Transcription factor Nrg1 mediates capsule formation, stress response, and pathogenesis in Cryptococcus neoformans

Abstract

The Cryptococcus neoformans NRG1 gene was identified using gene microarrays to define putative transcription factor genes regulated by the cyclic AMP (cAMP) signal transduction pathway. Disruption of NRG1 results in delayed capsule formation and mating, two phenotypes that are directly controlled by cAMP signaling. Putative targets of the Nrg1 transcription factor were identified using a second genome microarray to define differences in the transcriptomes of the wild-type and nrg1 mutant strains. These experiments implicate Nrg1 in the transcriptional control of multiple genes involved in carbohydrate metabolism and substrate oxidation, as well as the UGD1 gene encoding a UDP-glucose dehydrogenase required for polysaccharide capsule production and cell wall integrity. In addition to being under transcriptional control of the cAMP pathway, Nrg1 contains a putative protein kinase A phosphorylation site; mutation of this motif results in reduced Nrg1 activity. Consistent with prior studies in hypocapsular mutants, the nrg1 mutant strain is attenuated in an animal model of disseminated cryptococcal disease.

FIG. 1.

NRG1 is transcriptionally regulated by cAMP and glucose. (A) The wild-type (WT) and gpa1 mutant strains were incubated to mid-logarithmic phase in synthetic complete medium. The cells were then incubated in synthetic complete medium with either 2% glucose (+) or 0.1% glucose (−) for 30 min at 30°C. Total RNA was assessed by Northern analysis, using the NRG1 gene as a probe. Equal loading of RNA was assessed by ethidium bromide staining, with the rRNA bands demonstrated. (B) Total RNA was isolated from the following strains after 30 min of glucose deprivation of log-phase cells: wild type (WT; H99), gpa1 mutant (AAC1), nrg1 mutant (KLC27), two nrg1+NRG1 reconstituted strains (QGC31 and QGC32), and three nrg1+nrg1-C529G strains (QGC33, QGC34, and QGC35). Quantitative real-time PCR was performed on the corresponding cDNA samples to assess relative NRG1 transcript abundance compared to the wild-type strain ± one standard deviation.

FIG. 2.

Alignment of C. neoformans Nrg1 and S. cerevisiae Nrg1 and Nrg2 proteins. (A) The C2H2 DNA-binding domains of the C. neoformans (Cn), C. albicans (Ca), and S. cerevisiae (Sc) Nrg proteins demonstrate notable sequence similarity within in this functional region. (B) A graphical representation of the Nrg proteins of C. neoformans, C. albicans, and S. cerevisiae, demonstrating the relative positions of the Zn finger domains and PKA consensus sequence for phosphorylation.

FIG. 3.

Capsule formation induction defect of the nrg1 mutant. The following strains were incubated in capsule formation-inducing conditions (Dulbecco's modified Eagle's medium with 25 mM NaHCO₃, 5% CO₂) for 48 h: the NRG1 wild type (WT; strain H99), the nrg1 mutant (strain KLC27), the nrg1 mutant reconstituted with a wild-type copy of NRG1 (nrg1+NRG1 [strain QGC31]), and the nrg1 mutant reconstituted with an nrg1 allele with a mutation in the putative PKA phosphorylation sequence (nrg1+nrg1-C529G [strain QGC34]). Capsule size was assessed by counterstaining with India ink (×400).

FIG. 4.

Altered cell surface phenotypes of the nrg1 mutant. (A) Cell-cell adherence. The wild-type (WT), nrg1, nrg1+NRG1, and nrg1+nrg1-C529G strains were incubated in YPD medium for 24 and 72 h. The cultures were vortexed and examined microscopically (×400). (B) Flocculation. The wild-type (squares), nrg1 (circles), nrg1+NRG1 (diamonds), and nrg1+nrg1-C529G (triangles) strains were incubated for 24 h in synthetic complete medium, and each culture was normalized to an OD₆₀₀ of 1.5. The OD₆₀₀ of the resulting cell suspension was serially measured to assess cell flocculation. Data points represent the means of three data points ± standard deviations. (C) Susceptibility to cell wall stresses. The wild-type (WT), nrg1, nrg1+NRG1, and nrg1+nrg1-C529G strains were serially diluted and incubated on YPD medium containing no additives (control), 0.01% SDS (48-h incubation), 0.1% SDS, 2.5 M sorbitol, or 1.5 M NaCl (7-day incubation).

FIG. 5.

Mating defect of the nrg1 mutant. The wild-type, nrg1 mutant, and nrg1+NRG1 reconstituted strains were incubated in mating reaction mixtures on V8 medium with the congenic MATa wild-type strain KN99a. The edges of the mating patches were microscopically assessed for mating filaments at 7 days and photographed (×40).

FIG. 6.

Virulence defect of the nrg1 mutant. The wild-type, nrg1 mutant, and nrg1+NRG1 reconstituted strains were intranasally inoculated into A/Jcr mice. Animal survival was measured over the course of the 52-day experiment.