Hrk1 plays both Hog1-dependent and -independent roles in controlling stress response and antifungal drug resistance in Cryptococcus neoformans

Abstract

The HOG (High Osmolarity Glycerol response) pathway plays a central role in controlling stress response, ergosterol biosynthesis, virulence factor production, and differentiation of Cryptococcus neoformans, which causes fatal fungal meningoencephalitis. Recent transcriptome analysis of the HOG pathway discovered a Hog1-regulated gene (CNAG_00130.2), encoding a putative protein kinase orthologous to Rck1/2 in Saccharomyces cerevisiae and Srk1 in Schizosaccharomyces pombe. Its function is not known in C. neoformans. The present study functionally characterized the role of Hrk1 in C. neoformans. Northern blot analysis confirmed that HRK1 expression depends on the Hog1 MAPK. Similar to the hog1Δ mutant, the hrk1Δ mutant exhibited almost complete resistance to fludioxonil, which triggers glycerol biosynthesis via the HOG pathway. Supporting this, the hrk1Δ mutant showed reduced intracellular glycerol accumulation and swollen cell morphology in response to fludioxonil, further suggesting that Hrk1 works downstream of the HOG pathway. However, Hrk1 also appeared to have Hog1-independent functions. Mutation of HRK1 not only further increased osmosensitivity of the hog1Δ mutant, but also suppressed increased azole-resistance of the hog1Δ mutant in an Erg11-independent manner. Furthermore, unlike the hog1Δ mutant, Hrk1 was not involved in capsule biosynthesis. Hrk1 was slightly involved in melanin production but dispensable for virulence of C. neoformans. These findings suggest that Hrk1 plays both Hog1-dependent and -independent roles in stress and antifungal drug susceptibility and virulence factor production in C. neoformans. Particularly, the finding that inhibition of Hrk1 substantially increases azole drug susceptibility provides a novel strategy for combination antifungal therapy.

Figure 1. Identification of Hog1-regulated kinase, Hrk1, in C. neoformans.

(A) The relative expression levels of the HRK1 gene (CNAG_00130.2) from microarray data using total RNA isolated from the WT (H99) strain and ssk1Δ, skn7Δ, and hog1Δ mutants grown to the middle logarithmic phase at 30°C in YPD medium . (B) Northern blot analysis for measuring basal expression levels of HRK1 using total RNA of (A). (C) The phylogenetic analysis of Hrk1 orthologs between C. neoformans and other eukaryotes. The following proteins were compared for the analysis: S. cerevisiae Rck1 (YGL158W, SGDID: S000003126), S. cerevisiae Rck2 (YLR248W, SGDID: S000004238), Homo sapiens MAPKAPK-2, N. crassa Hrk1 (NCU09212.4), A. nidulans Hrk1 ortholog (AN4483, AspGDID: ASPL0000072431), U. maydis Hrk1 ortholog (UM02121), C. neoformans Hrk1 (CNAG_00130.2) and S. pombe Srk1 (SPCC1322.08). The phylogenetic tree was generated by CLUSTAL W (tree-building method, neighbor joining) with MacVector software (version 7.2.3; Accelrys). (D) Multiple sequence alignment of Hrk1 orthologs is depicted by Clustal W alignment from MacVector software.

Figure 2. Analysis of stress-dependent expression patterns of HRK1 in C. neoformans.

Total RNA was isolated from WT and the ssk1Δ and hog1Δ mutants grown in YPD medium containing 1 M NaCl for osmotic stress (A), 2.5 mM H₂O₂ for oxidative stress (B), or 40 µg/ml fludioxonil for antifungal drug treatment (C) at different time points (0, 30 and 60 min). For quantitative RT-PCR (qRT-PCR), data collected from three independent biological replicates with three technical replicates were normalized by using ACT1 as a control. Relative gene expression indicates HRK1 expression levels of each strain and time point compared to those of the wild-type strain at zero time point (unstressed condition).

Figure 3. The role of Hrk1 in osmotic stress response of C. neoformans.

Each C. neoformans strain was grown overnight at 30°C in liquid YPD medium, 10-fold serially diluted (1–10⁴ dilutions), and spotted (4 µl of dilution) on YPD (+glucose) or YP (−glucose) agar containing 1.5 M NaCl and 1.5 M KCl. Cells were incubated at 30°C for 72 h and photographed.

Figure 4. The role of Hrk1 in fludioxonil susceptibility and intracellular glycerol synthesis of C. neoformans.

(A) Each strain grown was 10-fold serially diluted (1–10⁴ dilutions), and spotted (4 µl of dilution) on YPD agar containing indicated concentrations of fludioxonil. Cells were incubated at 30°C for 72 h and photographed. (B–C) Each strain was grown to the middle logarithmic phase in liquid YPD medium, reincubated in YPD or YPD medium containing fludioxonil (10 µg/ml) at 30°C for 48 hrs with shaking, and then photographed (B). Diameter of each strain treated with or without fludioxonil was quantitatively measured by using SPOT image analysis software (Diagnostic Instrument Inc.). The Y-axis indicates average diameter of the cell (µm, micron). (D) To quantitatively measure accumulation of intracellular glycerol content, each strain grown as described in Fig. 4B was reincubated in YPD containing fludioxonil (10 µg/ml) for the indicated incubation time. Glycerol content in cell extracts was measured by a UV-glycerol assay kit as described in Materials and Methods. Three independent experiments were performed. Standard deviations are presented as error bars. Statistical differences in relative cell diameter (C) or intracellular glycerol content (D) between strains were determined by Bonferroni's multiple comparison test. Each symbol indicates the following: *, P<0.05; **, P<0.01; ***, P<0.0001; NS, not significant (P>0.05).

Figure 5. The role of Hrk1 in oxidative stress response of C. neoformans.

Each C. neoformans strain was 10-fold serially diluted (1–10⁴ dilutions), and spotted (4 µl of dilution) on YPD agar containing the indicated concentration of diamide or hydrogen peroxide (H₂O₂). Cells were incubated at 30°C for 72 hrs and photographed.

Figure 6. The role of Hrk1 in susceptibility to polyene and azole antifungal drugs of C. neoformans.

(A) Each C. neoformans strain was 10-fold serially diluted (1–10⁴ dilutions), and spotted (4 µl of dilution) on YPD agar containing indicated concentrations of amphotericin B, fluconazole, or ketoconazole. Cells were incubated at 30°C for 72 hrs and photographed. (B–C) Northern blot analysis for examining expression profiles of ERG11 in hog1Δ, hrk1Δ, and hrk1Δ hog1Δ mutants compared to the wild-type strain (H99) (B). The expression levels of ERG11 relative to ACT1 were measured via phosphorimager (Fuji BAS 2500) (C). The Y-axis indicates ERG11 expression levels relative to ACT1, which is percent ratio of ERG11 expression level vs. ACT1 expression level. The hog1Δ and hrk1Δ hog1Δ, but not hrk1Δ, mutants showed higher ERG11 expression levels compared to the wild-type strain.

Figure 7. The role of Hrk1 in capsule and melanin production and virulence of C. neoformans.

(A) Each C. neoformans strain were spotted and cultured on DME medium for capsule production at 37°C for 2 days. Capsule was visualized by staining with India ink and observed by microscopy (Bar, 10 µm). Quantitative measurements of the relative capsule volume (right panel). The packed volume of the cells 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 were determined by Bonferroni's multiple comparison test. Error bars indicate the standard deviations. *, P<0.001. (B) Each strain was spotted and grown on Niger seed medium (glucose 0.1, 0.5, and 1%) at 30°C or 37°C for 5 days. (C) For virulence assay, groups of six- to eight-week old female A/J mice were infected with 5×10⁴ cells in 0.05 ml PBS by intranasal inhalation. Percent survival (%) was monitored daily until all mice were sacrificed. The hrk1Δ mutant is as virulent as the WT strain.

Figure 8. The proposed model for function and regulation of C. neoformans Hrk1.

Basal and stress-induced expressions of HRK1 are governed by the Hog1 MAPK. An unknown signaling component upstream of the Ssk2 MAPKKK, other than the Ssk1 response regulator, may trigger the Hog1 MAPK module (Ssk2-Pbs2-Hog1) in response to osmotic and oxidative stresses. Hrk1 is involved in intracellular glycerol synthesis upon fludioxonil treatment via the HOG pathway. However, Hrk1 appears to play several Hog1-independent roles. Independent of the HOG pathway modulating ergosterol biosynthesis, Hrk1 promotes susceptibility to polyene and azole drugs. Furthermore, Hrk1 is partly involved in promoting resistance to oxidative damaging agents, such as diamide, and melanin biosynthesis, which is in stark contrast to Hog1. In conclusion, Hrk1 plays both Hog1-dependent and –independent roles in C. neoformans.