Fig 1.
Distribution into functional classes of the 183 determinants of resistance to 5-flucytosine.
A) Genes clustered according to the biological process taxonomy of Gene Ontology, using the GoToolBox software (http://genome.crg.es/GOToolBox/). The most highly ranked statistically significant (p-value<0.01) GO terms are displayed. The gene frequency within each class is indicated by the black bars, compared to the frequency registered for the S. cerevisiae whole genome, indicated by the grey bars, gene frequency being the percentage of the genes in a list associated to a specific GO terms. B) Classification based on the description of gene function registered in the Saccharomyces Genome Database (www.yeastgenome.org). The genes that fall into each class are detailed in supplementary S1 Table.
Fig 2.
Arginine supplementation decreases the inhibitory effect exerted by 5-flucytosine in C. glabrata.
The growth curves of parental C. glabrata KUE100 cells in the absence (□,◊) or presence (■,♦) of 0.5 mg/L 5-flucytosine are compared, upon supplementation (◊,♦) or not (□,■) with 60mg/L arginine. The displayed growth curves are the average of three independent experiments. Error bars represent the corresponding standard deviation. **indicates that there is a statistically significant (P<0.01) difference between the OD600nm reached in the presence and absence of arginine, at the indicated times points.
Fig 3.
Cell wall damage followed by remodeling occurs during adaptation to 5-flucytosine stress.
A) C. glabrata KUE100 cells exposed to lyticase stress were harvested in the exponential phase of growth in the absence of stress (♦) or upon 30 min of exposure to 0.7 mg/L 5-flucytosine (■), or in the exponential phase of growth reached upon adaptation to 0.7 mg/L 5-flucytosine (▲). The different cell populations were washed with water and resuspended in 0.1M sodium phosphate buffer at pH 7.5. After addition of 20mg/L lyticase, the decrease in the OD600nm of the cell suspension was measured periodically and indicated as a percentage of the initial OD600nm. The indicated values are averages of at least three independent experiments. Error bars represent the corresponding standard deviation. * P<0.05; ***P<0.001. B) Comparison, by spot assays, of the susceptibility of C. glabrata KUE100 cells harvested in the exponential phase of growth in the absence of stress (control) or upon 30 min of exposure to 0.5 mg/L 5-flucytosine (30’ 5-FC), or in the exponential phase of growth reached upon adaptation to 0.5 mg/L 5-flucytosine (exp 5-FC) to Calcofluor White, in MMB agar plates. The inocula were prepared as described in Materials and Methods, and the cell suspensions used to prepare the spots 1:5 (b) and 1:25 (c) dilutions of the cell suspension used for column a. The images are representative of at least three independent experiments.
Fig 4.
The acquaglyceroporins CgFps1 and CgFps2 confer resistance to 5-flucytosine.
A) Comparison, by spot assays, of the susceptibility of the C. glabrata KUE100, KUE100_Δcgfps1 and KUE100_Δcgfps2 strains (B) to 5-flucytosine, in MMB agar plates. B) Fluorescence of exponential-phase BY4741 S. cerevisiae cells, harboring the cloning vector pGREG576 (control) or the pGREG576_CgFPS1 plasmids (CgFps1_GFP), after 5h of galactose-induced recombinant protein expression. C) Comparison, by spot assays, of the susceptibility to 5-flucytosine of S. cerevisiae BY4741 and BY4741_Δfps1 cells, harboring the cloning vector pGREG576 (v) or the same plasmid expressing the CgFPS1 gene, in MMB agar plates. In A) and C) the inocula were prepared as described in Materials and Methods, and the cell suspensions used to prepare the spots 1:5 (b) and 1:25 (c) dilutions of the cell suspension used for column a. The images are representative of at least three independent experiments.
Fig 5.
CgFPS1 and CgFPS2 expression decreases the accumulation of 5-flucytosine in C. glabrata cells.
Time-course accumulation of [3H]-flucytosine in non-adapted KUE100 (♦), KUE100_Δcgfps1 (■) and KUE100_Δcgfps2 (▲) strains, during cultivation in BM liquid medium in the presence of 3mg/L of unlabelled 5-flucytosine. The indicated accumulation ratio values are averages of at least three independent experiments. Error bars represent the corresponding standard deviation. * P<0.05; **P<0.01.
Fig 6.
The expression of CgFPS1 and CgFPS2 is independent of 5-flucytosine exposure or CgPDR1 deletion.
Comparison of the variation of the CgFPS1 (A) and CgFPS2 (B) transcript levels in the 66032u C. glabrata wild-type strain and in the derived 66032u_Δcgpdr1 deletion mutant, before (control) and after 1h of exposure to 3.5mg/L 5-flucytosine (5-FC). The presented transcript levels were obtained by quantitative RT-PCR and are relative CgFPS1/CgACT1 or CgFPS2/CgACT1 mRNA, relative to the values registered in the 66032 parental strain in control conditions. The indicated values are averages of at least three independent experiments. Error bars represent the corresponding standard deviation.