Table 1.
Strains used in this study.
Table 2.
Primers used in this study.
Fig 1.
(A) The effect of clorgyline on fluconazole susceptibility in the C. glabrata CBS138 strain. Checkerboard susceptibility assay was performed as described in the Materials and Methods section. Percentages of cell growth relative to drug-free control are expressed as means ± SE. Clorgyline concentration: white bar, 0 μg/ml; light grey bar, 40 μg/ml; dark grey bar, 80 μg/ml; and black bar, 160 μg/ml. (B) Spot dilution assay. Logarithmic-phase cells were adjusted to 2 × 107 cells/ml, and 5 μl of serial 10-fold dilutions were then spotted onto SC plates containing FLCZ, clorgyline, or both. Plates were incubated at 30°C for 48 h. Final concentrations: clorgyline, 80 μg/ml; and FLCZ, 64 μg/ml.
Fig 2.
Effects of clorgyline on susceptibility to micafungin and amphotericin B in Candida wild-type strains.
Spot dilution tests were performed using MIN plates as described in the legend of Fig 1B. Clorgyline was added at a final concentration of 0 μg/ml or 80 μg/ml. Micafungin (MCFG) final concentrations: C. glabrata, 0.06 μg/ml; C. albicans, 0.08 μg/ml; C. parapsilosis, 0.25 μg/ml; C. tropicalis, 0.06 μg/ml; and C. krusei, 0.08 μg/ml. Amphotericin B (AMPH-B) final concentrations: C. glabrata, 0.5 μg/ml; C. albicans, 0.5 μg/ml; C. parapsilosis, 1 μg/ml; C. tropicalis, 0.5 μg/ml; and C. krusei, 2 μg/ml. Candida wild-type strains: C. glabrata, CBS138; C. albicans, SC5314; C. parapsilosis, ATCC 90018; C. tropicalis, ATCC 750; and C. krusei, ATCC 6258.
Table 3.
Interaction of micafungin and clorgyline against Candida species determined by fractional inhibitory concentration index.
Table 4.
Interaction of amphotericin B and clorgyline against Candida species determined by fractional inhibitory concentration index.
Fig 3.
(A) Time-course analysis of PDR1, CDR1, and CDR2 expression in the C. glabrata wild-type strain. Logarithmic-phase cells were prepared in YPD broth and clorgyline was added at the concentration of 80 μg/ml. Cells were incubated at 30°C with agitation and total RNAs were extracted at indicated time points. PDR1, CDR1, and CDR2 mRNA abundance was measured by real-time qRT-PCR and normalized by using 18S rRNA as an internal control. Data were expressed as expression ratio relative to the mRNA abundance immediately before the clorgyline addition (time point 0). The mean ± SE of three independent experiments are shown. (B) Effects of clorgyline on susceptibility to micafungin and amphotericin B in C. glabrata wild-type strain and CDR1, CDR2, and PDR1 mutant strains. Spot dilution tests were performed as described in the legend of Fig 1B. Final drug concentrations: clorgyline, 80 μg/ml; micafungin (MCFG), 0.03 μg/ml; and amphotericin B (AMPH-B), 1.25 μg/ml. C. glabrata strains: wild-type, CBS138; Δcdr1, TG-C1; Δcdr2, TG-C2; and Δpdr1, TG-C3. (C) Time-course analysis of Cdr1 and Cdr2 expression in the C. glabrata wild-type strain CBS 138. Logarithmic-phase cells were incubated at 30°C with 80 μg/ml of clorgyline in YPD broth. Total proteins were extracted at indicated time points, separated by SDS-PAGE, and blotted to a polyvinylidene difluoride membrane. Each protein was detected using the indicated antibodies. (D) Expression of Cdr1 protein in the wild-type control and CDR1-oversepressing (CDR1-OE) strains evaluated by western blot analysis as described above. C. glabrata strains: Wild type, TG11; and CDR1-OE, TG-C5. (E) Micafungin and amphotericin B susceptibility of the CDR1-oversepressing (CDR1-OE) strain. Spot dilution tests were performed using SC-trp plates as described in the legend of Fig 1B. Final drug concentrations: clorgyline, 80 μg/ml; micafungin (MCFG), 0.025 μg/ml; and amphotericin B (AMPH-B), 1.25 μg/ml. C. glabrata strains: Wild-type, TG11; and CDR1-OE, TG-C5.
Fig 4.
(A) Time-course analysis of FMS1 expression in C. glabrata wild-type strain. Cell culture, RNA extraction, real-time qRT-PCR, and data presentation were performed as described in the Materials and Methods section and Fig 3A legend. (B) Effects of clorgyline on susceptibility to micafungin and amphotericin B in the C. glabrata wild-type and FMS1 mutant strains. Spot dilution tests were performed as described in the legend of Fig 1B. Final drug concentrations: clorgyline, 80 μg/ml; micafungin (MCFG), 0.03 μg/ml; and amphotericin B (AMPH-B), 1.25 μg/ml. C. glabrata strains: Wild type, CBS138; and Δfms1, TG-C4.
Fig 5.
Effects of two known azole transporter inhibitors, carbonyl cyanide 3-chlorophenylhydrazone and milbemycin A4 oxime, on micafungin susceptibility in C. glabrata wild-type strain and CDR1, CDR2, and PDR1 mutant strains.
Spot dilution tests were performed as described in the legend of Fig 1B. Final drug concentrations: carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 40 μM; milbemycin A4 oxime (Milbemycin), 4 μM; micafungin (MCFG), 0.03 μg/ml. C. glabrata strains: Wild type, CBS138; Δcdr1, TG-C1; Δcdr2, TG-C2; and Δpdr1, TG-C3.