Figure 1.
Expression of CgCDR1, CgCDR2, and CgSNQ2 in C. glabrata azole-resistant isolates from groups of sequential clinical isolates.
Quantification was performed by real-time RT-PCR. The values, which are averages of four separated experiments, represent the increase in gene expression relative to the azole-susceptible parental strains (set at 1.00). Error bars show standard deviations.
Figure 2.
Localisation of 65 non-synonymous substitutions in CgPdr1p.
The eight polymorphisms present in CgPDR1 alleles from both azole-susceptible and azole-resistant isolates are indicated by grey bars. The 57 amino acid polymorphisms (from a total of 58 mutations) specific for CgPDR1 alleles from azole-resistant isolates are indicated by black bars. Putative DBD (DNA binding domain) and MHR (middle homology region) were located by Pfam analysis. ID (putative inhibitory domain) and AD (putative transcriptional activation domain) were deduced by similarity with Pdr1p and Pdr3p from S. cerevisiae [11]. The limits of these domains are indicated by amino acids position in CgPdr1p.
Figure 3.
Expression of CgPDR1 in matched pairs of azole-susceptible (S) and azole-resistant (R) isolates.
RNA was isolated from log phase cultures, slot-blotted to membranes and hybridized with the indicated gene probes. CgACT1 served as internal control. Signals obtained in blotted membranes were quantified by counting radioactivity by phosphor imaging. Signals obtained for CgPDR1 were normalized with CgACT1. Expression values represent the increase of CgPDR1 expression in azole-resistant isolates relative to their azole-susceptible parental strains. RNA was also used for real-time quantitative RT-PCR as described in Material and Methods. Values given on the right diagram are means (±SEM) of three separate experiments and express CgPDR1 relative expression in resistant isolates as compared to their matched susceptible parents.
Table 1.
Azole susceptibilities and CgPDR1 mutations from C. glabrata clinical isolates.
Figure 4.
Effect of CgPDR1 expression on azole resistance.
(A) Expression of CgPDR1 in a matched pair of azole-susceptible and azole-resistant isolates (DSY2235 and DSY2234), in revertant strains (“rev”) overexpressing CgPDR1 alleles from an episomal plasmid in a pdr1Δ mutant derived from DSY562 (SFY53). CgPDR1 alleles present in each strain (“DSY” for clinical strains and “rev” for revertant strains) were named according to their strain number origin. RNA was isolated from log phase cultures, slot-blotted to membranes, and hybridized with the indicated gene probes. CgACT1 served as internal control. Signals obtained in blotted membranes were quantified by counting radioactivity by phosphor imaging. Signals obtained for CgPDR1 were normalized with CgACT1 and substracted from background. Expression values represent the increase of CgPDR1 expression in revertant strains relative to the clinical isolates expressing the same CgPDR1 allele. (B) Fluconazole susceptibility testing of C. glabrata clinical isolates DSY2235 and DSY2234, revertant strains (rev) overexpressing CgPDR1 alleles and of a pdr1Δ mutant (SFY53). Isolates were grown on YPD medium containing the drug at the indicated concentration at 30°C for two days. (C) Immunodetection of CgCdr1p and CgCdr2p in C. glabrata clinical isolates DSY2235 and DSY2234, in revertant strains (rev) overexpressing CgPDR1 alleles in a pdr1Δ mutant (SFY53). Proteins extract were separated by SDS-10% PAGE and immunoblotted with rabbit polyclonal anti-CgCdr1p and anti-CgCdr2p antibodies as described previously [9]. MICs to fluconazole were determined by broth microdilution method in accordance with the CLSI M27-A2 document (National Committee for Clinical Laboratory Standards, 2002).
Figure 5.
Quantitative analysis of azole resistance in selected C. glabrata isolates.
(A) Fluconazole susceptibility testing of C. glabrata clinical isolates DSY562 and DSY565 and their derivative mutants. Isolates were grown on YPD medium containing the drug at the indicated concentration at 30°C for two days. The indicated genotypes correspond to the following strains: DSY562 pdr1Δ: SFY92; DSY565 pdr1Δ: SFY94; DSY562 pdr1Δ+562 (standing for re-introduction of CgPDR1 from DSY562): SFY114; DSY562 pdr1Δ+565L280F (standing for re-introduction of CgPDR1 from DSY565 with L280F substitution): SFY115; DSY565pdr1Δ+562: SFY118; DSY565 pdr1Δ+565L280F: SFY119. (B) Expression of CgCDR1, CgCDR2, and CgSNQ2 in C. glabrata clinical isolates DSY562 and DSY565 and their derivative mutants. Quantification was performed by real-time RT-PCR. The values, which are averages of four separated experiments, represent the increase in gene expression relative to DSY562 (set at 1.00). Error bars show standard deviations. (C) Immunodetection of CgCdr1p and CgCdr2p in C. glabrata clinical isolates DSY562 and DSY565 and their derivative mutants. Proteins extract were separated by SDS-10% PAGE and immunoblotted with rabbit polyclonal anti-CgCdr1p and anti-CgCdr2p antibodies as described previously [9]. MICs to fluconazole were determined by broth microdilution method in accordance with the CLSI M27-A2 document.
Figure 6.
Reconstitution of CgPDR1 GOF alleles in C. glabrata.
(A) Fluconazole susceptibility testing of DSY562 pdr1Δ mutant strain (SFY93) expressing different CgPDR1 alleles, which were named according to their strain number origin and by indicating the amino acid substitution (in superscript) associated with a specific strain number. The following strains correspond to the indicated genotypes: DSY562 pdr1Δ+486:SFY98; 489L328F: SFY99; 738: SFY100; 739R376W: SFY101; 2253: SFY102; 2254D1082G:SFY103; 2235: SFY104; 2234T588A: SFY105; 701: SFY106; 704T607S: SFY107; 529:SFY108; 530E1083Q: SFY109; 753: SFY110; 754Y584C: SFY111; 726: SFY112; 727D876Y: SFY113; BPY55P882L: SFY116. (B) Expression of CgCDR1, CgCDR2, and CgSNQ2 in the DSY562 pdr1Δ mutant strain (SFY93) expressing different CgPDR1 alleles, named according to their strain number origin. Quantification was performed by real-time RT-PCR. The values, which are averages of four separated experiments, represent the increase in gene expression relative to DSY562 (set at 1.00). Error bars show standard deviations.
Figure 7.
C. glabrata tissue burdens in murine infection models.
(A) Fungal tissue burdens in kidneys from immuno-competent BALB/c mice infected intravenously with 4×107 viable cells of C. glabrata strains. Mice were sacrificed at day 7 post-infection; and results, which are expressed as CFUs per gram of tissue, represent values recorded separately for each of the ten mice. Geometric means are indicated by horizontal bars and asterisks indicate statistically significant differences (*: P<0.05; **: P<0.01; ***: P<0.001). NS indicates no significance (P>0.05). The origin of each strain is indicated. Strain background (DSY562 or DSY565) is indicated by filled or empty symbols, respectively. The pdr1Δ mutants from strains DSY562 and DSY565 correspond to SFY92 and SFY94, respectively. Revertants constructed from pdr1Δ mutants are indicated by the re-introduced GOF mutation or by the re-introduced wild type CgPDR1 allele from DSY562. Prism 5.0 was used for statistical analysis and data were processed with non-parametric Wilocoxon Rank sum tests. Comparisons are indicated in the Figure (see Table S3 for details) and associate selected data points. (B) Fungal tissue burdens in kidneys from immuno-suppressed mice infected intravenously with 4×107 viable cells of C. glabrata strains. BALB/c mice were rendered neutropenic by intraperitoneal administration of cyclophosphamide (200 mg kg−1 of body weight per day) three days before challenge and on the day of infection. Mice were sacrificed at day 7 post-infection.
Figure 8.
Virulence of C. glabrata is dependent on CgPDR1 GOF mutations.
(A) Survival curves of mice infected with DSY562 and DSY565 and derived mutants and revertants. Statistical differences were performed using the Log-rank Mantel-Cox test (Prism 5.0) by comparing survival curves of mice infected by DSY562 and by other strains as indicated. The range of significant P values obtained is indicated for DSY565 and strains containing the CgPDR1 allele with a L280F substitution. (B) Survival curves of mice infected with DSY562 and DSY565 and strains reconstituted with different CgPDR1 alleles. The range of significant P values obtained is indicated for DSY565 and strains containing GOF mutations.
Figure 9.
Fitness assays between azole-resistant and azole-susceptible isolates.
(A) In vitro fitness assays. Strains SFY114 (DSY562 pdr1Δ-PDR1) and SFY115 (DSY562 pdr1Δ-L280F) were inoculated in YEPD in single or mixed (1∶1) cultures. Aliquots were taken from each culture in triplicate and population ratios were calculated from the proportion of azole-susceptible and azole-resistant colonies plated onto YEPD agar as described in Material and Methods. (B) In vivo fitness assays. SFY114 (DSY562 pdr1Δ-PDR1) and SFY115 (DSY562 pdr1Δ-L280F) were inoculated intravenously as single or mixed (1∶1) cultures to three groups of mice as described in Material and Methods. Kidneys were taken at given time points from sacrificed mice and population ratios were measured as described above.
Figure 10.
Efficacy of azole treatment in C. glabrata assessed by fungal tissue burden in kidneys.
Fluconazole (100 mg/Kg/day) was administered by intraperitoneal injection in immuno-suppressed mice as described in Material and Methods. Treatment was initiated 24 h after inoculation (day 1 post-infection) and continued until day 7 post-infection. Mice were injected with 4×107 CFU of each investigated strain and organ homogenates were obtained from ten mice per group that were sacrificed and necropsied on day 8 post-infection. Results, which are expressed as CFUs per gram of tissue, represent means of values recorded separately for each of the mice. Geometric means are indicated by horizontal bars and asterisks indicate statistically significant differences between two conditions (see legend of Figure 7 and Table S3 for details). Closed and empty circles indicate CFU from untreated and fluconazole-treated animals infected with a DSY562 background, respectively. Closed and empty diamonds indicate CFU from untreated and fluconazole-treated isolates with a DSY565 background, respectively.