Candida albicans Rim13p, a protease required for Rim101p processing at acidic and alkaline pHs

Abstract

Candida albicans is an important commensal of mucosal surfaces that is also an opportunistic pathogen. This organism colonizes a wide range of host sites that differ in pH; thus, it must respond appropriately to this environmental stress to survive. The ability to respond to neutral-to-alkaline pHs is governed in part by the RIM101 signal transduction pathway. Here we describe the analysis of C. albicans Rim13p, a homolog of the Rim13p/PalB calpain-like protease member of the RIM101/pacC pathway from Saccharomyces cerevisiae and Aspergillus nidulans, respectively. RIM13, like other members of the RIM101 pathway, is required for alkaline pH-induced filamentation and growth under extreme alkaline conditions. Further, our studies suggest that the RIM101 pathway promotes pH-independent responses, including resistance to high concentrations of lithium and to the drug hygromycin B. RIM13 encodes a calpain-like protease, and we found that Rim101p undergoes a Rim13p-dependent C-terminal proteolytic processing event at neutral-to-alkaline pHs, similar to that reported for S. cerevisiae Rim101p and A. nidulans PacC. However, we present evidence that suggests that C. albicans Rim101p undergoes a novel processing event at acidic pHs that has not been reported in either S. cerevisiae or A. nidulans. Thus, our results provide a framework to understand how the C. albicans Rim101p processing pathway promotes alkaline pH-independent processes.

FIG. 1.

Growth of rim13::Tn7 mutants at alkaline pH. The wild type (WT; DAY286), the rim101^−/− mutant strain (DAY5), rim13::Tn7::UAU1/rim13::URA3 homozygous strains (GKO88, GKO89, and DAY668), rim13::Tn7::UAU1/RIM13 heterozygous strains (DAY665 and DAY666), and the rim13::Tn7::UAU1/rim13::URA3/RIM13 triplication strain (DAY667) were grown for 2 days at 37°C on YPD (A) and on YPD at pH 10 (B).

FIG. 2.

Generation of RIM13 strains. Genomic DNAs from BWP17 and its derivatives were purified and used for PCRs with rim13 5-detect and rim13 3-detect. Lanes: 1, wild-type auxotrophic strain (BWP17); 2, wild-type prototrophic strain (DAY185); 3, RIM13^+/− strain (DAY67); 4, rim13^−/− strain (DAY349); 5, rim13^−/− strain (DAY224); 6, rim13^−/− RIM13 strain (DAY226); 7, rim13^−/− RIM101 strain (DAY128); 8, rim13^−/− RIM101-405 strain (DAY132).

FIG. 3.

Growth phenotypes of RIM101 pathway mutants. The wild type (DAY185), rim13^−/− homozygous mutant, complemented, and RIM101-405-rescued strains (DAY224, DAY226, and DAY132, respectively), the rim101^−/− mutant strain (DAY25), and rim8^−/− homozygous mutant, complemented, and RIM101-405-rescued strains (DAY117, DAY106, and DAY62, respectively) were grown overnight at 30°C in YPD plus Uri. Cells were diluted 10-fold in water, and 3-μl volumes of fivefold serial dilutions were spotted onto YPD at pH 8, YPD at pH 10, YPD plus 10 μM calcofluor, YPD plus 10 μM calcofluor at pH 8, YPD plus 150 mM LiCl, and YPD plus 150 μg of hygromycin B per ml. Growth was measured after 2 days at 30°C, except for the 10 μM calcofluor pH 8 plates and the 150 mM LiCl plates, which were measured after 4 days at 30°C.

FIG. 4.

Rim101-V5p Western blots. (A) Cartoon of the Rim101p sequence showing the three zinc finger domains, the D/E-rich C-terminal domain, and the position of the V5 insertion. The AgeI (A, top), BstEII (B, middle), and NgoMI (N, bottom) constructs are shown with the last residue before the insertion in parentheses. (B) Western blot assays of rim101^−/− mutant strains containing the RIM101-V5-AgeI (DAY492, lanes 1 and 4), RIM101-V5-BsteII (DAY499, lanes 2 and 5), and RIM101-V5-NgoMI (DAY504, lanes 3 and 6) constructs. (C) Western blot assays of RIM13^+/+ (DAY499, lanes and 1 and 2) and rim13^−/− (DAY626, lanes 3 and 4) strains containing the RIM101-V5-BsteII construct. (D) Western blot assays of wild-type (DAY492, lanes 1 and 2), rim20^−/− (DAY610, lanes 3 and 4), rim8^−/− (DAY615, lanes 5 and 6), and rim13^−/− (DAY643, lanes 7 and 8) strains containing the RIM101-V5-AgeI construct. All strains were grown for 4 h at pH 4 or 7, and proteins were purified and separated by SDS-8% PAGE. Gels were transferred to nitrocellulose and probed with anti-V5-horseradish peroxidase (Invitrogen). WT, wild type. The values on the left of panels B, C, and D are molecular sizes in kilodaltons.

FIG. 5.

Model of Rim101p processing. (A) Cartoon of C. albicans Rim101p showing the locations of the V5 insertions (A, AgeI; B, BstEII; N, NgoMI) and the predicted C-terminal processing sites at alkaline pHs (C-proc ALK) and acidic pHs (C-proc ACID) (arrows). (B) Scale model of Rim101p/PacC processing in C. albicans, S. cerevisiae, and A. nidulans showing predicted processing forms and pH and Rim13p/PalB dependence. In C. albicans, the 65- and 74-kDa forms are pH and Rim13p dependent. The possibility that the CaRim101p 65-kDa form arises from the 74-kDa intermediate is shown. The functional activities of these CaRim101p forms has not been determined. In S. cerevisiae, the single pH- and Rim13p-dependent processing step is shown. This is the only ScRim101p processing event described. In A. nidulans, PacC is processed in a pH- and PalB-dependent step to a 53-kDa intermediate. This intermediate is then a substrate for a PalB-independent processing event to the active 27-kDa form.