Dominant active alleles of RIM101 (PRR2) bypass the pH restriction on filamentation of Candida albicans

Abstract

Morphological development of the fungal pathogen Candida albicans is profoundly affected by ambient pH. Acidic pH restricts growth to the yeast form, whereas neutral pH permits development of the filamentous form. Superimposed on the pH restriction is a temperature requirement of approximately 37 degrees C for filamentation. The role of pH in development was investigated by selecting revertants of phr2Delta mutants that had gained the ability to grow at acid pH. The extragenic suppressors in two independent revertants were identified as nonsense mutations in the pH response regulator RIM101 (PRR2) that resulted in a carboxy-terminal truncation of the open reading frame. These dominant active alleles conferred the ability to filament at acidic pH, to express PHR1, an alkaline-expressed gene, at acidic pH, and to repress the acid-expressed gene PHR2. It was also observed that both the wild-type and mutant alleles could act as multicopy suppressors of the temperature restriction on filamentation, allowing extensive filamentation at 29 degrees C. The ability of the activated alleles to promote filamentation was dependent upon the developmental regulator EFG1. The results suggest that RIM101 is responsible for the pH dependence of hyphal development.

FIG. 1

Phenotypes of revertant CEM-2. (a) Genealogy of CEM-2. The relevant genotypes are shown. (b) Growth of wild-type strain SC5314, the phr2Δ mutant CFM-2, and the revertant CEM-2 after 48 h of incubation on YNB at pH 4.0 and 30°C. (c) Morphology of the indicated strains 3 h postinoculation into medium 199 at pH 4.0 or pH 8 and 37°C. (d) PHR1 expression as a function of ambient pH. RNA was isolated from the indicated strains cultured in medium 199 at the pH indicated below the panels. The upper panel shows the Northern blot hybridized with PHR1. The lower panel shows the corresponding ethidium bromide-stained agarose gel prior to blotting.

FIG. 2

Effect of RIM101 disruption in CEM-1. (a) Genealogy of revertant CEM-1 and transformants CEM-5A, CEM-5B, CEM-6A, and CEM-6B. The relevant genotypes are indicated. (b) Growth of strains on YNB at pH 4.0 or 7.0 following 48 h at 30°C. (c) PHR1 expression as a function of ambient pH. RNA was isolated from the indicated strains cultured in medium 199 at the pH indicated below the panels. The upper panel shows the Northern blot hybridized with PHR1. The lower panel was hybridized with ACT1. (d) Morphology of the indicated strains 3 h postinoculation into medium 199 at pH 4.0 or pH 8 and 37°C.

FIG. 3

Effect of mutant alleles of RIM101. (a) Genealogy of strains. Transformation of the mutant alleles RIM101-1426 and RIM101-1751 into CFM-4 produced strains CEM-16-1 and CEM-16-3, respectively. Introduction of the wild-type allele produced control strains CEM-31-1 and CEM-31-3. (b) Growth of strains on YNB at pH 4.0 or 7.0 after 48 h at 30°C. (c) Morphology of the indicated strains 3 h postinoculation into medium 199 at pH 4.0 and 37°C. (d) PHR1 expression as a function of ambient pH. RNA was isolated from the indicated strains cultured in medium 199 at the pH indicated below the panels. The upper panel shows the Northern blot hybridized with PHR1. The lower panel was hybridized with ACT1. (e) Effect of RIM101-1426 on PHR2 expression. RNA was isolated from CAF3-1-16 (lane 1), CAF3-1-31 (lane 2), and SC5314 (lane 3) grown in medium 199 at pH 4. The Northern blots were hybridized with PHR2 or ACT1, as indicated. CAF3-16-1 contains RIM101-1426. CAF3-31-1 contains the wild-type allele. SC5314 is a wild-type control.

FIG. 4

Multicopy suppression of temperature restriction. (a) Equal amounts of genomic DNA from strain CEM-8 [RIM101/(RIM101-1426)_n≥3], CEM-7 (RIM101/RIM101-1426/RIM101-1426), and the parental strain CEM-1 (RIM101/RIM101-1426) were digested with either PstI (left panel) or EcoRI (right panel). The Southern blots were hybridized with RIM101 or ACT1, as indicated. (b) Morphology of CEM-1, CEM-7, and CEM-8 following 3 h of incubation in medium 199 at pH 4.0 or pH 7.0 and 29 or 37°C.

FIG. 5

Effect of mutations in CPH1 and EFG1 on expression of RIM101-1426. (a) Genealogy of strains. (b) Morphology of strains 3 h postinoculation into medium 199 at p/H 4.0 and 37°C. (c) Colony morphology following 6 days of incubation on medium 199 at pH 4.0 and 37°C.

FIG. 5

Effect of mutations in CPH1 and EFG1 on expression of RIM101-1426. (a) Genealogy of strains. (b) Morphology of strains 3 h postinoculation into medium 199 at p/H 4.0 and 37°C. (c) Colony morphology following 6 days of incubation on medium 199 at pH 4.0 and 37°C.

FIG. 6

Effect of EFG1 on expression of RIM101-1426. RNA was isolated from HLC67-16-1 (efg1Δ CPH1 RIM101-1426), CDB1-16-1 (efg1Δ cph1Δ RIM101-1426), and CEM-16-1 (EFG1 CPH1 RIM101-1426) 3 h postinoculation into medium 199 at pH 4 and 37°C. The Northern blot was hybridized with PHR1, HWP1, or ACT1, as indicated.