Candida albicans ferric reductases are differentially regulated in response to distinct forms of iron limitation by the Rim101 and CBF transcription factors

Abstract

Iron is an essential nutrient that is severely limited in the mammalian host. Candida albicans encodes a family of 15 putative ferric reductases, which are required for iron acquisition and utilization. Despite the central role of ferric reductases in iron acquisition and mobilization, relatively little is known about the regulatory networks that govern ferric reductase gene expression in C. albicans. Here we have demonstrated the differential regulation of two ferric reductases, FRE2 and FRP1, in response to distinct iron-limited environments. FRE2 and FRP1 are both induced in alkaline-pH environments directly by the Rim101 transcription factor. However, FRP1 but not FRE2 is also induced by iron chelation. We have identified a CCAAT motif as the critical regulatory sequence for chelator-mediated induction and have found that the CCAAT binding factor (CBF) is essential for FRP1 expression in iron-limited environments. We found that a hap5Delta/hap5Delta mutant, which disrupts the core DNA binding activity of CBF, is unable to grow under iron-limited conditions. C. albicans encodes three CBF-dependent transcription factors, and we identified the Hap43 protein as the CBF-dependent transcription factor required for iron-limited responses. These studies provide key insights into the regulation of ferric reductase gene expression in the fungal pathogen C. albicans.

FIG. 1.

Predicted FRP1 and FRE2 regulatory sites. Predicted Rim101 (lines) and Sfu1 (asterisk) regulatory sites within the FRP1 and FRE2 promoter regions are shown.

FIG. 2.

EMSA of promoter regions containing putative Rim101 binding sites. Protein extracts from the wild-type (WT) (DAY286; lanes 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, and 17) or rim101Δ/rim101Δ (DAY5; lanes 3, 6, 9, 12, 15, and 18) strain grown at pH 8 were incubated with radiolabeled DNA probes for endogenous (+) (lanes 1, 3, 4, 6, 7, and 9) or mutated (−) (lanes 2, 5, and 8) FRE2 (−876, −443, −22) oligomers or endogenous (+) (lanes 10, 12, 13, 15, 16, and 18) or mutated (−) (lanes 11, 14, and 17) FRP1 (−981, −626, −164) oligomers and analyzed by EMSA.

FIG. 3.

FRE2 and FRP1 promoter lacZ fusions identify important Rim101 binding sites. β-Galactosidase assays were performed on P_FRE2-lacZ and P_FRP1-lacZ reporter strains grown at pH 4 or pH 8 or at pH 4 with 200 μM BIP. At least three independent transformants were used to determine average Miller units and standard deviation. n-fold induction was determined by comparison of the expression of a given construct in either pH-8- or BIP-grown cells to expression in pH-4-grown cells. rim101Δ/Δ, rim101Δ/rim101Δ.

FIG. 4.

Deletion analysis of the FRP1 promoter. β-Galactosidase assays were performed with the P_FRP1-lacZ and derivative reporter strains grown at pH 4 or pH 8 or at pH 4 with 200 μM BIP. At least three independent transformants were used to determine average Miller units and standard deviation. n-fold induction was determined by comparison of the expression a given construct in either pH-8- or BIP-grown cells to expression in pH-4-grown cells.

FIG. 5.

EMSAs of the region predicted to contain the BIP-dependent site. Thirty-base-pair oligomers spanning −220/−190, −200/−170, −180/−150, −160/−130, −140/−110, and −120/−90 in relation to the FRP1 START codon were incubated with protein extracts from wild-type C. albicans (DAY286) grown at pH 4 (lanes 1, 4, 7, 10, 13, and 16), pH 4 with 200 μM BIP (lanes 2, 5, 8, 11, 14, and 17), or pH 8 (lanes 3, 6, 9, 12, 15, and 18). Free probe (open arrowhead), a nonspecific gel shift (closed arrowhead), and specific gel shifts (arrows A to C) are indicated.

FIG. 6.

Competition assays between the −220/−190 and −160/−130 regions. Radiolabeled probes for the −160/−130 region (lanes 1 to 7) and −220/−190 region (lanes 8 to 14) were incubated with protein extracts from wild-type (DAY286) cells and −160/−130 region (lanes 2 to 4 and 12 to 14) or −220/−190 region (lanes 5 to 7 and 9 to 11) cold competitor. Competitor was added at 50-fold (lanes 2, 5, 9, and 12), 100-fold (lanes 3, 6, 10, and 13), or 500-fold (lanes 4, 7, 11, and 14) excess. Control reactions lacking competitor are included (lanes 1 and 8).

FIG. 7.

The FRP1 promoter is bound by Hap5 via the CCAAT site. (A) EMSAs of the −160/−130 region containing the endogenous CCAAT site (lanes 1 to 4) or mutated TGCGC site (lanes 5 to 7). Protein extracts were obtained from wild-type (WT) (DAY286) cells grown in M199 (pH 4) (lanes 2 and 5), M199 (pH 4) plus 200 μM BIP (lanes 3 and 6), or M199 (pH 8) medium (lanes 4 and 7). (B) EMSAs of the −160/−130 region using protein extracts from wild-type (DAY286; lanes 2 to 4) or hap5Δ/hap5Δ (DAY1062; lanes 5 to 7) cells grown as for panel A. fp, free probe lane with no protein extract.

FIG. 8.

Hap5 via the CCAAT site governs FRP1 promoter activity. β-Galactosidase assays were performed on wild-type (WT) cells containing the P_FRP1-8-lacZ (data also shown in Fig. 4) and P_FRP1-8*TGCGC-lacZ reporters and on hap5Δ/hap5Δ cells containing the P_FRE2-lacZ and P_FRP1-8-lacZ reporters grown at pH 4 or pH 8 or at pH 4 with 200 μM BIP. At least three independent transformants were used to determine average Miller units and standard deviation. Induction (n-fold) was determined by comparison of the expression of a given construct in either pH 8- or BIP-grown cells to expression in pH 4-grown cells.

FIG. 9.

Hap43 is required for iron limitation-specific promoter binding. EMSAs of the −160/−130 probe incubated with protein extracts derived from wild-type (WT) (DAY286; lanes 1 to 3), hap5Δ/hap5Δ (DAY1062; lanes 4 to 6), hap41Δ/hap41Δ (DAY1083; lanes 7 to 9), or hap43Δ/hap43Δ (DAY1085; lanes 10 to 12) cells are shown. Protein extracts were obtained from cells grown in M199 (pH 4) (lanes 1, 4, 7, and 10), M199 (pH 4) plus 200 μM BIP (lanes 2, 5, 8, and 11), or M199 (pH 8) medium (lanes 3, 6, 9, and 12).

FIG. 10.

Growth of the wild-type (WT) (DAY286), rim101Δ/rim101Δ (DAY5), hap5Δ/hap5Δ (DAY1062), ftr1Δ/ftr1Δ (DAY609), hap41Δ/hap41Δ (DAY1083), and hap43Δ/hap43Δ (DAY1085) strains on YPG plates with or without 150 μM BPS after 3 days of incubation at 37°C.