Transcriptional expression of PHR2 is positively controlled by the calcium signaling transcription factor Crz1 through its binding motif in the promoter

Abstract

The fungal cell wall consists of glucans, mannoproteins, and chitin and is essential for cell viability, morphogenesis, and pathogenesis. The enzymes of the GH72 family are responsible for ß-(1,3)-glucan elongation and branching, which is crucial for the formation of the glucan-chitin polymer at the bud neck of yeast cells. In the human fungal pathogen Candida albicans, there are five GH72 enzyme-encoding genes: PHR1, PHR2, PHR3, PGA4, and PGA5. It is known that expression of PHR1 and PHR2 is controlled by the pH-responsive Rim101 pathway through the transcription factor Rim101. In this study, we have demonstrated that the transcription expression of PHR2 is also controlled by the transcription factor Crz1 through its binding motif in the promoter. Therefore, we have uncovered a dual-control mechanism by which PHR2 expression is negatively regulated via CaRim101 through the pH-responsive pathway and positively modulated by CaCrz1 through the calcium/calcineurin signaling pathway.

Keywords: Candida albicans; Crz1; PHR2; calcium.

FIG 1

Roles of PHR2 in the sensitivity to calcium (A) as well as to SDS, antifungal drugs, and cell wall-perturbing agents (B) C. albicans strains were grown overnight at 30°C in liquid SD-URA medium, and cultures were serially diluted 10 times and spotted onto YPD plates indicated. Plates were incubated at 30°C for 2–3 days before photos were taken. CFW, Calcofluor white; KCZ, ketoconazole; Teb, terbinafine; Flu, fluconazole; Cas, caspofungin.

FIG 2

Functions of PHR2 in cell growth (A), precipitation rate (B), constriction formation, and nuclear segregation (C). C. albicans strains were grown overnight at 30°C in liquid SD-URA medium, and cultures were inoculated and grown in YPD media with pH 4.0 or pH 6.0 for up to 12 h. Cell samples were taken every 2 h for the OD_600nm measurement (A). Log-phase growing cells of indicated strains in YPD media with pH 4.0 or pH 6.0 were allowed to precipitate in test tubes, and photos were taken at 0, 15, 30, 60, and 180 min (B). After log-phase growing cells of the indicated strains in YPD media with pH 4.0 were stained with Calcofluor white for chitin (top panel) or DAPI for nuclei (bottom panel), they were examined under conventional fluorescence microscopy (C). Scale bars, 5 µm.

FIG 3

CaCrz1 binds in vitro and in vivo to the motif PHR2(H) in the promoter of PHR2. (A) Locations of two potential Crz1-binding motifs (boxed) in the PHR2 promoter. The 5′-TGGTG-3′ region (corresponding to the 5′ A_-458CCAC_-454 3′ region in the reverse complementary sequence) is the motif PHR2(M) predicted in the previous study (16), and the 5′ A_-315TAGGCACAG_-306 3′ region is the motif PHR2(H) identified in another previous study (11). Locations of EMSA_PHR2_F/R probes are indicated with dark solid lines. Locations of the ChIP PCR primer pair [CHIP_PHR2_F and CHIP_PHR2_R] are indicated with broken lines. (B) DIG-labeled probe EMSA_PHR2_F/R [PHR2(H)] was added to samples in Lanes 1–3. Purified His6-Crz1 protein of 1 µg was added in Lanes 2, 3, and 4. Unlabeled probe PHR2(H) was added to samples in Lane 3. (C) Detection of Crz1 binding to the PHR2 promoter in vivo by ChIP analysis. The wild-type SN148 strain expressing Crz1-HA and the control strain integrated with the CIp10 vector (no tag control) were grown to log-phase in the SD-URA medium, and cells were collected and treated with formaldehyde. Whole-cell extractions were obtained, and immunoprecipitation was done with anti-HA monoclonal antibodies. Immunoprecipitated pellets were used as templates for PCR with the primer pair ChIP_PHR2_F/R. PCR products were separated on a 1% agarose gel.

FIG 4

The Crz1-binding motif in the PHR2 promoter plays a role in the regulation of PHR2 expression. β-galactosidase activities of the wild-type promoter PHR2-lacZ and the mutated promoter PHR2(HΔ)-lacZ in the wild-type strain SN148 in the absence or presence of 0.2 M CaCl₂. The asterisk (*) indicates the statistically significant difference (P < 0.05) in the β-galactosidase activity of the wild-type promoter in the wild-type SN148 cells between the absence and the presence of supplemented 0.2 M CaCl₂.

FIG 5

Schematic model. The positive and negative regulation of PHR2 expression by the calcium/calcineurin signaling and the pH-responsive Rim101 pathways, respectively. Crz1-binding site (CDRE) and Rim101-binding site (shadowed) are indicated in the promoter of PHR2.