A unique fungal two-component system regulates stress responses, drug sensitivity, sexual development, and virulence of Cryptococcus neoformans

Abstract

The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal "two-component" system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component-like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen.

Figure 1.

The two-component system in C. neoformans. The domain structure of seven hybrid histidine kinases (Tco1–Tco7) and two response regulators (Ssk1 and Skn7). The letter “H” in the histidine kinase A phosphoreceptor domain indicates the histidine residue, and the letter “D” in the receiver domain indicates the aspartate residue. Conserved protein domains were identified by Pfam (version 18.0) HMM analysis at Washington University (St. Louis, MO).

Figure 2.

Response regulators Ssk1 and Skn7 govern Hog1-dependent and -independent stress responses and sensitivity to fludioxonil and MG. Each C. neoformans strain (serotype A WT [H99], hog1Δ [YSB64], pbs2Δ [YSB123], ssk1Δ [YSB261], ssk1Δ +SSK1 complemented [YSB428], and skn7Δ [YSB349]) was grown to mid-logarithmic phase in YPD medium, 10-fold serially diluted (1–10⁴ dilutions), and 3 μl of each cell suspension was spotted on YP medium containing 1 or 1.5 M NaCl or KCl (A), or 2 or 2.5 mM of H₂O₂, 100 μg/ml fludioxonil, and 15 or 20 mM MG (B). The hypothetical signaling cascade between response regulators and the Hog1 MAPK module is illustrated in A. To test high temperature and UV sensitivity (B), cells on YPD plates were incubated at 40°C and exposed to UV for 0.3 min (720 J/m²). Cells were further incubated for 2–4 d and photographed. (C) WT [H99], ssk1Δ [YSB261], and skn7Δ [YSB349] strains were grown to mid-logarithmic phase and exposed to 1 M NaCl, 20 mM MG, or 20 μg/ml fludioxonil in YPD medium for the time indicated, and total protein extracts were prepared for Western blot analysis. The dual phosphorylation status of Hog1 (T171 and Y173) was monitored using Phospho-p38 MAPK antibody (P-Hog1). The blots were stripped and reprobed with polyclonal anti-Hog1 antibody for the loading control (Hog1).

Figure 3.

Ssk1 and Skn7 response regulators negatively regulate sexual reproduction. (A) The following α and a strains were cocultured on V8 medium (pH 5.0) at room temperature in the dark: WT α × WT a (H99 × KN99a), hog1 α × hog1 a (YSB64 × YSB81), pbs2 α × pbs2 a (YSB123 × YSB125), ssk1 α × ssk1 a (YSB261 × YSB429), ssk1+SSK1 α × ssk1 a (YSB428 × YSB429), and skn7 α x skn7 a (YSB349 × YSB434). Representative edges of the mating patches were photographed at 100× magnification after 2-, 5-, or 7-d incubation. (B) The α WT (H99) or cac1Δ (YSB42) strains were cocultured as described (A) for 1 wk with the a WT (KN99a), hog1Δ (YSB81), pbs2Δ (YSB125), ssk1Δ (YSB429), and skn7 (YSB433) strains and photographed. (C) The α WT (H99) and crg1Δ (H99 crg1) strains were confronted with the a WT (KN99a), crg1Δ (PPW196), pbs2Δ (YSB125), ssk1Δ (YSB429), or skn7Δ (YSB434) strains, incubated for 7 d at room temperature in the dark, and photographed at 40× magnification.

Figure 4.

Enhanced capsule and melanin production by ssk1Δ and skn7Δ mutants. (A) For capsule production, WT [H99], hog1Δ [YSB64], pbs2Δ [YSB123], ssk1Δ [YSB261], ssk1Δ +SSK1 complemented [YSB428], and skn7Δ [YSB349] strains were grown overnight (∼16 h) in YPD medium, spotted on solid DMEM, incubated at 37°C for 2 d, and visualized by India ink staining. Bar, 10 μm. (B) For melanin production, the same strains were spotted on Niger seed medium containing 0.1, 1, or 2% glucose, incubated at 37°C for 3 d, and photographed.

Figure 5.

Two sensor histidine kinases, Tco1 and Tco2, govern a subset of Hog1-dependent stress responses and protect against fludioxonil and MG. (A) Multistress responses of WT (H99), hog1Δ (YSB64), ssk1Δ (YSB261), tco1Δ (YSB278), tco1Δ +TCO1 complemented (YSB355), tco2Δ (YSB281), tco2Δ +TCO2 complemented (YSB366), tco1Δ tco2Δ (YSB324), tco3Δ (YSB284), tco4Δ (YSB417), tco5Δ (YSB286), and tco7Δ (YSB348) were monitored as described in Figure 2 in YPD medium containing 2.5 or 3 mM H₂O₂, 100 μg/ml fludioxonil, or 15 or 20 mM MG. The hypothetical signaling cascade between the two-component system and the Pbs2-Hog1 MAPK module is illustrated.

Figure 6.

Sensor histidine kinases play redundant roles in activation of the Hog1 MAPK in response to fludioxonil and MG. Hog1 dual phosphorylation status was monitored by Western blot analysis in WT (H99), tco1Δ (YSB278), tco2Δ (YSB281), and tco1Δ tco2Δ (YSB324) in YPD medium containing 1.0 M NaCl (A), 20 mM MG (B), and 20 μg/ml fludioxonil (C) for the indicated times.

Figure 7.

Disruption of TCO1 impairs sexual reproduction of C. neoformans. (A) The following α and a strains were cocultured on V8 medium, pH 5.0, at room temperature in the dark: α × a (H99 × KN99a), tco1α × a (YSB278 × KN99a), tco1α × tco1a (YSB278 × YSB321), tco1 +TCO1α × tco1a (YSB355 × YSB321), tco1 + TCO1α × a (YSB355 × KN99a), tco2α × tco2a (YSB281 × YSB412), tco3α × tco3a (YSB284 × YSB416), tco4α × tco4a (YSB417 × YSB437), tco5α × tco5a (YSB286 × YSB419), and tco7α × tco7a (YSB348 × YSB423). Representative edges of the mating patches were photographed at 100x magnification after 1- or 2-wk incubation. (B) Cell fusion assays were performed with α × a (YSB119 and YSB121), tco1α × a (YSB278 × YSB121), tco1α × tco1a (YSB278 × YSB321). A mixture of equal numbers of NAT-marked α and NEO-marked a cells (5 × 10⁴ cells each) was incubated on V8 medium for 24 h at room temperature. Cells were harvested, spread, incubated at room temperature on YPD medium containing nourseothricin and G418 for 6 d, and photographed. (C) Morphology of dikaryotic colonies after cell fusion was photographed at 100× magnification after incubation at room temperature for 6 d. For tco1α × tco1a, small filamentous colonies are shown in the top right panel, and larger smooth colonies are shown in the bottom right panel.

Figure 8.

Tco1 negatively regulates melanin production. Melanin production in tco1Δ (YSB278), tco1Δ +TCO1 complemented (YSB355), tco2Δ (YSB281), tco1Δ tco2Δ (YSB324), tco3Δ (YSB284), tco4Δ (YSB417), tco5Δ (YSB286), and tco7Δ (YSB348) strains were monitored, compared with WT (H99) and hog1Δ (YSB64) strains, on the same Niger seed medium that was described in Figure 4. The plate was incubated at 37°C for 3 d and photographed.

Figure 9.

Virulence of tco1 and tco2 mutants. A/Jcr mice were infected with 10⁵ cells of WT (H99), tco1Δ (YSB278), tco1Δ +TCO1 complemented (YSB355), tco2Δ (YSB281), and tco2Δ +TCO2 complemented (YSB366) by intranasal inhalation. Percentage of survival (%) was monitored for 33 d after inoculation.

Figure 10.

Proposed model for the functional connection of the Pbs2-Hog1 MAPK pathway with the fungal two-component system in C. neoformans. Under normal conditions, the Hog1 MAPK is constitutively phosphorylated (inactive form) via the Pbs2 MAPKK and an unknown MAPKKK. Phosphorylated Hog1 MAPK functions to repress capsule and melanin production and sexual development under normal growth conditions. Because the Tco1 and Tco2 sensor kinases do not significantly affect the constitutive phosphorylation of Hog1, another sensor or internal signaling enables Ssk1 to trigger constitutively phosphorylate the Hog1 MAPK. In response to diverse stresses or exposure to the antifungal drug fludioxonil or the toxic metabolic by-product MG, a variety of sensor kinases, including Tco1 and Tco2, or sensor-independent internal signaling further activates Ssk1, which in turn activates a Hog1-specific phosphatase to dephosphorylate and activate the Hog1 MAPK.