Transcriptional responses of Candida glabrata biofilm cells to fluconazole are modulated by the carbon source

Abstract

Candida glabrata is an important human fungal pathogen known to trigger serious infections in immune-compromised individuals. Its ability to form biofilms, which exhibit high tolerance to antifungal treatments, has been considered as an important virulence factor. However, the mechanisms involving antifungal resistance in biofilms and the impact of host niche environments on these processes are still poorly defined. In this study, we performed a whole-transcriptome analysis of C. glabrata biofilm cells exposed to different environmental conditions and constraints in order to identify the molecular pathways involved in fluconazole resistance and understand how acidic pH niches, associated with the presence of acetic acid, are able to modulate these responses. We show that fluconazole treatment induces gene expression reprogramming in a carbon source and pH-dependent manner. This is particularly relevant for a set of genes involved in DNA replication, ergosterol, and ubiquinone biosynthesis. We also provide additional evidence that the loss of mitochondrial function is associated with fluconazole resistance, independently of the growth condition. Lastly, we propose that C. glabrata Mge1, a cochaperone involved in iron metabolism and protein import into the mitochondria, is a key regulator of fluconazole susceptibility during carbon and pH adaptation by reducing the metabolic flux towards toxic sterol formation. These new findings suggest that different host microenvironments influence directly the physiology of C. glabrata, with implications on how this pathogen responds to antifungal treatment. Our analyses identify several pathways that can be targeted and will potentially prove to be useful for developing new antifungals to treat biofilm-based infections.

Keywords: Antimicrobials; Biofilms; Cellular microbiology; Next-generation sequencing; Pathogens.

Fig. 1. Global transcriptional response of C. glabrata biofilm cells to fluconazole treatment when grown in the presence of glucose (RPMI + fluconazole versus RPMI) or in the presence of both glucose and acetate (RPMI + acetate + fluconazole versus RPMI + acetate).

a Heatmap of all genes differentially expressed (p < 0.05) in the presence versus the absence of fluconazole in both growth conditions. b Venn diagrams of downregulated (decrease of twofold or lower) and upregulated (greater than twofold) genes in C. glabrata biofilm cells due to fluconazole treatment in both growth conditions.

Fig. 2. Transcriptional response of C. glabrata biofilm cells to fluconazole when grown in the presence of acetate.

Network visualization of enriched pathways with the systematic names of C. glabrata (downregulated, left panel; and upregulated, right panel) when grown in the presence of acetate and fluconazole was performed by ClueGO analysis. The size of the nodes represents the statistical significance of the terms. The systematic names of C. glabrata genes and respective orthologs in S. cerevisiae associated with each biological process are shown in red and black, respectively.

Fig. 3. Transcriptional response of C. glabrata biofilm cells to fluconazole, grown in the presence of glucose, as sole carbon source.

Network visualization of enriched pathways with the systematic names of C. glabrata (downregulated, left panel; and upregulated, right panel) when grown in the presence of glucose and fluconazole was performed by ClueGO analysis. The size of the nodes represents the statistical significance of the terms. The systematic names of C. glabrata genes and respective orthologs in S. cerevisiae associated with each biological process are shown in red and black, respectively.

Fig. 4. Validation by qRT-PCR of genes that were differentially expressed (CAT5, COQ6, ERG9, ERG11, FTR1, and ATX1) in response to fluconazole in C. glabrata biofilm cells grown with glucose as sole carbon source (RPMI; pH 7.0) or supplemented with acetate (RPMI + Ace; pH 5.0).

Graphs show percentage expression of each gene compared to a housekeeping gene, PGK1. The error bars show standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were considered statistically significant relative to untreated C. glabrata biofilm cells.

Fig. 5. Fluconazole susceptibility is modulated by pH, carbon source and MGE1 expression in C. glabrata.

a Log₂ fold-change comparison of RNA-seq (blue bars) and quantitative RT-PCR (yellow bars) for MGE1 expression in response to fluconazole treatment when C. glabrata cells are growing either using glucose as sole carbon source (RPMI pH 7.0) or in the presence of acetate (RPMI pH 5.0 + Ace). b Serial dilutions of wild-type strain (WT; 2001HTL strain transformed with the empty plasmid as a control) and overexpression strain (MGE1; 2001HTL strain transformed with a plasmid expressing CgMGE1 ORF, together with its terminator, from the CgTDH3 promoter) were spotted on RPMI medium containing 0.2% glucose at pH 7.0 or pH 5.0, and/or containing acetate (ace; 0.5% v/v), and/or fluconazole (flu; 8 or 24 µg/mL) and/or doxycycline (dox; 100 µg/mL). Pictures were taken after 48 h and 72 h of incubation at 37 °C.

Fig. 6. MGE1 overexpression modulates the aberrant flux of sterols following fluconazole treatment.

a Schematic representation of sterol biosynthetic pathways. Erg11 is the primary target of fluconazole. Red names represent toxic fungistatic sterols. b CgMGE1 overexpression reduces toxic sterol formation. Cells were grown in RPMI medium containing 0.2% glucose at pH 7.0, or pH 5.0, and/or containing 0.5% acetate, in the presence or absence of fluconazole for 24 h. Sterol levels were determined by GC-MS and are displayed for lanosterol, ergosterol, and 14-methylergosta-8,24(28)-dien-3β,6α-diol. The values were calculated relative to the internal standard (ITS; cholestane). The error bars show standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were considered statistically significant relative to C. glabrata WT cells. Gray dotted grid lines in each condition represent the mean obtained for ergosterol content for the same MGE1-overexpressing cells.

Fig. 7. Schematic overview of the transcriptional responses of C. glabrata biofilms to fluconazole.

Differentially expressed genes upon fluconazole treatment when cells are grown in the presence of a glucose and acetate or b glucose.