RIM101-dependent and-independent pathways govern pH responses in Candida albicans

Abstract

Growth and differentiation of Candida albicans over a broad pH range underlie its ability to infect an array of tissues in susceptible hosts. We identified C. albicans RIM101, RIM20, and RIM8 based on their homology to components of the one known fungal pH response pathway. PCR product-disruption mutations in each gene cause defects in three responses to alkaline pH: filamentation, induction of PRA1 and PHR1, and repression of PHR2. We find that RIM101 itself is an alkaline-induced gene that also depends on Rim20p and Rim8p for induction. Two observations indicate that a novel pH response pathway also exists. First, PHR2 becomes an alkaline-induced gene in the absence of Rim101p, Rim20p, or Rim8p. Second, we created strains in which Rim101p activity is independent of Rim20p and Rim8p; in these strains, filamentation remains pH dependent. Thus, pH governs gene expression and cellular differentiation in C. albicans through both RIM101-dependent and RIM101-independent pathways.

FIG. 1

Confirmation of the rim8⁻/rim8⁻ mutant. The figure shows a gel following PCR with flanking detection primers of genomic DNA of wild type (sample 1), rim8::ARG4 transformants (samples 2 to 5), and rim8::URA3 transformants (samples 6 and 7). Note integration at both RIM8 copies in one case (sample 5) following tranformation with rim8::ARG4.

FIG. 2

ClustalW alignments of C. albicans Rim101p (A), Rim20p (B), and Rim8p (C) with the S. cerevisiae homolog (A and C) and the A. nidulans homolog (B). Alignments were done by using the server at http://dot.imgen.bcm.tmc.edu:9331/multi-align/Options/clustalw.html. C. albicans RIM101, RIM20, and RIM8 sequences have been deposited in GenBank (see Materials and Methods). S. cerevisiae sequences were obtained from the S. cerevisiae database at http://www-genome.stanford.edu/Saccharomyces/. The A. nidulans sequence was obtained from the GenBank database at http://www.ncbi.nlm.nih.gov/. Ca, C. albicans; Sc, S. cerevisiae; An, A. nidulans.

FIG. 3

Morphology of strains grown at acidic and alkaline pH. The figure shows wild-type cells (A and B) and rim101⁻/rim101⁻ (C and D), rim20⁻/rim20⁻ (E), rim8⁻/rim8⁻ (F), rim101⁻/rim101⁻, pRIM101 (G), rim101⁻/rim101⁻, pRIM101-405 (H), rim20⁻/rim20⁻ pRIM101-405 (I), and rim8⁻/rim8⁻ pRIM101-405 (J) cells grown at acidic (A and C) or alkaline (B and D to J) pH.

FIG. 4

Northern blot analyses of strains grown at acidic and alkaline pH. RNA was prepared from RIM101/RIM101 (samples 1 and 7), RIM101/rim101⁻ (samples 2 and 8), rim101⁻/rim101⁻ (samples 3 and 9), rim101⁻/rim101⁻ pRIM101 (samples 4 and 10), rim20⁻/rim20⁻ (samples 5 and 11), and rim8⁻/rim8⁻ (samples 6 and 12) cells grown at acidic (samples 1 to 6) or alkaline (samples 7 to 12) pH. Northern blots were visualized with a phosphorimager.

FIG. 5

Quantitation of Northern blots from Fig. 4. Samples were normalized for loading with the TEF1 signal. Note that the scale of PHR2 has been expanded to permit comparison of maximal wild-type and mutant levels.

FIG. 6

Model for RIM101-dependent and RIM101-independent control of alkaline responses. Alkaline pH stimulates Rim101p activity through increased expression and proteolytic activation, both of which require Rim8p and Rim20p. Full-length Rim101p-long does not have a known activity. Processed Rim101p-short is required for the alkaline response, which includes activation of alkaline-induced genes, repression of alkaline-repressed genes, and filamentation. Since RIM101 is an alkaline-induced gene, its expression may depend on autoregulation by Rim101p-short. Alkaline pH also stimulates a RIM101-independent pathway. (We have diagrammed one RIM101-independent pathway for simplicity, but there may be several.) This pathway activates PHR2 expression (in the absence of a functional RIM101 pathway) and stimulates filamentation in conjunction with Rim101p.