Introduction. Biofilms are the natural growth state for most microorganisms. C. albicans biofilms are composed of multiple cell types (round budding yeast-form cells, oval pseudohyphal cells, and elongated hyphal cells) encased in an extracellular matrix. C. albicans biofilms are notorious for resistance to antimicrobial treatments, a property that might be determined by complex mechanisms. Exogenous farnesol exerts a certain antifungal activity against C. albicans with medical implications. Different from other microbes, C. albicans biofilms can tolerate exogenous farnesol at high concentration with some cells still surviving and even maintaining proliferation, but the mechanism is unclear.
Hypothesis. The study hypothesizes that C. albicans resists farnesol by activating the PKC signalling pathway.
Aim. The study aims to discuss the molecular mechanism of C. albicans resistance to farnesol.
Methodology. The ROS levels, the genes and proteins of the PKC pathway were compared between the farnesol-tolerant and non-tolerant groups using ROS levels assay, q-RT PCR and Western blot, respectively. Further, the mutant strains (pkc1Δ/Δ and mkc1Δ/Δ) were constructed, then the survival rates and ROS levels of biofilms exposed to farnesol were compared between mutant and wild strains. The morphological changes were observed using TEM.
Results. The survival rates of C. albicans biofilms decreased rapidly under the lower concentration of farnesol (P<0.05), and kept stable (P>0.05) as the concentration rose up to 200 µM. The gene expression of the PKC pathway increased, while ROS levels remained stable and even decreased in the farnesol-tolerant biofilms, compared with those in the farnesol-nontolerant biofilms after farnesol treatment (P<0.05); pkc1 and mkc1 were significantly upregulated by C. albicans during the development of biofilm tolerance to farnesol. The cell wall and cytoplasm of pkc1Δ/Δ and mkc1Δ/Δ were damaged, and the ROS level increased (P<0.05); meanwhile, the survival rate of biofilms decreased compared with that of wild-type strain under the same farnesol concentrations (P<0.05). ROS inhibitors reversed these changes in pkc1Δ/Δ and mkc1Δ/Δ when the mutant strains exposed to farnesol.
Conclusion.C. albicans biofilms can tolerate high concentrations of farnesol by activating pkc1 and mkc1 of the PKC pathway and stabilizing ROS levels. The pkc1 and mkc1 are two key genes regulated by C. albicans in the process of biofilm tolerance to farnesol.
KullbergBJ, VasquezJ, MootsikapunP, NucciM, PaivaJ-A et al. Efficacy of anidulafungin in 539 patients with invasive candidiasis: a patient-level pooled analysis of six clinical trials. J Antimicrob Chemother2017; 72:2368–2377 [View Article] [PubMed]
YuL-H, WeiX, MaM, ChenX-J, XuS-B. Possible inhibitory molecular mechanism of farnesol on the development of fluconazole resistance in Candida albicans biofilm. Antimicrob Agents Chemother2012; 56:770–775 [View Article] [PubMed]
EgbeNE, DornellesTO, PagetCM, CastelliLM, AsheMP. Farnesol inhibits translation to limit growth and filamentation in C. albicans and S. cerevisiae. Microb Cell2017; 4:294–304 [View Article] [PubMed]
HasimS, VaughnEN, DonohoeD, GordonDM, PfiffnerS et al. Influence of phosphatidylserine and phosphatidylethanolamine on farnesol tolerance in Candida albicans. Yeast2018; 35:343–351 [View Article] [PubMed]
XiaJ, QianF, XuW, ZhangZ, WeiX. In vitro inhibitory effects of farnesol and interactions between farnesol and antifungals against biofilms of Candida albicans resistant strains. Biofouling2017; 33:283–293 [View Article] [PubMed]
da Silva DantasA, DayA, IkehM, KosI, AchanB et al. Oxidative stress responses in the human fungal pathogen, Candida albicans. Biomolecules2015; 5:142–165 [View Article] [PubMed]
LaFayetteSL, CollinsC, ZaasAK, SchellWA, Betancourt-QuirozM et al. PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90. PLoS Pathog2010; 6:e1001069 [View Article] [PubMed]
Navarro-GarcíaF, EismanB, FiuzaSM, NombelaC, PlaJ. The MAP kinase Mkc1p is activated under different stress conditions in Candida albicans. Microbiology (Reading)2005; 151:2737–2749 [View Article] [PubMed]
MachidaK, TanakaT, FujitaK, TaniguchiM. Farnesol-induced generation of reactive oxygen species via indirect inhibition of the mitochondrial electron transport chain in the yeast Saccharomyces cerevisiae. J Bacteriol1998; 180:4460–4465 [View Article] [PubMed]
FairnGD, MacDonaldK, McMasterCR. A chemogenomic screen in Saccharomyces cerevisiae uncovers a primary role for the mitochondria in farnesol toxicity and its regulation by the Pkc1 pathway. J Biol Chem2007; 282:4868–4874 [View Article] [PubMed]
PierceCG, UppuluriP, TummalaS, Lopez-RibotJL. A 96 well microtiter plate-based method for monitoring formation and antifungal susceptibility testing of Candida albicans biofilms. JoVE201044 [View Article]
KuhnDM, BalkisM, ChandraJ, MukherjeePK, GhannoumMA. Uses and limitations of the XTT assay in studies of Candida growth and metabolism. J Clin Microbiol2003; 41:506–508 [View Article] [PubMed]
PumeesatP, MuangkaewW, AmpawongS, LuplertlopN. Candida albicans biofilm development under increased temperature. New Microbiol2017; 40:279–283 [PubMed]
TaskovaRM, ZornH, KringsU, BouwsH, BergerRG. A comparison of cell wall disruption techniques for the isolation of intracellular metabolites from Pleurotus and Lepista sp. Z Naturforsch C J Biosci2006; 61:347–350 [View Article] [PubMed]
JaneczkoM. Emodin reduces the activity of (1,3)-β-D-glucan Synthase from Candida albicans and does not interact with Caspofungin. Pol J Microbiol2018; 67:463–470 [View Article] [PubMed]
ZengX, YeG, TangW, OuyangT, TianL et al. Fungicidal efficiency of electrolyzed oxidizing water on Candida albicans and its biochemical mechanism. J Biosci Bioeng2011; 112:86–91 [View Article] [PubMed]
ChenS, XiaJ, LiC, ZuoL, WeiX. The possible molecular mechanisms of farnesol on the antifungal resistance of C. albicans biofilms: the regulation of CYR1 and PDE2. BMC Microbiol2018; 18:203. [View Article] [PubMed]
FuchsBB, MylonakisE. Our paths might cross: the role of the fungal cell wall integrity pathway in stress response and cross talk with other stress response pathways. Eukaryot Cell2009; 8:1616–1625 [View Article] [PubMed]
CápM, VáchováL, PalkováZ. Reactive oxygen species in the signaling and adaptation of multicellular microbial communities. Oxid Med Cell Longev2012; 2012:976753. [View Article] [PubMed]
FrostDJ, BrandtKD, CugierD, GoldmanR. A whole-cell Candida albicans assay for the detection of inhibitors towards fungal cell wall synthesis and assembly. J Antibiot1995; 48:306–310 [View Article]
RománE, Alonso-MongeR, GongQ, LiD, CalderoneR et al. The Cek1 MAPK is a short-lived protein regulated by quorum sensing in the fungal pathogen Candida albicans. FEMS Yeast Res2009; 9:942–955 [View Article] [PubMed]
NobleSM, JohnsonAD. Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell2005; 4:298–309 [View Article] [PubMed]