Cryptococcal titan cell formation is regulated by G-protein signaling in response to multiple stimuli

Abstract

The titan cell is a recently described morphological form of the pathogenic fungus Cryptococcus neoformans. Occurring during the earliest stages of lung infection, titan cells are 5 to 10 times larger than the normal yeast-like cells, thereby resisting engulfment by lung phagocytes and favoring the persistence of infection. These enlarged cells exhibit an altered capsule structure, a thickened cell wall, increased ploidy, and resistance to nitrosative and oxidative stresses. We demonstrate that two G-protein-coupled receptors are important for induction of the titan cell phenotype: the Ste3a pheromone receptor (in mating type a cells) and the Gpr5 protein. Both receptors control titan cell formation through elements of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. This conserved signaling pathway, in turn, mediates its effect on titan cells through the PKA-regulated Rim101 transcription factor. Additional downstream effectors required for titan cell formation include the G(1) cyclin Pcl103, the Rho104 GTPase, and two GTPase-activating proteins, Gap1 and Cnc1560. These observations support developing models in which the PKA signaling pathway coordinately regulates many virulence-associated phenotypes in diverse human pathogens.

Fig. 1.

Titan cell signaling in vivo is regulated by the Gpr5 receptor. Mice were infected intranasally with 2 × 10⁷ cells of wild-type KN99a or KN99α or with 2 × 10⁷ cells of the gpr4Δ, gpr5Δ, or gpr4Δ gpr5Δ mating type a or α mutant. At 3 days postinfection, BAL was performed. (A) Small cells (cell body diameter, <10 μm) and titan cells (cell body diameter, >10 μm) were quantified by microscopy. More than 300 cells per treatment per mouse were counted. Error bars indicate standard deviations for 3 to 5 mice per treatment. Statistical analysis of the difference between each mutant strain and the wild-type strain of the same mating type was performed. *, P < 0.02. (B) In vitro cells were grown overnight in rich medium (YPD), fixed in 3.7% formaldehyde, and examined by microscopy. In vivo BAL samples isolated at 3 days postinfection were fixed in 3.7% formaldehyde and were examined by microscopy. Bar, 20 μm.

Fig. 2.

Signaling via the Gα protein Gpa1 regulates titan cell production. Mice were infected intranasally with 2 × 10⁷ cells of wild-type KN99a or KN99α or with 2 × 10⁷ cells of the GPA1^Q284L, gpa2Δ, gpa3Δ, or gpa2Δ gpa3Δ mating type a or α mutant. At 3 days postinfection, BAL samples were collected. (A) Small cells (cell body diameter, <10 μm) and titan cells (cell body diameter, >10 μm) were quantified by microscopy. More than 300 cells were counted per treatment per mouse. Error bars indicate standard deviations for 3 to 5 mice per treatment. Statistical analysis of the difference between each mutant strain and the wild-type strain of the same mating type was performed. *, P < 0.02. (B) In vitro cells were grown overnight in rich medium (YPD), fixed in 3.7% formaldehyde, and examined by microscopy. In vivo BAL samples isolated at 3 days postinfection were fixed in 3.7% formaldehyde and were examined by microscopy. Bar, 20 μm.

Fig. 3.

Ste3a interacts with Gpa1 in the split-ubiquitin yeast two-hybrid system. The C-terminal half of ubiquitin (Cub) was fused to the C termini of the full-length cDNAs of Ste3a (Ste3a::Cub), Ste3α (Ste3α::Cub), and Gpr5 (Gpr5::Cub). The N-terminal half of ubiquitin (NubG) was fused to the C termini of full-length cDNAs of Gpa1 (Gpa1::NubG), Gpa2 (Gpa2::NubG), Gpa3 (Gpa3::NubG), Gpa1^G283A (Gpa1^G283A::NubG), and Gpa1^Q284L (Gpa1^Q284L::NubG). The interaction of Ste3a::Cub with the control vector pAI-Alg5 served as a positive control to ensure the correct topology of the Ste3a::Cub fusion protein; the interaction of Ste3a::Cub with the empty vector pDL2-Alg5 served as a negative control. Yeast transformants contained both a Cub fusion and a NubG fusion construct and were grown on selective medium lacking histidine or adenine after serial dilution. β-Galactosidase activity assays were performed to further verify interactions. Interactions between Ste3a (top) or Ste3α (center) and the Gα proteins are shown on selective media, as are control interactions (bottom).

Fig. 4.

Effects of coinfection on titan cell formation. Mice were intranasally infected with 2 × 10⁷ wild-type (KN99a or KN99α) or gpr4Δ gpr5Δ mutant cells labeled with Alexa Fluor 488 (Green) or were coinfected with one labeled and one unlabeled strain. Cells obtained by BAL were fixed and examined by microscopy for green fluorescence (cell type) and cell size. More than 300 cells were examined per animal, and the small cells (cell body diameter, <10 μm) and titan cells (cell body diameter, >10 μm) were quantified. Error bars indicate standard deviations for 3 to 4 mice per treatment.

Fig. 5.

Regulators of titan cell signaling. Mice were infected intranasally with 2 × 10⁷ cryptococcal cells. At 3 days postinfection, BAL samples were collected and fixed. Small cells (cell body diameter, <10 μm) and titan cells (cell body diameter, >10 μm) were quantified by microscopy. More than 300 cells were counted per treatment per mouse. (A) The rim101Δ mutant and a complemented (rim101Δ + RIM101) strain were assessed for titan cell formation. Error bars indicate standard deviations for 3 to 5 mice per treatment. The asterisk indicates a significant difference (P < 0.02) from the wild type. (B) cdc420Δ, cdc420Δ + CDC420, and cdc42Δ strains were assessed for titan cell formation. Error bars indicate standard deviations for 3 to 5 mice per treatment. The asterisk indicates a significant difference (P < 0.02) from the wild type. (C) Thirty-two mutant strains were assayed for titan cell formation. The standard deviation (dotted lines) for the wild type (open bar) was used to identify mutant strains that exhibit altered titan cell formation. (D) Four of the mutant strains for which results are shown in panel C were further analyzed for titan cell formation. For all these strains, the levels of titan cell formation are statistically significantly different from that of the wild type (P < 0.02). Error bars indicate standard deviations for 3 to 5 mice per treatment.