Physiological and Transcriptional Responses of Candida parapsilosis to Exogenous Tyrosol

Abstract

Tyrosol plays a key role in fungal morphogenesis and biofilm development. Also, it has a remarkable antifungal effect at supraphysiological concentrations. However, the background of the antifungal effect remains unknown, especially in the case of non-albicans Candida species such as Candida parapsilosis We examined the effect of tyrosol on growth, adhesion, redox homeostasis, virulence, as well as fluconazole susceptibility. To gain further insights into the physiological consequences of tyrosol treatment, we also determined genome-wide gene expression changes using transcriptome sequencing (RNA-Seq). A concentration of 15 mM tyrosol caused significant growth inhibition within 2 h of the addition of tyrosol, while the adhesion of yeast cells was not affected. Tyrosol increased the production of reactive oxygen species remarkably, as revealed by a dichlorofluorescein test, and it was associated with elevated superoxide dismutase, glutathione peroxidase, and catalase activities. The interaction between fluconazole and tyrosol was antagonistic. Tyrosol exposure resulted in 261 and 181 differentially expressed genes with at least a 1.5-fold increase or decrease in expression, respectively, which were selected for further study. Genes involved in ribosome biogenesis showed downregulation, while genes related to the oxidative stress response and ethanol fermentation were upregulated. In addition, tyrosol treatment upregulated the expression of efflux pump genes, including MDR1 and CDR1, and downregulated the expression of the FAD2 and FAD3 virulence genes involved in desaturated fatty acid formation. Our data demonstrate that exogenous tyrosol significantly affects the physiology and gene expression of C. parapsilosis, which could contribute to the development of treatments targeting quorum sensing in the future.IMPORTANCECandida-secreted quorum-sensing molecules (i.e., farnesol and tyrosol) are key regulators in fungal physiology, which induce phenotypic adaptations, including morphological changes, altered biofilm formation, and synchronized expression of virulence factors. Moreover, they have a remarkable antifungal activity at supraphysiological concentrations. Limited data are available concerning the tyrosol-induced molecular and physiological effects on non-albicans Candida species such as C. parapsilosis In addition, the background of the previously observed antifungal effect caused by tyrosol remains unknown. This study reveals that tyrosol exposure enhanced the oxidative stress response and the expression of efflux pump genes, while it inhibited growth and ribosome biogenesis as well as several virulence-related genes. Metabolism was changed toward glycolysis and ethanol fermentation. Furthermore, the initial adherence was not influenced significantly in the presence of tyrosol. Our results provide several potential explanations for the previously observed antifungal effect.

Keywords: Candida parapsilosis; RNA-Seq; oxidative stress; quorum sensing; tyrosol.

FIG 1

Effect of tyrosol on growth of C. parapsilosis. Growth of C. parapsilosis CLIB 214 in YPD medium was monitored by measurement of the absorbance (OD₆₄₀). Tyrosol was added at a 4-h incubation time at a 15 mM final concentration. Data represent mean values ± SD calculated from 10 independent experiments. The asterisk indicates a significant difference between control and tyrosol-treated cultures calculated by paired Student’s t test.

FIG 2

Results of in vivo experiments. Shown are kidney tissue burdens of permanently neutropenic BALB/c mice infected intravenously with C. parapsilosis strain CLIB 214. Daily intraperitoneal tyrosol (15 mM) treatment was started at 24 h postinoculation. Fungal kidney tissue burden was determined at the end of the experiments on day 6. Bars represent means ± SD. The level of statistical significance compared with the untreated control group on day 6 is indicated (**, P < 0.01).

FIG 3

Correlation between RT-qPCR and transcriptome data. RNA-Seq data are presented as log₂(FC) values, where FC is “fold change.” Relative transcription levels were quantified as ΔΔCP = ΔCP_control − ΔCP_treated, where ΔCP_treated = CP_{tested gene} − CP_{reference gene}, measured from treated cultures, and ΔCP_control = CP_{tested gene} − CP_{reference gene}, measured from control cultures. CP values represent the qRT-PCR cycle numbers of crossing points. The ACT1 gene was used as a reference gene. ΔΔCP values significantly (P < 0.05 by Student’s t test; n = 3) higher or lower than zero (up- or downregulated genes) are marked in red and blue, respectively. Pearson’s correlation coefficient between the RT-qPCR and RNA-seq values was 0.88.

FIG 4

Genome-wide transcriptional changes induced by tyrosol in C. parapsilosis. Upregulated (red) and downregulated (dark blue) genes were defined as differentially expressed genes (corrected P value of <0.05), where the log₂(FC) was greater than 0.585 or less than −0.585, respectively, and FC is the fold change of FPKM values (tyrosol treated versus untreated). On the sides of the volcano plot are representative genes upregulated or downregulated by tyrosol treatment.