Figure 1.
Identification of Genes that Are Differentially Expressed in MDR1 Overexpressing Clinical C. albicans Isolates
(A, B) Venn Diagrams showing the number of genes that are upregulated (A) or downregulated (B) in the MDR1 overexpressing, drug-resistant isolates F5, G5, and 6692 as compared with their matched drug-susceptible isolates F1, G1, and 5833, respectively.
(C) Heat map showing genes that are upregulated (red) or downregulated (green) in all three MDR1 overexpressing isolates. Results are from three independent experiments (Exp. 1 to Exp. 3) for each comparison. The genes are ordered according to their relative degree of up- and downregulation, as indicated by the brightness of the red and green fields (see Table S1 for details about the expression data). Gene names were taken from CandidaDB (http://genolist.pasteur.fr/CandidaDB), except for MDR1 and MRR1, and their orf19 names are given.
Figure 2.
Construction of mrr1Δ Mutants and Complemented Strains
(A) Structure of the deletion cassette from plasmid pZCF36M2 (top), which was used to delete the MRR1 ORF in strains SC5314, F5, G5, and CAG48B, and genomic structure of the MRR1 locus in the parental strains (bottom). The MRR1 coding region is represented by the white arrow and the upstream and downstream regions (5′MRR1 and 3′MRR1) by the solid lines. The SAT1 flipper cassette (SAT1-FLIP), in which the caFLP gene is expressed from the inducible SAP2 promoter [30], is represented by the grey rectangle bordered by FRT sites (black arrows). The 34 bp FRT sites are not drawn to scale. The probes used for Southern hybridization analysis of the mutants are indicated by the black bars.
(B) Structure of the DNA fragments from plasmids pZCF36K2, pZCF36K3, pZCF36K4, and pZCF36K5 (top), which were used for integration the MRR1F2–1, MRR1F5, MRR1G2–2, and MRR1G5 alleles, respectively, into the disrupted mrr1 locus of homozygous and heterozygous mrr1Δ mutants (bottom) using the caSAT1 selection marker (grey arrow). TACT1, transcription termination sequence of the ACT1 gene.
Only relevant restriction sites are given in (A) and (B): A, ApaI; B, BglII; E, EcoRI; N, NsiI; Nd, NdeI; P, PstI; S, SalI; ScI, SacI; ScII, SacII; X, XhoI. The PstI and NsiI sites shown in parenthesis were destroyed by the cloning procedure.
(C) Southern hybridization of NsiI-digested genomic DNA of mrr1Δ mutants derived from strain SC5314 and of strains with reinserted MRR1 alleles with the MRR1-specific probe 1. The sizes of the hybridizing fragments (in kb) are given on the left side of the blot and their identities are indicated on the right. The genotype of the strains is given above the respective lanes. Only one of the two independently constructed series of strains is shown. Inactivation of MRR1 in the clinical isolates F5 and G5 and the reporter strain CAG48B and reinsertion of different MRR1 alleles in mrr1 mutants of the reporter strain occurred in an analogous fashion.
Figure 3.
MRR1 Mediates Drug Resistance of MDR1 Overexpressing Clinical C. albicans Isolates
Minimal inhibitory concentration (MIC) of the indicated metabolic inhibitors (in μg ml−1) for the drug-sensitive clinical isolates F2 and G2, the matched drug-resistant isolates F5 and G5, and two independently constructed heterozygous and homozygous mrr1Δ mutants as well as mdr1Δ mutants of the drug-resistant isolates F5 and G5. The susceptibilities of the isolates F2, F5, and its mutant derivatives are shown in the left panels, and those of isolates G2, G5, and its mutant derivatives are shown in the right panels.
Figure 4.
MRR1 Is Required for Constitutive and Induced MDR1 Expression
(A) Constitutive MDR1 promoter activity in the drug-resistant clinical isolates F5 and G5 and in heterozygous and homozygous mrr1Δ mutants. The mean fluorescence of two independently constructed derivatives of each parental strain carrying a PMDR1-GFP reporter fusion and grown to log phase in YPD medium was determined by flow cytometry. The parental strains F5 and G5, which do not contain the GFP gene, were included as negative controls.
(B) Benomyl- and H2O2-induced MDR1 promoter activity in strain SC5314 and its mrr1Δ derivatives. Two independently constructed reporter strains of each parental strain carrying a PMDR1-GFP reporter fusion were grown in the absence (-) or presence (+) of benomyl or H2O2 as detailed in the experimental procedures and the mean fluorescence of the cells was determined by flow cytometry. The parental strain SC5314, which does not contain GFP, was included to control for background fluorescence.
Figure 5.
MRR1 Gain-of-Function Alleles Cause Constitutive MDR1 Overexpression and Multidrug Resistance
(A) MICs (in μg ml−1) of the indicated metabolic inhibitors for the wild-type parental strain SC5314, two independently constructed homozygous mrr1Δ mutants, and derivatives carrying the indicated mutant MRR1 alleles in the absence or presence of a wild-type MRR1 allele.
(B) MDR1 promoter activity in a wild-type reporter strain, an mrr1Δ mutant, and derivatives carrying the indicated mutant MRR1 alleles in the absence or presence of a wild-type MRR1 allele. The strains were grown to log phase in YPD medium and the mean fluorescence of the cells was determined by flow cytometry.
Figure 6.
Identification of Mrr1p Target Genes by DNA Microarray Analysis
(A) Genes that were upregulated by the P683S mutation in the SC5314 genetic background (MRR1F5 versus MRR1F2–1). The genes are ordered according to their average degree of upregulation in four repeat experiments performed with two independent transformants (strains A and B). The changes in the expression level of these genes in the drug-resistant clinical isolate F5 as compared with a matched susceptible isolate (F5 versus F1) and in two independently constructed mrr1Δ mutants (strains A and B) as compared with their wild-type progenitor F5 (F5mrr1Δ versus F5) are shown for comparison.
(B) Genes that were upregulated by the G997V mutation in the SC5314 genetic background (MRR1G5 versus MRR1G2–2). The genes are ordered according to their average degree of upregulation in four repeat experiments performed with two independent transformants (strains A and B). The changes in the expression level of these genes in the drug-resistant clinical isolate G5 as compared with a matched susceptible isolate (G5 versus G1) and in two independently constructed mrr1Δ mutants (strains A and B) as compared with their wild-type progenitor G5 (mrr1Δ versus G5) are shown for comparison.
The fold increase or decrease in the expression of the genes is given for each experiment. Upregulated genes are highlighted in red, downregulated genes are highlighted in green, and genes that were not differentially expressed are shown as white boxes in all pairwise comparisons.
Table 1.
Genes Commonly Downregulated by MRR1 Deletion in Isolates F5 and G5