Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite- and same-sex mating in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1alpha/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell-cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central to fungal development and virulence.

Figure 1. Deletion of either the MAT2 or the ZNF2 gene impairs filamentation during a-α and α-α mating.

(A) JEC21α, XL280α, and corresponding mat2Δ and znf2Δ mutants (XL XL926, XL576, XL942, and XL574) were individually incubated on V8 medium for 1 week in the dark at 22°C to examine the ability to differentiate. (B) Appropriate α and a mating partners of wild type (JEC21 and JEC20), and mat2Δ (XL926 and XL961) and znf2Δ (XL576 and XL879) mutants in the JEC21α background were mixed and co-cultured on filamentation agar medium for 48 hours in the dark at 22°C to examine filamentation during unilateral and bilateral matings.

Figure 2. Znf2 is required for hyphal formation after cell-cell fusion during a-α mating.

The schematic diagram depicts the mating process of wild-type strains. Haploid α and a yeast cells were co-incubated and the cell-cell fusion products, when selected at high temperatures, will undergo nuclear fusion and become heterozygous α/a diploid yeast cells. When the environment becomes favorable for filamentation (by lowering the temperature), the α/a diploid cells will undergo a morphological transition, produce hyphae, and eventually undergo meiosis and sporulate (not shown here). Appropriately marked α and a strains of wild type (XL877 and XL878) and znf2Δ mutants (XL874 and XL875) were paired, mixed, and co-cultured on V8 medium for 15 hours at room temperature in the dark. The cocultures were collected and transferred to YNB minimal medium to select for fusion products as shown in the left panel. Microscopic images of the colony derived from the fusion event are shown in the right panel. Scale bar, 200 micrometers.

Figure 3. Mat2 is required for pheromone sensing and production, whereas Znf2 is dispensable for the response during a-α mating.

(A) Northern blot analysis of the expression pattern of the MFα gene during a-α mating in wild type, mat2Δ, znf2Δ, and ste7Δ mutants in JEC21 background at 0 hours, 6 hours, 15 hours, and 24 hours post coinoculation of the a and α mating partners. The expression level of the actin gene (ACT1) serves as a control. (B) Confrontation assays of the effect of mat2 and znf2 mutations on the ability of a and α cells to produce and respond to pheromones. Scale bar, 200 micrometers.

Figure 4. Znf2 and the Sxi1α/2a complex do not regulate Mat2 or the Cpk1 pathway at the transcript level.

The expression pattern of the CPK1, MAT2, SXI1α, and ZNF2 genes during bilateral matings in the following strain pairs (α × a, α znf2Δ × a znf2Δ, α mat2Δ × a mat2Δ, α ste7Δ × a ste7Δ, and sxi1αΔ × sxi2 aΔ in JEC21 background) that had been cocultured on V8 medium (pH = 7.0) for 24 hr.

Figure 5. Mat2 and the Cpk1 MAPK cascade are not required for hyphal morphogenesis whereas Znf2 is a hyphal morphology determinant.

The schematic diagram shows that if haploid a cells that harbor the SXI2 a gene in their genome are transformed with the SXI1α gene (indicated by the Sxi1α above the arrow), both the SXI1α and SXI2 a genes will be expressed in the a cells. The protein complex formed by these two cell-identity proteins renders the haploid a cells competent to produce hyphae like a/α diploid or a-α dikaryons without the requirement for the fusion of α and a cells. The a cells with ste7Δ, mat2Δ, or znf2Δ mutation in JEC21 background and the corresponding mutants bearing the P_GPD1-SXI1α transgene were cultured on filamentation agar medium and incubated in the dark for 48 hours at 22°C.

Figure 6. Gene expression profiles of ste7Δ, mat2Δ, and znf2Δ mutants during bilateral mating.

The top panel shows the nearly identical profiles between ste7Δ and mat2Δ mutants with 96% overlap in contrast to only 47% congruence with genes differentially expressed in znf2Δ mutants. The bottom panel shows a classification of the genes that were differentially expressed in the mutants based on GO ontology.

Figure 7. Mat2 is dispensable for virulence, while Znf2 is a negative regulator of pathogenicity.

Animals (10 each group) were intranasally infected with 5×10⁴ yeast cells of the wild type (H99), ste7Δ (YSB345), mat2Δ (XL1598), znf2Δ (XL1601), and znf2Δ-ZNF2 (XL1643) strains. The survival rates of animals were plotted against time after inoculation, and P values compared to the wild type control are: ste7Δ (P = 0.68), mat2Δ (P = 0.048), znf2Δ (P = 0.0026), znf2Δ+ZNF2 (P = 0.00034).

Figure 8. Mat2 and Znf2 operate cellular circuits orchestrating opposite- and same-sex mating in C. neoformans.

(A) Mat2 is depicted as the direct target of the MAPK pathway and it further serves to regulate the homodomain complex and Znf2. Genes encoded by the MAT locus are underlined. The dotted arrow indicates predicted interactions. (B) HMG domain proteins serve as the transcription factor downstream of the MAPK in the pheromone sensing pathways of divergent fungal species. Cn: C. neoformans. Um: U. maydis. Sp: S. pombe. Sc: S. cerevisiae.