Candida albicans lacking the gene encoding the regulatory subunit of protein kinase A displays a defect in hyphal formation and an altered localization of the catalytic subunit

Abstract

The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.

FIG. 1.

Restriction map of the C. albicans BCY1 locus. The thick line represents a 7.4-kb EcoRV C. albicans genomic fragment containing the BCY1 gene from fosmid 4A12. The white arrow indicates the position of the C. albicans BCY1 open reading frame. pMP1 and pMP3 refer to the constructions made to delete the C. albicans BCY1 alleles. Endonuclease restriction sites: E, EcoRI; EV, EcoRV; K, KpnI; P, PinAI; S, StyI.

FIG. 2.

Sequence alignment of the deduced amino acid sequence of C. albicans BCY1 with those of R subunits from several fungal species. Identities are in black boxes. Gaps introduced for alignment are indicated by dashes. The alignment was performed with Clustal W (38) and edited with GeneDoc (version 2.6.002). Potential phosphorylation sites are indicated by an arrowhead (▾). C.a., Candida albicans; S.c., Saccharomyces cerevisiae (accession number M15756); B.e., Blastocladiella emersonii (accession number M81713); A.n., Aspergillus nidulans (accession number AF043231); M.g., Magnaporthe grisea (accession number AF024633); N.c., Neurospora crassa (accession number L78009).

FIG. 3.

Effect of TPK2 overexpression on growth of the BCY1 null strain. A C. albicans TPK1/TPK1 tpk2/tpk2 bcy1/bcy1 mutant was transformed with plasmid pLS4. Four different transformants were tested (•, ⧫, ▴, ▾). The control strain was CAI4 transformed with plasmid pBI (▪). Strains were grown in noninducing medium SD (A) and in CSAA inducing medium (B) at 30°C with agitation (150 rpm). The optical density of the cultures was measured at 600 nm.

FIG. 4.

Western blot analysis of crude extract from strains CAI4, H2D, and bcy1 tpk2. Equal amounts of protein were subjected to SDS-10% PAGE, transferred to a polyvinylidene difluoride membrane, and reacted with anti-C. albicans Bcy1p antiserum as described in Materials and Methods. The arrow on the left indicates the migration position of Bcy1p.

FIG. 5.

Protein kinase A activity in crude extracts from CAI4, H2D and bcy1 tpk2 strains. Protein kinase A (PKA) activity was measured in aliquots of crude extracts from the three strains in the absence and in the presence of 10 μM cAMP as described in Materials and Methods. Values are means ± standard deviation from three independent experiments.

FIG. 6.

Growth of strains CAI4, H2D, and bcy1 tpk2. Cells were grown in liquid YPD supplemented with uridine (50 μg/ml). The optical density at 600 nm (OD₆₀₀) of the cultures was adjusted to 0.1 with the same medium, and 5-μl aliquots from the cultures and from 10-fold serial dilutions were spotted onto YPD plates supplemented with uridine (50 μg/ml). Plates were incubated at 30°C for 2 days.

FIG. 7.

Germinative behavior of strains CAI4, H2D, and bcy1 tpk2. Stationary-phase yeast cells were induced to germinate for 2 h at 37°C in MnCl₂/imidazole medium in the presence of 10% horse serum or 10 mM GlcNAc. Bar, 5 μm.

FIG. 8.

Coimmunoprecipitation of Tpk1-GFP with Bcy1p. S-100 fractions from strains CAI4, H2D, bcy1 tpk2, ASM1, ASM2, and ASM3 containing the same amounts of protein were subjected to immunoprecipitation with anti-C. albicans Bcy1p antiserum as described in Materials and Methods. Immunoprecipitates were resolved in SDS-7.5% PAGE, transferred to polyvinylidene difluoride membranes, and developed with a monoclonal anti-GFP antibody. Lanes: 1, parental strain CAI4; 2, mutant H2D strain; 3, bcy1 tpk2 double mutant strain; 4, ASM1 strain; 5, ASM2 strain; 6, ASM3 strain. The positions of molecular mass markers are indicated on the left. The arrow indicates the position of the 96-kDa fused protein band. The low-molecular-mass band observed in all lanes corresponds to a nonspecific reaction of the antibody.

FIG. 9.

Subcellular localization of Tpk1-GFP in wild-type and mutant strains. Localization of the fusion protein was visualized by fluorescence microscopy in strains ASM1, ASM2, and ASM3 (upper panels). Nuclei were visualized by DAPI staining (lower panels). Bar, 5 μm.