Evidence for novel pH-dependent regulation of Candida albicans Rim101, a direct transcriptional repressor of the cell wall beta-glycosidase Phr2

Abstract

Candida albicans is a commensal fungus of mucosal surfaces that can cause disease in susceptible hosts. One aspect of the success of C. albicans as both a commensal and a pathogen is its ability to adapt to diverse environmental conditions, including dramatic variations in environmental pH. The response to a neutral-to-alkaline pH change is controlled by the Rim101 signal transduction pathway. In neutral-to-alkaline environments, the zinc finger transcription factor Rim101 is activated by the proteolytic removal of an inhibitory C-terminal domain. Upon activation, Rim101 acts to induce alkaline response gene expression and repress acidic response gene expression. Previously, recombinant Rim101 was shown to directly bind to the alkaline-pH-induced gene PHR1. Here, we demonstrate that endogenous Rim101 also directly binds to the alkaline-pH-repressed gene PHR2. Furthermore, we find that of the three putative binding sites, only the -124 site and, to a lesser extent, the -51 site play a role in vivo. In C. albicans, the predicted Rim101 binding site was thought to be CCAAGAA, divergent from the GCCAAG site defined in Aspergillus nidulans and Saccharomyces cerevisiae. Our results suggest that the Rim101 binding site in C. albicans is GCCAAGAA, but slight variations are tolerated in a context-dependent fashion. Finally, our data suggest that Rim101 activity is governed not only by proteolytic processing but also by an additional mechanism not previously described.

FIG. 1.

Diagram of the putative Rim101 binding sites within the PHR2 promoter. The sites at positions −575 and −124 are in the opposite orientation to the start codon. All three sequences are listed from the sense strand.

FIG. 2.

EMSA of promoter regions containing putative Rim101 binding sites. Protein extracts from wild-type (DAY286) (lanes 2, 3, 6, 7, 10, 11, 14, and 15) and rim101^−/− (DAY5) (lanes 4, 8, 12, and 16) strains grown at pH 8 were incubated with radiolabeled DNA probes for endogenous (+) (lanes 2 and 4) or mutated (−) (lane 3) PHR1 oligomers or endogenous (+) (lanes 6, 8, 10, 12, 14, and 16) or mutated (−) (lanes 7, 11, and 15) PHR2 (−575, −124, and −51) oligomers and analyzed by EMSA. Lanes 1, 5, 9, and 13 contain free probe (fp) without protein extracts.

FIG. 3.

Competition assays for Rim101-dependent binding to the putative Rim101 binding sites. Protein extracts from wild-type (DAY286) cells grown at pH 8 were incubated with radiolabeled probe for PHR1 (A) or PHR2 −124 (B) in the absence (lane 1) of competitor, in the presence of 30- to 1,000-fold excess wild-type (WT) (lanes 2 to 5) cold competitor, or in the presence of 30- to 1,000-fold excess mutant (mut) (lanes 6 to 9) cold competitor.

FIG. 4.

Supershift assays demonstrate that Rim101 binds to promoter regions containing the Rim101 binding site. Protein extracts from rim101^−/− + RIM101-V5 (DAY492) cells grown at pH 8 incubated with anti-V5-HRP antibody (V5) (lanes 3, 6, 9, and 12) or without anti-V5-HRP antibody (+) (lanes 2, 5, 8, and 11) were allowed to bind to DNA probes for wild-type PHR1 or PHR2 (−575, −124, and −51) promoters. Lanes 1, 4, 7, and 10 contain free probe (fp) without protein extract.

FIG. 5.

The −124 site acts as a classical and divergent Rim101 binding site by EMSA. Protein extracts from wild-type (DAY286) cells grown at pH 8 were incubated with DNA probes for the endogenous (+), the mutated (−), the classical-site mutated (ΔC), and the divergent-site mutated (ΔD) sequences of the −124 PHR2 site.

FIG. 6.

Rim101 processing is not required for DNA binding by EMSA. Protein extracts from rim13^−/− + RIM101-V5 (DAY643) cells grown at pH 8 were incubated with radiolabeled DNA probes for the endogenous (+) (lanes 1, 3, 5, and 7) or mutated (−) (lanes 2, 4, 6, and 8) PHR1 promoter or PHR2 (−575, −124, and −51) promoters.

FIG. 7.

EMSAs show that Rim101 DNA binding ability is affected by environmental pH. Protein extracts from rim101^−/− + RIM101-V5 (DAY492) (A) and rim13^−/− + RIM101-V5 (DAY643) (B) cells grown at pH 4 (lanes 1 to 8) and pH 8 (lanes 9 to 16) were incubated with radiolabeled DNA probes for endogenous (+) PHR1 (lanes 1 and 9); mutated (−) PHR1 (lanes 2 and 10); endogenous (+) PHR2 at the −575 (lanes 3 and 11), −124 (lanes 5 and 13), and −51 (lanes 7 and 15) sites; or mutated (−) PHR2 at the −575 (lanes 4 and 12), −124 (lanes 6 and 14), and −51 (lanes 8 and 16) sites and analyzed by EMSA. Protein samples (250 μg) from rim101^−/− + RIM101-V5 (A, lanes 17 and 18) and rim13^−/− + RIM101-V5 (B, lanes 17 and 18) grown at pH 4 (lane 17) or pH 8 (lane 18) were analyzed by Western blotting. The 75-kDa molecular mass marker, full-length (FL) Rim101-V5, and processed (P1 and P2) Rim101-V5 are noted.