Fungal quorum sensing molecules: Role in fungal morphogenesis and pathogenicity

Quorum sensing in fungal morphogenesis

An effect of inoculum size on morphogenesis has been observed for several dimorphic fungi 7. Budding yeast cells were formed at cell densities ≥10⁶ cells/ml and mycelia were produced at densities <10⁶ cells/ml. This phenomenon is controlled by QSMs 2, 23. Farnesol, the first QSM identified in a eukaryotic microorganism, was discovered in C. albicans 8. Farnesol inhibits yeast-to-hypha switching but it cannot block the elongation of preexisting hyphae 7. However, Lindsay et al. 8 showed that farnesol could promote the hypha-to-yeast transition as well. Moreover, pseudohyphae and hyphae development were inhibited by farnesol in C. dublinensis 9. Kebaara et al. 4 showed that Tup1, a transcriptional repressor of hypha-specific genes, was increased both at the TUP1 mRNA and protein levels after treatment of C. albicans cells with farnesol. Hyphal growth is regulated through several signaling pathways, in particular the cAMP pathway 24-27. Farnesol blocked hyphal formation by inhibiting the cAMP signaling pathway through inhibition of the activity of Candida adenylyl cyclase, Cyr1p 28. Additionally, Sato et al. 29 observed that the mitogen-activated protein (MAP) kinase cascade was inhibited in farnesol-treated C. albicans cells. Moreover, C. albicans ATCC 10231 released farnesoic acid into the culture medium, which also inhibited filamentous growth 30. Farnesol also had an effect on the conidiation, germination, and hyphal morphology of several fungi 31, 32. In Aspergillus niger, high concentrations of farnesol inhibited conidiation and reduced intracellular cAMP levels, but did not inhibit aerial hyphae production 31. In addition, the development and germination of macroconidia of Fusarium graminearum, a plant fungal pathogen, were impaired by farnesol treatment 12. The amount of ergosterol decreased in farnesol-treated cells and affected the integrity of the fungal plasma membrane in Coccidioides posadasii 33. Moreover, Derengowski et al. 34 showed that the fungicidal properties of farnesol were associated with the degradation of cytoplasmic organelles but not cell walls or plasma membrane. Farnesol also affects C. albicans metabolic pathways, including several pathways associated with morphogenesis and pathways associated with other functions 35.

Tyrosol is another quorum-sensing molecule secreted by C. albicans, and is continuously released into the culture medium during growth, playing a role in the promotion of germ tube formation 5. Additionally, several alcohols have been found in planktonic cells and biofilm supernatants from C. albicans and C. dubliniensis. These include isoamyl alcohol, 2-phenylethanol, 1-dodecanol, E-nerolidol, and E,E-farnesol, which are species, culture mode and growth time specific, and play a role in morphogenesis 36. Additionally, the production of autosignaling alcohols, tryptophol, and phenylethylalcohol, which promote pseudohyphal growth in Saccharomyces cerevisiae, was regulated by nitrogen availability and cell density and could not stimulate filamentation in C. albicans; this indicates that these signals are species-specific 37. Moreover, several alcohols were able to inhibit yeast-to-hypha switching of C. albicans in a concentration-dependent manner 38. Phenylethanol and tyrosol were also detected in Debaryomyces hansenii depending on the cell density and environmental conditions 39.

Effect of quorum sensing in fungal biofilm development

Biofilms are well-structured microbial communities attaching to biotic or abiotic surfaces surrounded by extracellular polymeric substance (EPS) 40, 41. Several fungi have been reported to form biofilms, especially on medical devices. This leads to an increase in infection and mortality because biofilms are more resistant than planktonic cells to many antifungal drugs 42. Fungal biofilm formation has been widely studied in C. albicans 41, 43-47. During biofilm development of C. albicans, yeast cells play a role in the initial attachment to the surface; this is followed by formation of filaments, and finally a mature biofilm forms, comprising yeast, true hyphae, pseudohyphae, and EPS 45-47. This implies that morphology plays a role in biofilm development. QSMs also control the formation of C. albicans biofilms. Farnesol inhibited biofilm development concomitant with a decrease in HWP1 (encoding hypha-specific wall protein) expression, reflecting the morphological characteristics of the cells 48. Moreover, supernatant recovered from biofilms can inhibit hyphal formation of C. albicans planktonic cells, which demonstrate the presence of autoregulatory molecules produced by biofilms 48. Under anaerobic conditions, farnesol is not produced 29, but other alcohol-based autoregulating substances produced in these conditions can promote the dissemination of yeast cells from the biofilm, thereby inhibiting biofilm development 38. The attachment of fungal cells on surfaces is the first step of biofilm development and QSMs play an important role in this. Phenylethanol and tyrosol increased the ability of D. hansenii to attach to polystyrene while tryptophol and farnesol reduced this ability and also influenced the sliding motility of this organism 39, demonstrating the influence these molecules have on biofilm formation.

QSMs regulate defense against fungal invasion during infection

QSMs play a role in the defense against fungal invasion. QSMs can induce oxidative stress in macrophages. Effector activity of macrophages against C. albicans was reduced in farnesol-treated murine macrophages and farnesol also induced cell death and apoptosis via intracellular reactive oxygen species (ROS) production 15. Joo et al. mentioned that apoptosis in lung carcinoma cells was induced by farnesol in cooperation with endoplasmic reticulum (ER) stress 49. They also reported that farnesol induced inflammatory genes through the stimulation of the NF-kB pathway, including MEK1/2-ERK1/2-MSK1-dependent phosphorylation of p65/RelA(Ser²⁷⁶) 50, 51. In in vitro studies, farnesol is able to protect C. albicans from ROS 52. Deveau et al. proposed that farnesol increased ROS resistance in C. albicans through suppression of the Ras1-cAMP pathway 51, 53. Other QSMs have also been shown to have an effect on immune cells: tyrosol altered neutrophilic killing by inhibiting the respiratory burst 54. Thus, these experiments support the suggestion that QSMs play an important role in virulence in fungal infection.

Ghosh et al. explained that wild-type C. albicans can escape from macrophages within 6 h via stimulation of arginine biosynthesis, which induces yeast-to-hypha formation 16. They also evaluated cytokine production in the murine RAW264.7 macrophage cell line and showed that the expression of IL-6 was decreased in farnesol-treated cells 17. Therefore, farnesol plays a key role as a virulence factor of fungal infection. Moreover, in a mouse model of Candida infection, farnesol also inhibited IFN-γ and IL-12 (Th1 cytokines), which are produced by macrophages to promote systemic candidiasis immunity 18. Navarathna et al. found that pathogenicity of systemic candidiasis was reduced when endogenous farnesol was reduced. This was due to the deficiency of a phosphatase-encoding gene, which prevented the conversion of farnesyl pyrophosphate to farnesol 55. Additionally, Leonhardt et al. addressed another characteristic of farnesol in C. albicans virulence. Farnesol activated innate immune cells, including neutrophils and monocytes, leading to enhanced inflammation, but inhibited the differentiation of monocytes into immature dendritic cells 56.

Farnesol induces apoptosis in yeasts and filamentous fungi

In addition to its effects on immune cells, exogenous farnesol has been shown to block the yeast-to-hypha transition and biofilm development at the premature stages in a concentration-dependent manner (high concentrations) 48, 57, 58. Jabra-Rizk et al. showed that high concentrations of farnesol also induce cell death in C. albicans 57. As a result, farnesol was able to induce apoptosis in several fungi. In C. albicans, Shirtliff et al. used a proteomic approach coupled with detection of apoptosis markers on farnesol-treated cells. They found that apoptosis was induced via caspase stimulation 13. Additionally, Léger et al. investigated the role of the metacaspase Mca1p in the mechanism of farnesol-induced apoptosis. Mca1p was involved in the apoptosis pathways of both mca1 disruption and farnesol-treated cells 14. Furthermore, farnesol may interact at another point in the programmed cell death pathway by altering the levels of intracellular glutathione S-conjugate (F-GS) combined with transporter protein (Cdr1-p). This results in the decrease of intracellular reduced glutathione (GSH), leading to the activation of apoptosis pathways and fungal cell death 59.

A number of studies have investigated the mechanisms of farnesol treatment of other filamentous fungi. Similar to in C. albicans, farnesol induced apoptosis in A. nidulans 10, A. flavus 11, P. expansum 32, F. graminerum 12, Scedosporium boydii (unpublished TW, PP, NL), and Lomentospora prolificans (unpublished TW, PP, NL). In A. nidulans, Semighini et al. demonstrated that apoptosis was activated through ROS and the FadA heterotrimeric G protein complex 10. Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase and NADH-ubiquinone oxidoreductases (NdeA-B and NdiA) have both been implicated in compensatory pathways of farnesol-induced ROS and apoptosis 60, 61. Furthermore, Wang et al. detected apoptosis markers (nuclear condensation, phosphatidylserine, externalization, DNA fragmentation, ROS, metacaspase activation, and cellular ultrastructure changes) in A. flavus treated with farnesol 11. There is only one study in a fungal plant pathogen, F. graminerum, where farnesol also stimulated apoptosis 12. In our study (data not shown, submitted), we found that farnesol-treated cells of S. boydii and L. prolificans led to the condensation of chromatin and orange staining of cells by ethidium bromide/acridine orange, which indicated apoptosis. Thus, farnesol may activate the apoptosis pathway in both S. boydii and L. prolificans; however, more work needs to be done to determine the precise mechanism by which this is achieved.

QSMs as antifungal agents

QSMs have been investigated as antifungal agents. Shama et al. showed that farnesol could modulate drug extrusion, mediated by ABC transporters such as CaCdr1p and CaMdr1p, without affecting the major facilitator superfamily (MFS) transporters such as CaMdr1p. This synergistic effect was also observed with farnesol and antifungal drugs (azoles and polyenes) in C. albicans 62. Therefore, a nontoxic concentration of farnesol may be used as an alternative anti-C. albicans. As in Candida species, when farnesol was combined with antifungal agents, the MIC was decreased compared with antifungal agents alone 20. A number of studies have demonstrated the requirement for a high MIC for treatment of C. albicans biofilms (reviewed in Taff et al. 63 and Chandra et al. 64). Katragkou et al. showed a synergistic effect of farnesol combined with a number of antifungal agents (fluconazole, amphotericin B, and micafungin) against C. albicans biofilms 21. Sterol biosynthesis mechanisms may be involved in reducing fluconazole resistance in farnesol-treated C. albicans biofilms by repressing the ERG11, ERG25, ERG6, ERG3, and ERG1 genes regulating ergosterol biosynthesis 65. Similar to farnesol, tyrosol was investigated in combination with antifungal agents and showed a synergistic effect when combined with amphotericin B (90% reduction in C. krusei biofilm and C. tropicalis biofilm when 80 µM tyrosol was coupled with 4 mg/L amphotericin B) 22. Cordeiro et al. reported that exogenous tyrosol alone or combined with amphotericin B inhibited planktonic cells and Candida biofilm growth. Interestingly, mature biofilms were also inhibited by tyrosol alone or combined with amphotericin B, but tyrosol combined with azoles increased the biofilm activity in a dose-dependent manner 19. In dimorphic fungi, Derengowski et al. showed the in vitro activity of farnesol against P. brasiliensis (an average MIC around 25 µM and minimal lethal concentration around 30 µM) 34. The combination of farnesol with antifungal drugs showed synergistic effects (fractional inhibitory concentration index [FICI] ≤0.5) in C. posadasii 33 and H. capsulatum 66.