protein [18]. Furthermore, farnesol directly inhibits Cyr1 activity by binding to the cyclase domain of Cyr1, disturbing cAMP signaling, thus farnesol represses filamentation and supports the hypha-to-yeast transition [17]. The supplementation of exogenous cAMP into media containing farnesol completely restored filamentation [3,19]. Cyr1 can integrate a diverse range of external signals [20], generating a pulse of cAMP essential for hyphal initiation. Studies by Lu et al. [21] indicated the activation of the cAMP-dependent protein kinase A (PKA) pathway was required to down-regulate the transcription of NRG1 during hyphal growth as Nrg1 protein level did not show an obvious reduction in cyr1 and tpk2 mutants. Tpk1 and Tpk2 are PKA catalytic subunits, and deletion mutants of TPK2 block hyphal formation [22,23]. The mutants lacking pde2 showed increased cAMP signaling and more resistant to farnesol-mediated induction of hypha-to-yeast transitions [24]. CYR1 and PDE2 regulate a pair of enzymes associated with cAMP synthesis and degradation, and PDE2 regulation by farnesol was subordinate to CYR1 regulation [25]. In addition, farnesol can induce hydrogen peroxide resistance. Strains lacking either Ras1 or Cyr1 would no longer exhibit increased protection against hydrogen peroxide upon preincubation with farnesol [14], demonstrating that farnesol induces oxidative-stress resistance via Ras1-Cyr1 signaling pathway.

Efg1, a key transcriptional activator of HSGs, is activated through the Ras-cAMP signal transduction pathway to induce hyphal growth [26]. Thus, farnesol blocks activation of the HSGs regulated by Efg1 by inhibiting the Ras-cAMP pathway. However, farnesol does not affect the EFG1 mRNA level. Langford et al. [27] identified that Czf1 played an essential role as downstream of Efg1 in the response to farnesol since the mutation in CZF1 was resistant to farnesol, Efg1 was required for filamentation in aerobic condition while Czf1 was required in hypoxia. CZF1 expression is regulated by Efg1 and negatively regulated by Czf1 [28].

Blocking Cup9 degradation to inhibit the Sok1-mediated degradation of Nrg1

Nrg1 is a transcriptional regulator and the major repressor of hyphal development by binding to the DNA with the Tup1 complexes [4]. By chromatin immunoprecipitation of C-terminal Myc-tagged Nrg1, Lu et al. [21] found that Nrg1 was binding to the promoters of HSGs including HWP1, ALS3, and ECE1 during yeast growth. As the level of Nrg1 repressor decreases, less Nrg1 occupancy at hypha-specific gene promoters [20]. Farnesol inhibits hyphal initiation mainly through blocking the protein degradation of Nrg1 [5]. Lu et al. identified Sok1 as a kinase required for Nrg1 degradation during inoculation, and overexpression of SOK1 overcame the farnesol-mediated inhibition of hyphal initiation. They found Cup9 was responsible for the repression of SOK1 expression and farnesol inhibited Cup9 degradation mediated by Ubr1 [5]. By the way, hyphal initiation requires both the cAMP-PKA pathway-dependent transcriptional down-regulation of NRG1 and Sok1-mediated degradation of Nrg1 protein, neither pathway alone is enough for hyphal initiation [5].

Increasing Tup1 levels

Tup1, a transcriptional repressor, functions with the DNA binding proteins Nrg1 and Rfg1 to inhibit the HSGs expression, negatively regulating the yeasts to hyphae transition [4]. Tup1 is a crucial component in response to farnesol in C. albicans, as the tup1/tup1 and nrg1/nrg1 mutants, not the rfg1/rfg1 mutant, fails to respond to farnesol [4]. Interestingly, they [4] also observed that tup1 and nrg1 mutants elevated farnesol synthesis 17~19 fold. In addition, farnesol causes a small but consistent increase of TUP1 mRNA, a ~2.5-fold increase in Tup1 protein level [18], and even the expression of two Tup1-regulated HSGs, HWP1 and RBT1.

Regulating MAP kinase pathway

Farnesol was considered to function through the Cek1-MAPK pathway to inhibit morphological switching, since the addition of farnesol represses the expression of CPH1, HST7 and GAP1 [2] but CST20 was not affected [29]. Some researchers found C. albicans mutants lacking the histidine kinase Chk1 were refractory to the inhibitory effect of farnesol [30], Farnesol acts on two-component signaling via Chk1.

Correlation between farnesol and EED1

EED1 (Epithelial Escape and Dissemination 1), a unique gene of C. albicans, plays a crucial role in the expression of the hyphy-specific genes, the extension of germ tubes into elongated hyphae and hyphal maintenance [31]. Expression of EED1 is regulated by the transcription factor Efg1 and is negatively regulated by Nrg1 and Tup1 as well [26]. Farnesol might reduce EED1 expression mediated by Nrg1, promoting lateral yeast formation and the hypha-to-yeast transition [12]. Ume6 is a transcription factor that controls the expression of a wide range of hyphal-associated genes (HAGs), affects biofilm formation, and promotes hyphal development [32]. The expression of UME6 is affected by EED1 [26] and supports the maintenance of HGC1 expression, which is involved in hyphal maintenance. Polke et al. [12] have identified the first mutant (EED1Δ/Δ) with hypersecretion of farnesol and hypersensitivity to farnesol, which contributes to the reverse morphogenesis and is not affected by hyperactivation of the RAS1-cAMP-pathway, or deletion of Czf1 and Nrg1, thus farnesol might acts on EED1 via a novel mechanism associated with a regulated farnesol transporter such as Ste3p, Ste6p or another unidentified yet, which still need further researches [13]. The mutants (EED1Δ/Δ) still can form germ tubes to invade epithelium but fail to elongate filaments, and the lateral yeast formation from preexisting hyphae is observed [10,26]. The C. albicans mutant (EED1Δ/Δ) is a good study model for the investigation on fungal dimorphism as well as the characteristics of farnesol in C. albicans.

Farnesol as an immunomodulatory molecule

Abe et al. indicated farnesol decreased murine macrophages phagocytic and anti-Candida activity in vitro with nuclei morphological change and DNA fragmentation, perhaps via induction of ROS [33]. Hargarten et al. [34] discovered that C. albicans white cells secrete farnesol as a potent chemoattractive stimulator of macrophages, resulting in an 8.5-fold increase in macrophage migration in vitro and a 3-fold increase in the peritoneal infiltration of macrophages in vivo. Farnesol induces the activation of neutrophils and monocytes and promotes oxidative burst and the release of proinflammatory cytokines TNF-α and MIP-1α, without enhanced fungal phagocytosis or killing [35]. The study by Zawrotniak et al. showed that farnesol activated the neutrophil extracellular traps (NETs) production, and the Mac-1 and TLR2 receptors were responsible for farnesol recognizing, promoting neutrophils to release ROS quickly after activation by farnesol [36]. In addition, farnesol significantly affects the differentiation of monocytes into immature dendritic cells (iDC) and the ability of iDC to induce proper T cell responses, thereby mitigating the Th1 response [35].

Although farnesol plays a protective role in superficial C. albicans infections such as oral candidiasis [37] with decreasing filamentation rates, less tissue damage and a protective epithelial response, farnesol acts as a virulence factor in systemic infection possibly by modulating immune surveillance towards a non-protective Th2 response, promoting tissue damage and the dispersion of yeast cell [10]. Farnesol modulates human dendritic cells (DC) function via multiple signaling pathways during DC maturation to overcome DC-mediated immunity surveillance. Farnesol increases the expression of the Ag-presenting glycoprotein CD1d by activating nuclear receptors PPARγ and RARα, as well as p38 MAPK. Farnesol reduces IL-12 secretion while enhances the release of IL-10 via MAPK and NF-κB signaling pathways, impairing their ability to activate invariant NKT, Th1, and FOXP3⁺ regulatory T cells [38]. Exposing C. albicans to acidic environments results in the exposure of cell wall pathogen-associated molecular patterns (PAMPs, including β-glucan and chitin) and increases the detection of the yeast by macrophages. Then farnesol regulates the remasking of β-glucan, while chitin remasking is regulated by Efg1 through regulation of CHT2, influencing the host’s innate immune responses [39]. Disrupting the masking of surface markers so that they can be recognized by the innate immunity of the host can be one of the follow-up research directions. Farnesol promotes intestinal epithelial barrier function by activating JAK/STAT3 signaling pathway in differentiated Caco-2 cells and inducing the Zonula Occludens-1 Protein (ZO-1) expression [40]. The immunomodulatory mechanisms still need more further studies.

Farnesol-mediated apoptosis

The influence of farnesol on C. albicans yeast cells is concentration-dependent, while higher farnesol level (more than 200 μM) is stressful for yeasts, lower level (about 40 μM) protects them from stress [41,42]. It has been already proved that farnesol promotes apoptosis (Figure 2).

Figure 2.

Farnesol-mediated cell death. Red T-shaped lines indicate inhibition.

Langford et al. discovered Efg1 and Czf1 coordinate the farnesol-mediated cell death in C. albicans synergistically [27]. Farnesol might trigger apoptosis via accumulation of reactive oxygen species (ROS) which activates intracellular caspases and damages essential cellular compartments but the mechanisms are still unclear [10,41]. Zhu et al. [43] demonstrated farnesol conjugated with intracellular glutathione combined with Cdr1p-mediated extrusion of glutathione-farnesol complexes, resulting in glutathione depletion, higher susceptibility of the cell to oxidative stress and ultimately cell apoptosis. The only metacaspase Mca1 of C. albicans is required and activated during farnesol-induced cell apoptosis by affecting the glycosylation of several critical proteins such as Cdc48 and Ssb1 [44]. Edc3 is a key regulator of CaMca1 expression [45]. It was observed that the expression of the apoptosis genes (CARD-9 and NOXa) increased and the expression of the anti-apoptosis genes (Bcl-2) decreased in the farnesol-treated Galleria mellonella [46]. Farnesol also has anti-inflammatory and anticancer properties by modulating Ras protein and NF-κB activation to downregulate the expression of COX-2, TNF-α, IL-6, and modulating various tumorigenic proteins and signal transduction cascades [47-49]. Farnesol inhibits tumor growth in multiple myeloma by modulating the signal transducer transcriptional activator 3 (STAT3) pathway [48].

The effects of farnesol on polymicrobial biofilms and other species

C. albicans can establish and persist complex polymicrobial biofilms with other different human microbial pathogens, including Staphylococcus aureus (S. aureus), Streptococcus mutans (S. mutans) etc. presenting enhanced pathogenicity and drug resistance by impeding drug penetration and immune cell access. Farnesol activates drug efflux pumps in S. aureus, enhancing tolerance to antibacterials [50,51]. Farnesol might inhibit staphyloxanthin biosynthesis by binding to the CrtM enzyme competitively and enhance tolerance to oxidative stressors by activating the thiol-based oxidative-stress response [52]. However, the effect of farnesol is a dose-dependent manner. Farnesol at high concentrations inhibits the growth of S. aureus and induce the leakage of potassium ions, causing the cytoplasmic membrane of S. aureus to be disordered and eventual decomposed [53]. A 30 mM concentration of farnesol appears to reduce the rate of colonization and inhibit biofilm formation of S. aureus in vitro and in vivo [54].

A study [42] found that 25-50 μM farnesol enhanced S. mutans Gtf activity, promoting microcolony development and biofilm formation. Whereas Cao [55] reported 500-1000 μM farnesol inhibited biofilm formation by 26.4% to 37.1% and downregulated the expression of virulence-associated genes including luxS, brpA, ffh, recA, nth, and smx. And when the concentrations above 12.5 mM, farnesol behaves similarly to chlorhexidine gluconate [56]. Farnesol at 0.78 mM and 1.56 mM significantly reduced the acid production of S. mutans biofilm [57]. Rocha [58] reported that the water-soluble exopolysaccharide in the extracellular matrix (ECM) was significantly reduced in the combination therapy of myricetin, farnesol, and fluoride. Cernakova [59] determined 200 μM farnesol was the minimum inhibitory concentration that inhibited the growth of C. albicans SC5314 biofilm by 50%, and observed C. albicans mainly existed as blastospores but the growth of S. mutans was not influenced in a dual biofilm with the addition of 200 μM farnesol.

Egbe et al. [60] found that farnesol reduced the 48S preinitiation ribosomal complex levels in Saccharomyces cerevisiae by affecting the interaction of the mRNA with the small ribosomal subunit, inhibiting the initiation step of translation and ultimately the filamentous growth. Farnesol inhibits Fusarium keratoplasticum biofilm formation by destructing hyphae and the extracellular matrix in preformed biofilm and preventing conidia adhesion and filamentation [61].

HOG pathway responsible for repression of the yeast-to-hypha transition

The Hog pathway in C. albicans, mediated by the MAPK Hog1, plays a central role in the response and adaptation to osmotic and oxidative stress and is also involved in morphogenesis, cell wall biogenesis, and virulence. And Hog1 plays a repressive role in hyphal development during hyphal elongation [11] (Figure 3).

Figure 3.

Morphological switching Inhibitor, HOG pathways in Candida albicans. Arrows indicate stimulation, T-shaped lines indicate inhibition, and gray lines represent relationships that are inactive.

The HOG MAPK pathway

Hog1 participates in two distinct morphogenetic processes, as a repressor (yeast-to-hypha transition) and as an inducer (chlamydospore formation) [62]. In contrast to the Hog1 pathway in Saccharomyces cerevisiae, Cheetham et al. [63] demonstrated that Hog1 in C. albicans was regulated by a single Ssk2. And Ssk2 regulates Hog1 via activation of Pbs2. Annotation of the C. albicans genome sequence [63] revealed four putative osmosensor proteins: Chk1, Nik1 (Cos1), Sln1, and Ssu81 (Sho1). In C. albicans, the SLN1 branch is responsible for the activation of Hog1 upon oxidative stress. Sho1p activates Pbs2p via Ste11p [64]. A stimulus is perceived by the Sln1 sensor at the plasma membrane, the signal is transmitted to the MAPK central core via the Sln1-Ypd1-Ssk1 signaling branch, triggering the MAPKKK Ssk2 activation, which sequentially phosphorylates the MAPKK Pbs2 and, consequently, the MAPK Hog1 [11]. Phosphorylated Hog1 is repressive to hyphal elongation. Sln1 and Nik1 are thought as upstream of Chk1 [65]. The severely reduced hyphal formation was observed in ssk1 mutants even on serum agar [66]. After phosphorylated transiently upon osmotic stress, Hog1 translocates into the nucleus, regulates gene transcription, and mediates intracellular glycerol accumulation [67]. Hog-mediated repression of the yeast-to-hypha switch is independent of the Efg1 and Cph1 transcription factors [2].

The three mutants with the deletion of HOG1, PBS2, or SSK2 showed the strongest hyphal elongation under nutrient-rich conditions, which demonstrates the HOG pathway plays a repressive role on hyphal elongation in C. albicans. And the mutants expressed the GATA transcription factor Brg1 to induce hyphal elongation [68]. Phosphorylated Hog1 represses the expression of BRG1 indirectly but via the transcriptional repressor Sko1 [9], C. albicans with the deletion of sko1 reverts virulence [69]. Only a basal level of phosphorylated Hog1 is required for the promoter association of Sko1 to repress the hyphal transcriptional program.

Function in the TOR pathway

The target of rapamycin (TOR) protein kinase pathway is the major nutrient-sensing pathway. C. albicans has a single TOR kinase, Tor1, which is rapamycin-sensitive and a central regulator of cell growth in response to nitrogen and amino acid availability. Tor1 can be activated in a nutrient-rich medium inducing hyphae cells to convert to yeast cells [70]. Tor1 inhibits cell adhesion by repressing the expression of adhesin genes ALS1, ALS3, HWP1, and ECE1 [64]. Diminishing Tor1 activity upon rapamycin or in nutrient-limiting media activates the two Hog1 tyrosine phosphatases, Ptp2 and Ptp3, that down-regulate Hog1 basal activity. The inactivation of Hog1 is important for sustained hyphal elongation. Sko1 dissociates from the BRG1 promoter when the level of phosphorylated Hog1 is lowered (by rapamycin) or abolished (in HOG1 deletion mutant), and ultimately activate BRG1 expression, keeping Nrg1 protein off the promoters of HSGs and inducing sustained hyphal elongation [68,71]. Besides, it seems that Hog1 phosphorylation in response to stresses plays a dominant role in the regulation via Tor1 signaling [68].

Brg1 plays an importation role in hyphal elongation and BRG1 expression requires both the removal of Nrg1 and a sub-growth inhibitory level of rapamycin [71]. The Nrg1 protein, the major transcription inhibitor of hyphal development, is temporarily removed from promoters of HSGs upon activation of the cAMP/PKA pathway [21]. Nrg1 binding sites are in nucleosome-free regions in yeast cells, whereas Brg1 binding sites are occupied by nucleosomes. Nucleosome disassembly during hyphal initiation exposes the binding sites for Nrg1 and Brg1. But Brg1 recruits the histone deacetylase Hda1 to promoters of HSGs, and Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to the eviction of the NuA4 acetyltransferase module, nucleosome repositioning and flocclusion of Nrg1 binding sites that prevents Nrg1 from binding to the promoters. However, it is only when Nrg1 is gone that promoter recruitment of Hda1 for hyphal maintenance happens and Brg1 can bind to the promoters [21]. Furthermore, the hypha-specific regulator Ume6 is a key downstream target of Brg1 and it functions after Brg1 as a built-in positive feedback regulator [68].

Mds3, a regulator of pH-dependent morphogenesis, functions in parallel with the classic Rim101 pH-sensing pathway. As Mds3 Delta/Delta cells have an expression profile indicative of a hyperactive TOR pathway, and the transcriptional and morphological defects of the mutant are rescued by rapamycin, Mds3 is believed to be a new element of the TOR pathway that contributes to morphogenesis in C. albicans [72]. Sit4 and Mds3 may function similarly in the TOR pathway due to their coimmunoprecipitation.

Function in the Cek1 pathway

In C. albicans, there are four MAPKs identified that play important roles in cell physiology, including Mkc1, Cek1, Cek2, and Hog1. The Cek1-mediated pathway was discovered that implicated in mating, invasive hyphal growth, cell wall formation, virulence, biofilm formation, quorum sensing, and so on. It was reported that Cek1 regulates the β-glucan exposure on the cell wall [64]. The C. albicans SHO1 branch is involved in the activation of Cek1. The Sho1 adaptor protein links oxidative stress to cell wall biogenesis and morphogenesis, and Shol plays a minor role in response to oxidative stress, which mainly occurs through a putative Sln1-Ssk1 branch of the HOG pathway [73]. Msb2, a mucin-like protein, mediates activation of Cek1 mitogen-activated protein kinase and controls the cell wall construction [74]. Cst20, a protein kinase of the PAK family, belongs to the Cst20-Hst7-Cek1-Cph1 MAPK pathway [75]. The transmembrane proteins Sho1, Msb2, and Opy2 sense external signals [11] and may form a protein complex that may interact with Cdc42, Cst20 and Ste50, transmitting signals to Cek1-mediated pathway and triggering Cek1 phosphorylation. In Addition, Sho1 and Opy2 may play a major role in the formation of the complex, which may be necessary to recruit all the elements required to polarization/depolarization of the cytoskeleton and other associated structures, such as the septin ring, under osmotic stress [67]. Hog1 represses the activation of the Cek1 MAPK under basal conditions [76], and hog1, pbs2, or ssk2 mutants display enhanced hyphal formation and hyperphosphorylation of Cek1 MAPK, which demonstrate an intrinsic connection between the HOG and the Cek1-mediated pathways [11].

Other signal components and functions

The endosomal sorting complex required for transport (ESCRT) system is involved in hyphal induction in a neutral-alkaline environment via the Rim101 pathway, and ESCRT protein Vps4 is essential for polarity maintenance during hyphal formation and the proper localization of Cdc42 and Cdc3. Deletions of the ESCRT proteins cause delayed germination, a decreased recovery rate of GFP-Ras1, and downregulation of HSGs such as HGC1 [77]. Mu C et al. [78] identified phosphatidate phosphatase Pah1 as a crucial role in hyphal growth and virulence of C. albicans, as the pah1Δ/Δ mutants display hyphal defects due to the reduced expression of UME6.

The development of novel antimicrobial agents

Farnesol could be used as an adjuvant, potential antimicrobial agent to contribute to the prevention of candidiasis and caries with great therapeutic potential. Farnesol also can be found in essential oils [49]. Chitosan (CS) nanogel could be a useful nanocarrier for the pharmaceutical application of farnesol with little cytotoxicity. And CS nanogel containing farnesol decreased the expression of HWP1 and SAP6 genes in C. albicans [79]. Biosurfactant AC7, a lipopeptide from Bacillus subtilis, coats material surfaces in combination with farnesol, contributing to the inhibition of initial adhesion and biofilm growth of C. albicans and the prevention of medical device-associated infection [80]. Farnesol isomers, sources of separation, and preparation methods influence compound pharmacological properties and/or toxicological effects directly, which is one of the limitations for future pharmaceuticals development [49].

Farnesol might alter the drug resistance of the resistant C. albicans and other microbial species by inhibiting biofilm formation and regulating drug transportation. Farnesol in combination with fluconazole (FLC) decreases the minimal inhibitory concentrations (MIC) of FLC [81] by reducing the efflux activity of both Cdr1 and Cdr2 pumps, the ATP-binding cassette efflux transporters [82], which is dependent on farnesol concentrations and changes the resistance to azoles in the FLC-resistant C. albicans. Interestingly, farnesol induced CDR1 expression by activating the Zn cluster transcription factors Tac1 and Znc1 [83]. Moreover, it was observed that 200 μmol/L farnesol downregulated the expression of the ergosterol genes ERG20 and ERG11 [84]. Farnesol showed synergistic effects with fluconazole or 5-flurocytosine against biofilms, but antagonistic effects with terbinafine and itraconazole [85]. The combination of farnesol and carvacrol displayed synergistic inhibitory effects against amphotericin-B-resistant C. albicans in single- and mixed-species biofilms [86].

Monteiro [87] and Sebaa [88] suggested farnesol and tyrosol might as novel adjuvants in oral hygiene contributing to the prevention of dental caries and candidiasis with an antibiofilm effect on S. mutans and Candida species. 200 mM tyrosol can cause cell damage and significantly reduce metabolic activity and biofilms formation [89]. Barot [90] fabricated flowable resin composites incorporated with farnesol loaded halloysite nanotubes (Fa-HNTs) successfully without cytotoxicity on mouse embryonic fibroblast cells, and the composite with 7-13 wt% Fa-HNT showed promising physicochemical properties and significant antimicrobial activity, might beneficial for preventing secondary caries. However, long-term antibacterial properties still need further study. Codelivery of farnesol and ciprofloxacin inhibits biofilms of resistant Pseudomonas aeruginosa at significantly lower ciprofloxacin concentration in vitro [91].

Kalopanaxsaponin A [92], trans-cinnamaldehyde [93], Eucarobustol E [94], Biatriosporin D [95], Shikonin [96] upregulate the expression of Dpp3 to promote farnesol secretion, but further experiments in vivo are required. Zerumbone, a monocyclic sesquiterpene derived from Zingiber zerumbet (L.) Smith, decreases hyphal growth and suppresses biofilm formation by significantly downregulating the expression of biofilm-related genes and HSGs, including HWP1 and ALS3 [97]. Moreover, zerumbone remarkably inhibits the dual-species biofilms of C. albicans and S. aureus [98].

Further explorations on the regulation of farnesol synthesis, potential receptor, and transporters on C. albicans are required, beneficial for developing new antifungal drugs that promote more farnesol synthesis, increase the farnesol intercellular concentration and enhance the antifungal effects. The involved molecules above may be potential and effective drug targets of antifungal treatment. The combination of conventional therapeutic agents with new adjuvants such as farnesol might provide effective anti-infective treatment strategies, especially against the multidrug-resistant microorganisms. However, most experiments are only limited to in vitro, and further studies in vivo and clinical are required to evaluate the efficacy and safety.

Conclusions

Farnesol inhibits NRG1 and TUP1 transcriptional down-regulation through the Ras1-Cyr1/cAMP-PKA pathway and the Sok1-mediated degradation of Nrg1 protein to repress hyphal initiation, and inhibits hyphal elongation through Cek1-MAPK pathway. The HOG MAPK pathway in C. albicans is not only an osmosensing pathway, but also involved in regulating cell wall biogenesis and morphogenesis, playing a repressive role on filamentation. The activated Hog1 is also regulated by TOR pathway and inhibits Cek1 function (Figure 4). Multiple and intricate signal transduction pathways in C. albicans interact and coordinate with each other under different conditions, which are relative to its pathogenesis. Although the network has never been annotated so exhaustively, researchers have identified more signal molecules and pathways involved in morphogenesis and get more knowledgeable about the network, extraordinarily how morphologic switching and virulence or difference are regulated in or between vivo and vitro. An increased understanding of the molecular mechanism of morphologic inhibitors benefits research on pathological mechanisms and development of clinical therapy and novel antifungals.

Figure 4.

Morphological switching inhibitor farnesol and HOG pathways in Candida albicans. Farnesol inhibits hyphal growth by repressing NRG1 and TUP1 transcriptional down-regulation through the Ras1-Cyr1/cAMP-PKA pathway, by blocking the Cup9 degradation to inhibit the Sok1-mediated degradation of Nrg1 and by regulating Cek1-MAPK pathway. The HOG pathway plays a repressive role on filamentation and is also regulated by TOR pathway and inhibits Cek1 function. Arrows indicate stimulation, T-shaped lines indicate inhibition, and gray lines represent relationships that are inactive.

Disclosure of conflict of interest

None.

Abbreviations

C. albicans

Candida albicans

cAMP

cyclic adenosine monophosphate

ChIP

chromatin immunoprecipitation

CPH1

Candida Pseudo Hyphal regulator

CS

Chitosan

Czf1

C. albicans zinc finger 1

DC

dendritic cells

ECM

the extracellular matrix

EED1

Epithelial Escape and Dissemination 1

ESCRT

the endosomal sorting complex required for transport

Fa-HNTs

farnesol loaded halloysite nanotubes

FLC

fluconazole

HAGs

hyphal-associated genes

HOG

high-osmolarity and glycerol

HSGs

hypha-specific genes

iDC

immature dendritic cells

LRR

leucine-rich repeat

MAPK

mitogen-activated protein kinase

MAPKK

mitogen-activated protein kinase kinase

MAPKKK

mitogen-activated protein kinase kinase kinase

MIC

the minimal inhibitory concentrations

MIP-1α

macrophage inflammatory protein 1 alpha

mRNA

messenger Ribonucleic Acid

NETs

the neutrophil extracellular traps

PAMPs

pathogen-associated molecular patterns

PKA

protein kinase A

QSM

quorum sensing molecule

ROS

reactive oxygen species

STAT3

the signal transducer transcriptional activator 3

TNF-α

tumor necrosis factor alpha

TOR

target of rapamycin

ZO-1

the Zonula Occludens-1