Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway

Abstract

The mitogen-activated protein kinase (MAPK) pathways control diverse cellular functions in pathogenic fungi, including sexual differentiation, stress response, and maintenance of cell wall integrity. Here we characterized a Cryptococcus neoformans gene, which is homologous to the yeast Ste50 that is known to play an important role in mating pheromone response and stress response as an adaptor protein to the Ste11 MAPK kinase kinase in Saccharomyces cerevisiae. The C. neoformans Ste50 was not involved in any of the stress responses or virulence factor production (capsule and melanin) that are controlled by the HOG and Ras/cAMP signaling pathways. However, Ste50 was required for mating in both serotype A and serotype D C. neoformans strains. The ste50Δ mutant was completely defective in cell-cell fusion and mating pheromone production. Double mutation of the STE50 gene blocked increased production of pheromone and the hyper-filamentation phenotype of cells deleted of the CRG1 gene, which encodes the RGS protein that negatively regulates pheromone responsive G-protein signaling via the MAPK pathway. Regardless of the presence of the basidiomycota-specific SH3 domains of Ste50 that are known to be required for full virulence of Ustilago maydis, Ste50 was dispensable for virulence of C. neoformans in a murine model of cryptococcosis. In conclusion, the Ste50 adaptor protein controls sexual differentiation of C. neoformans via the pheromone-responsive MAPK pathway but is not required for virulence.

Fig. 1

Comparison of Ste50 orthologs between C. neoformans and other fungi. (A) Each Ste50 ortholog diagram shows functional protein domains, which were identified by the Pfam 24.0 database (http://pfam.sanger.ac.uk/). Each domain indicates the following: SAM, sterile alpha motif; RA, Ras- associated; SH3, Src homology 3. (B) Multiple sequence alignment of Ste50 orthologs is depicted by Clustal W alignment from MacVector software (versions 7.2.3, Accelrys). Protein sequences of Ste50 orthologs were retrieved from the following database: C. neoformans Ste50 - CNAG_07507 from the C. neoformans var. grubii H99 database of the Broad Institute (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html); Ustilago maydis Ubc2 - GenBank accession number AAK4932; Coprinus cinereus - locus CC1G_00975.3 from the C. cinereus Sequencing Project Database of the Broad Institute. (http://www.broadinstitute.org/annotation/genome/coprinus_cinereus/MultiHome.html); Phanerochaete chrysosporium Ste50 ortholog - protein ID 2321 at locus Phchr1/scaffold_3:1012644-101605 from the sequencing data produced by the United States Department of Energy, Joint Genome Institute (http://genome.jgi-psf.org/Phchr1/Phchr1.home.html); Neurospora crassa Ste50 ortholog - GenBank accession number XP_956774; Magnaporthe grisae Mst50 - GenBank accession number XP_359578; Candida albicans Ste50 - GenBank accession number XP_721713; Saccharomyces cerevisiae Ste50 - GenBank accession number NP_009898; Schizosaccharomyces pombe Ste4; GenBank accession number CAB38684.

Fig. 2

Ste50 interacts with Ste11 in C. neoformans. Two-hybrid assay was performed with C. neoformans Ste50 and Ste11. β-galactosidase activity of a LacZ reporter gene in the PJ69-4A strain co-transformed with indicated vectors were measured with extracts of two independent Leu⁺ Trp⁺ transformants cultured in SD-Leu-Trp medium. Error bar indicates standard deviation.

Fig. 3

Ste50 is not involved in the C. neoformans stress response. C. neoformans strains (the wild-type strain H99 and hog1Δ (YSB64), ste11Δ (YSB313), and ste50Δ (YSB317, YSB318, and YSB319) mutants) was grown overnight at 30°C in liquid YPD medium, 10-fold serially diluted (1–10⁴ dilutions), and spotted (3 μl of dilution) on YPD or YP agar containing the indicated concentrations of KCl, NaCl, SDS, Methylglyoxal, Congo red (A), hydrogen peroxide (H₂O₂), diamide, CdSO₄ (B), amphotericin B, ketoconazole, fluconazole, fludioxonil, and hydroxyurea (C). Cells were incubated at 30°C for 72 h and photographed. For the thermotolerance test (B), cells were spotted on YPD medium and incubated at 40°C for 4 days.

Fig. 4

Ste50 is not involved in capsule and melanin production of C. neoformans. (A) The following strains were spotted and grown on L-DOPA medium (glucose 0.1%) and Niger seed medium (glucose 0.1%) at 30°C or 37°C for 5 days: WT (H99) and ste50Δ (YSB317), hog1Δ (YSB64) and cac1Δ (YSB42) mutant strains. (B) The WT strain H99, hog1Δ (YSB64), cac1Δ (YSB42), ste50Δ (YSB317) and ste50Δ + STE50 (YSB564) strains were spotted and cultured on DME medium for capsule production at 37°C for 2 days. Cells were scraped, resuspended in distilled water, and visualized by India ink staining The packed volume of the cells (the wild-type strain [H99] and ste50Δ [YSB317] and ste50Δ + STE50 [YSB564] strains) was measured by calculating the ratio of the length of packed cell volume phase/length of total volume phase. Statistical differences in relative capsule size between strains was determined by Bonferroni’s multiple comparison test.

Fig. 5

Ste50 is required for sexual differentiation in both serotype A and serotype D C. neoformans strains. (A) Serotype A MATα and MATa strains were co-cultured on V8 medium (pH 5.0) for 2 weeks at room temperature in the dark: WT α × WT a (H99 and KN99), hog1Δ (α) × hog1Δ (a) (YSB64 and YSB81), ste11Δ (α)× WT (a) (YSB313 and KN99), ste50Δ (α) × WT (a) (YSB317 and KN99), ste50Δ (α) × ste50Δ (a) (YSB317 and YSB523), and ste50Δ + STE50 (α) × WT (a) (YSB564 and KN99). The images were photographed after 8 days and 14 days. (B) The following serotype D strains were co-cultured on V8 medium in the dark at room temperature up to 5 days and photographed after 2 days: JEC21 and JEC20 (α × a), YSB593 and JEC20 (ste50Δ × a)

Fig. 6

Ste50 governs sexual differentiation via the pheromone-responsive Cpk1 MAPK pathway. (A) Cell-cell fusion assays were performed with the following strains: WT α (NAT) × WT a (NEO) (YSB119 and YSB121), ste50Δ α (NAT) × WT a (NEO) (YSB317 and YSB121), WT α (NAT) × ste50Δ a (NEO) (YSB119 and YSB522), ste50Δ α (NAT) × ste50Δ a (NEO) (YSB317 and YSB522). Cell fusion efficiency for each experimental set was calculated relative to the control strains (WT α (NAT) × WT a (NEO)). (B) Confrontation assays were performed with the following strains: MATα crg1Δ (H99 crg1), MATa crg1Δ (PPW 196), MATα crg1Δ aca1Δ (YSB96), MATα crg1Δ ste50Δ (YSB632), and MATa crg1Δ ste50Δ (YSB637). Indicated strains were streaked in confrontation with each other on V8 agar medium and incubated at room temperature in the dark. Images were photographed after 10 days. (C) Mating reactions were initiated for the following strains: The crg1Δ ste50Δ double mutants showed mating defect in both unilateral crossing and bilateral crossing. crg1Δ α × crg1Δ a (H99 crg1 and PPW 196), crg1Δ α × crg1Δ ste50Δ a (H99 crg1 and YSB637), crg1Δ ste50Δ α × crg1Δ a (YSB632 and PPW 196), crg1Δ ste50Δ α × crg1Δ ste50Δ a (YSB632 and YSB637). The images were photographed after 12 days. (D) Northern blot analysis for monitoring pheromone gene expression was performed with total RNA isolated from solo- or co-cultures of the indicated strain(s) grown for 24 hr under mating conditions: WTα (H99), WTa (KN99a), ste50Δα (YSB317), and ste50Δa (YSB522). The blot was probed with the MFα1 gene.

Fig. 7

Ste50 is dispensable for virulence and titan cell formation of C. neoformans. (A) For virulence assays, groups of ten C57/BL6 or groups of six-ten A/J mice (see Materials and Methods) were infected with 5×10⁴ cells of MATα WT (●: H99), ste50Δ (■: YSB317), and ste50Δ+STE50 complemented (▴: YSB564) strains by intranasal inhalation. Percent survival (%) was monitored daily until all mice were sacrificed. The ste50Δ mutant is as virulent as the WT strain. In both experiments, the P-values for the C57/BL6 experiment were: 0.35, 0.001, and 0.001 for H99/ste50Δ, ste50Δ/ste50Δ+STE50, and H99/ste50Δ+STE50, respectively. P-values for the A/J experiment were 0.57, 0.03, and 0.23 for H99/ste50Δ, ste50Δ/ste50Δ+STE50, and H99/ste50Δ+STE50, respectively. (B) For titan cell formation assay, A/J mice were infected with 1×10⁶ cells of the wild-type (H99) and ste50Δ mutant (YSB317) strains and were sacrificed at 3 days post-infection. Cells in the lung lavage fluid were fixed with 3.7% formaldehyde and photographed. Bar = 10 μm, arrows denote titan cells.