Fig 1.
Schematic of PDR1 (CAGL0A00451g).
The 4 different domains are indicated by the dark grey boxes – DBD – DNA binding domain; ID – inhibitory domain; MHR- middle homology region and AD – activation domain. Each of the individual light grey and black vertical lines represents a PDR1 gain of function mutation that has been identified in a clinical isolates resulting in resistance and/or tolerance to azoles.
Table 1.
PDR1 clinical isolates and location of gain-of-function mutation used in this study.
Fig 2.
Genetic interaction network map of C. glabrata PDR1WT and PDR1L280F.
Genome-wide synthetic interaction SGA screens were performed using query strains expressing either the wild type or PDR1+L280F C. glarbata ORF. Genes are represented by nodes that are colour coded corresponding to their cellular roles (www.yeastgenome.org and www.candidagenome.org) and/or assigned through review of the literature. Interactions are represented by edges. A comprehensive list of all interactions can be found in the supplementary information.
Fig 3.
FLZ resistant clinical isolates of C. glabrata containing PDR1+ alleles are sensitive to ɣ-butyrolacetone.
C. glabrata FLZ resistant clinical isolates containing PDR1+ mutations were grown on synthetic complete (top panel) or synthetic complete +2mM ɣ-butylroacetone (bottom panel). The ɣ-butylroacetone was synthetic lethal to 20/31 FLZ resistant C. glabrata clinical isolates containing PDR1+ alleles. Plate layout showing which gain-of-function mutants screened shown below the gel images.
Fig 4.
Confirmation that expression of PDR1+L280F in C. glabrata gcn5Δ cells is synthetically lethal.
A C. glabrata gcn5 pdr1 strain was transformed with pCU-MET3 (A) or pCU-MET3 containing PDR1L280F (B) and cultured on synthetic complete media (left panel) or synthetic complete media lacking methionine (right panel). Induction of PDR1L280F is lethal in C. glabrata gcn5 cells thus confirming the synthetic lethal interaction identified in S. cerevisiae.
Fig 5.
Evolution of wildtype, gcn5 null and gcn5 chemically inhibited C. glabrata cells in the presence of fluconazole.
C. glabrata cells grown in increasing concentrations of FLZ, with the PDR1 gene sequenced after each round to determine when and in which domain gain of function mutations are identified in. (A) Schematic of experiment. 10 individual flasks of C. glabrata cells – wildtype, Δgcn5 and chemically inhibited Gcn5 were exposed to increased concentrations of FLZ. The flask where inhibition of growth was first observed was used as the started culture for the subsequence round of drug exposure until 10 rounds of drug exposure was completed. At each pitching of cells, PDR1 was sequenced to identified when a gain of function mutation first emerged. (B) In wildtype C. glarbata cells, after 3 rounds of exposure to increasing FLZ concentrations, PDR1 mutations were identified and mapped to the activation domain. (C) C. glabrata Δgcn5 cells, after 6 rounds of exposure to increasing FLZ concentrations, PDR1 gain of function mutations were isolated and mapped to the activation domain and the middle homology domain. (D) C. glabrata cells that had Gcn5p chemically inhibited through with the addition of ɣ-, after 7 rounds of exposure to increasing FLZ concentrations, the emergence of PDR1 gain of function mutations was observed in the activation domain and DNA binding domain. The number of gain of function mutations observed is dramatically reduced in both the Δgcn5 and chemically inhibited Gcn5 FLZ exposures.
Table 2.
Gain-of-function mutations in the activation domain of CgPDR1 observed during evolution in presence of FLZ.