STB5 is a negative regulator of azole resistance in Candida glabrata

Abstract

The opportunistic yeast pathogen Candida glabrata is recognized for its ability to acquire resistance during prolonged treatment with azole antifungals (J. E. Bennett, K. Izumikawa, and K. A. Marr. Antimicrob. Agents Chemother. 48:1773-1777, 2004). Resistance to azoles is largely mediated by the transcription factor PDR1, resulting in the upregulation of ATP-binding cassette (ABC) transporter proteins and drug efflux. Studies in the related yeast Saccharomyces cerevisiae have shown that Pdr1p forms a heterodimer with another transcription factor, Stb5p. In C. glabrata, the open reading frame (ORF) designated CAGL0I02552g has 38.8% amino acid identity with STB5 (YHR178w) and shares an N-terminal Zn(2)Cys(6) binuclear cluster domain and a fungus-specific transcriptional factor domain, prompting us to test for homologous function and a possible role in azole resistance. Complementation of a Δyhr178w (Δstb5) mutant with CAGL0I02552g resolved the increased sensitivity to cold, hydrogen peroxide, and caffeine of the mutant, for which reason we designated CAGl0I02552g CgSTB5. Overexpression of CgSTB5 in C. glabrata repressed azole resistance, whereas deletion of CgSTB5 caused a modest increase in resistance. Expression analysis found that CgSTB5 shares many transcriptional targets with CgPDR1 but, unlike the latter, is a negative regulator of pleiotropic drug resistance, including the ABC transporter genes CDR1, PDH1, and YOR1.

Fig 1

Complementation with CgSTB5 abrogates the sensitivities to caffeine and cold in the Δyhr178w mutant. (A) Growth on YPD agar plates at 30°C. (B) Growth in the presence of 0.15% caffeine. (C) Growth on YPD agar plates at 20°C. For strains, see Table 1. Cells were diluted to 1 × 10⁵ cells per 5 μl, followed by four 1:10 dilutions, and 5 μl of diluted cell culture was spotted on YPD or YPD with 0.15% caffeine agar plates and incubated at 30°C or 20°C for 48 h.

Fig 2

S. cerevisiae and C. glabrata stb5 mutants are sensitive to hydrogen peroxide. Overexpression of CgSTB5 in 84u had no effect. (A) Growth on YPD agar plates. (B) Growth on YPD agar plates with hydrogen peroxide. Strains were diluted to 1 × 10⁵ cells per 5 μl, followed by four 1:10 dilutions, and 5 μl of diluted cell culture was spotted on medium. S. cerevisiae strains were plated on YPD with 5 mM hydrogen peroxide, and C. glabrata strains were plated on YPD with 10 mM hydrogen peroxide. All plates were incubated at 30°C for 48 h.

Fig 3

The deletion of CgSTB5 causes a growth defect. Deletion of CgSTB5 resulted in poor growth in minimal media. The doubling times for the Cg84, CgSTB5OE, Δcgstb5, and 84uP strains were determined to be 2.3, 2.5, 3.3, and 2.2 h, respectively.

Fig 4

Decreased azole susceptibility of the Δcgstb5 mutant. The deletion of STB5 resulted in a decrease in susceptibility, and overexpression caused an increase in susceptibility to voriconazole and fluconazole. This finding was also reproducible in a pdr1 mutant.

Fig 5

Real-time PCR verification of microarray analysis. The deletion of CgSTB5 resulted in the upregulation of CgCDR1, CgPDH1, and CgYOR1, all members of the ABC transporter family of proteins, as well as CgPDR1, the transcriptional regulator of ABC transporters (upper panel). The same effect was seen in the Δpdr1 background but at a lower magnitude (lower panel). Overexpression of CgSTB5 did not significantly decrease transcription of these transporters despite increasing azole susceptibility. The standard error of the mean is shown for each gene.

Fig 6

Alignment of encoded amino acids in CgSTB5 homologues in 7 other yeasts. Numbers shown in the alignment are not identical to their order in CgSTB5p, which are as follows: (A) 32 to 59; (B) 408 to 657; (C) 881 to 908.