Comparative transcriptional analysis of Candida auris biofilms following farnesol and tyrosol treatment

Abstract

Candida auris is frequently associated with biofilm-related invasive infections. The resistant profile of these biofilms necessitates innovative therapeutic options, where quorum sensing may be a potential target. Farnesol and tyrosol are two fungal quorum-sensing molecules with antifungal effects at supraphysiological concentrations. Here, we performed genome-wide transcript profiling with C. auris biofilms following farnesol or tyrosol exposure using transcriptome sequencing (RNA-Seq). Since transition metals play a central role in fungal virulence and biofilm formation, levels of intracellular calcium, magnesium, and iron were determined following farnesol or tyrosol treatment using inductively coupled plasma optical emission spectrometry. Farnesol caused an 89.9% and 73.8% significant reduction in the calcium and magnesium content, respectively, whereas tyrosol resulted in 82.6%, 76.6%, and 81.2% decrease in the calcium, magnesium, and iron content, respectively, compared to the control. Genes involved in biofilm events, glycolysis, ergosterol biosynthesis, fatty acid oxidation, iron metabolism, and autophagy were primarily affected in treated cells. To prove ergosterol quorum-sensing molecule interactions, microdilution-based susceptibility testing was performed, where the complexation of farnesol, but not tyrosol, with ergosterol was impeded in the presence of exogenous ergosterol, resulting in a minimum inhibitory concentration increase in the quorum-sensing molecules. This study revealed several farnesol- and tyrosol-specific responses, which will contribute to the development of alternative therapies against C. auris biofilms.

Importance: Candida auris is a multidrug-resistant fungal pathogen, which is frequently associated with biofilm-related infections. Candida-derived quorum-sensing molecules (farnesol and tyrosol) play a pivotal role in the regulation of fungal morphogenesis and biofilm development. Furthermore, they may have remarkable anti-biofilm effects, especially at supraphysiological concentrations. Innovative therapeutic approaches interfering with quorum sensing may be a promising future strategy against C. auris biofilms; however, limited data are currently available concerning farnesol-induced and tyrosol-related molecular effects in C. auris. Here, we detected several genes involved in biofilm events, glycolysis, ergosterol biosynthesis, fatty acid oxidation, iron metabolism, and autophagy, which were primarily influenced following farnesol or tyrosol exposure. Moreover, calcium, magnesium, and iron homeostasis were also significantly affected. These results reveal those molecular and physiological events, which may support the development of novel therapeutic approaches against C. auris biofilms.

Keywords: Candida auris; biofilm; calcium; ergosterol; iron; magnesium; quorum-sensing; transcriptome.

Fig 1

Summary of RNA-Seq data and main gene enrichment analyses. (A) The effects of tyrosol (Tyr vs Cont) and farnesol (Farn vs Cont) treatment on the transcriptomes are depicted in the Venn diagrams. (B–D) Bubble charts of GO terms of CGD (http://www.candidagenome.org/cgi-bin/GO/goTermFinder) and results of the Fisher’s exact test generated by different expression genes. Bubble charts represent up- (Δ) and downregulated (•) genes belonging to gene groups farnesol-treated vs untreated (B), tyrosol-treated vs untreated (C), and farnesol- vs tyrosol-treated (D) comparisons where the enrichment was significant (P < 0.05). The color of the bubble means the significance of the corresponding pathway (in green color, low P values; in red color, high P values). In addition, the size of the bubble means the number of different expression genes in this pathway. Only the differentially expressed genes (corrected P value of <0.05) exhibiting more than a 1.5-fold increase or decrease in their transcription are shown. The full list of the significantly enriched GO terms is available in Table S2. Significant enrichment in the appropriate gene set according to Fisher’s exact test (P < 0.05). The full data set is available in Table S3.

Fig 2

The effects of quorum-sensing compounds, farnesol and tyrosol, on the expression of selected genes of Candida auris biofilms. The heat map demonstrates the expression profiles of representative genes according to the color scale that indicates gene expression changes in fold change units. Table S3 summarizes the data that were used for the construction of the heat map. Bold names indicate the genes that were selected for the RT-qPCR analysis. The data set for genes is available in Table S4 in the supplemental material.