Journal: mBio

Article Title: Unraveling Melanin Biosynthesis and Signaling Networks in Cryptococcus neoformans

doi: 10.1128/mBio.02267-19

Figure Lengend Snippet: Melanin-regulating core transcription factors in C. neoformans . (A) C. neoformans WT and mutant strains on Niger seed, dopamine, and epinephrine medium. Darker cultures had more effective melanin synthesis than those with lighter colors. (B to F) Quantitative reverse transcription-PCR (RT-PCR) was performed using total RNA of each strain under nutrient-rich (R; YPD) or nutrient-starved (S; YNB without glucose) conditions. The induction of (B) LAC1 , (C) BZP4 , (D) USV101 , (E) HOB1 , and (F) MBS1 by nutrient starvation was measured in WT and mutant strains. Three biologically independent experiments were performed with three technical replicates each. Error bars indicate standard errors of the means (SEM). Statistical differences among gene expression levels were calculated by one-way analysis of variance (ANOVA) multiple comparisons performed with Bonferroni’s correction (*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant). (G) Each strain was spotted on medium plates containing dopamine and epinephrine. Darker cultures had more effective melanin synthesis than those with lighter colors. (H) Proposed regulatory relationships among Hob1, Mbs1, Usv101, and Bzp4 for melanin production.

Article Snippet: Each strain was spotted (3 μl) on Niger seed medium (70 g Niger seed and 20 g Bacto agar per liter), dopamine medium, or epinephrine agar medium (1 g l -asparagine, 3 g KH 2 PO 4 , 250 mg MgSO 4 , 1 mg thiamine, 5 μg biotin, and 100 mg l -DOPA or epinephrine hydrochloride per liter) with a limited glucose concentration (0.1% or 0.2%).

Techniques: Mutagenesis, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: mBio

Article Title: Unraveling Melanin Biosynthesis and Signaling Networks in Cryptococcus neoformans

doi: 10.1128/mBio.02267-19

Figure Lengend Snippet: Cellular localization of the melanin-regulating core TFs in C. neoformans . (A to C) C. neoformans WT, lac1 Δ, and pka1 Δ with (A) bzp4 Δ, bzp4 Δ:: BZP4-mCherry , bzp4 Δ:: BZP4-mCherry pka1 Δ, (B) usv101 Δ, usv101 Δ:: USV101-mCherry , usv101 Δ:: USV101-mCherry pka1 Δ, and (C) mbs1 Δ, mbs1 Δ:: MBS1-mCherry , mbs1 Δ:: MBS1-mCherry pka1 Δ mutant strains on Niger seed, dopamine, and epinephrine medium. Darker cultures had more effective melanin synthesis than those with lighter colors. (D to F) Localization of the mCherry-tagged (D) Bzp4, (E) Usv101, and (F) Mbs1 with or without PKA1 deletion was observed. The cells were harvested under basal (nutrient-rich, YPD) conditions and nutrient-starved (YNB without glucose) conditions and stained with Hoechst 33342 to visualize the nucleus. The cells were observed by fluorescence microscopy. Bars, 10 μm.

Article Snippet: Each strain was spotted (3 μl) on Niger seed medium (70 g Niger seed and 20 g Bacto agar per liter), dopamine medium, or epinephrine agar medium (1 g l -asparagine, 3 g KH 2 PO 4 , 250 mg MgSO 4 , 1 mg thiamine, 5 μg biotin, and 100 mg l -DOPA or epinephrine hydrochloride per liter) with a limited glucose concentration (0.1% or 0.2%).

Techniques: Mutagenesis, Staining, Fluorescence, Microscopy

Journal: mBio

Article Title: Unraveling Melanin Biosynthesis and Signaling Networks in Cryptococcus neoformans

doi: 10.1128/mBio.02267-19

Figure Lengend Snippet: Melanin-regulating kinases in C. neoformans . (A) C. neoformans WT and mutant strains on Niger seed, dopamine, and epinephrine medium. Darker cultures had more effective melanin synthesis than those with lighter colors. (B to F) Quantitative RT-PCR was performed using total RNA of each strain under nutrient-rich (R; YPD) or nutrient-starved (S; YNB without glucose) conditions. The induction of (B) LAC1 , (C) BZP4 , (D) HOB1 , (E) USV101 , and (F) MBS1 by nutrient starvation was measured in WT and mutant strains. Three biologically independent experiments were performed with three technical replicates each. Error bars indicate SEM, and statistical differences among gene expression levels were calculated by one-way ANOVA multiple comparisons with Bonferroni’s correction (*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant).

Article Snippet: Each strain was spotted (3 μl) on Niger seed medium (70 g Niger seed and 20 g Bacto agar per liter), dopamine medium, or epinephrine agar medium (1 g l -asparagine, 3 g KH 2 PO 4 , 250 mg MgSO 4 , 1 mg thiamine, 5 μg biotin, and 100 mg l -DOPA or epinephrine hydrochloride per liter) with a limited glucose concentration (0.1% or 0.2%).

Techniques: Mutagenesis, Quantitative RT-PCR, Gene Expression

Journal: mBio

Article Title: Unraveling Melanin Biosynthesis and Signaling Networks in Cryptococcus neoformans

doi: 10.1128/mBio.02267-19

Figure Lengend Snippet: Bzp4-mCherry and Usv101-mCherry constitutively localized in the nucleus. (A and B) C. neoformans WT, lac1 Δ, gsk3 Δ, and kic1 Δ with (A) bzp4 Δ, bzp4 Δ:: BZP4-mCherry , and bzp4 Δ:: BZP4-mCherry pka1 Δ strains and (B) usv101 Δ, usv101 Δ:: USV101-mCherry , and usv101 Δ:: USV101-mCherry pka1 Δ mutant strains on Niger seed, dopamine, and epinephrine medium. Darker cultures had more effective melanin synthesis than those with lighter colors. (C and D) Localization of the mCherry-tagged (C) Bzp4 and (D) Usv101 with GSK3 or KIC1 deletion. The cells were harvested under basal (nutrient-rich, YPD) and nutrient-starved (YNB without glucose) conditions and stained with Hoechst 33342 to visualize the nucleus. The cells were observed by fluorescence microscopy. Bars, 10 μm.

Article Snippet: Each strain was spotted (3 μl) on Niger seed medium (70 g Niger seed and 20 g Bacto agar per liter), dopamine medium, or epinephrine agar medium (1 g l -asparagine, 3 g KH 2 PO 4 , 250 mg MgSO 4 , 1 mg thiamine, 5 μg biotin, and 100 mg l -DOPA or epinephrine hydrochloride per liter) with a limited glucose concentration (0.1% or 0.2%).

Techniques: Mutagenesis, Staining, Fluorescence, Microscopy

Journal: mBio

Article Title: Unraveling Melanin Biosynthesis and Signaling Networks in Cryptococcus neoformans

doi: 10.1128/mBio.02267-19

Figure Lengend Snippet: Vesicle trafficking-related genes were involved in melanin production in C. neoformans . (A) Graphic illustration of predicted Vps15/34/30 complex based on the function of S. cerevisiae . CPY, carboxypeptidase Y. (B) C. neoformans WT and mutant strains on Niger seed, dopamine, and epinephrine medium. Darker cultures had more effective melanin synthesis than those with lighter colors. (C) Localization of VPS15-mCherry, VPS30-mCherry, and VPS34-GFP under basal (nutrient-rich, YPD) conditions. LysoSensor or FM4-64 was used as a vacuole-staining dye. The cells were observed by fluorescence microscopy. Bars, 10 μm. (D) Phenotypic heat map of vps15 Δ, vps30 Δ, and vps34 Δ mutants (see data in Fig. S5 ). T25, T30, T37, and T39, growth rates at 25°C, 30°C, 37°C, and 39°C; MEL, melanin production levels; KCR, YPD plus KCl; NCR, YPD plus NaCl; SBR, YPD plus sorbitol; KCS, YP plus KCl; NCS, YP plus NaCl; SBS, YP plus sorbitol; DIA, diamide; HPX, hydrogen peroxide; MD, menadione; TBH, tert -butyl hydroperoxide; CDS, cadmium sulfide; HU, hydroxyurea; MMS, methyl methanesulfonate; TM, tunicamycin; DTT, dithiothreitol; CFW, calcofluor white; CR, Congo red; SDS, sodium dodecyl sulfate; AMB, amphotericin B; FCZ, fluconazole; 5FC, 5-flucytosine; FDX, fludioxonil.

Article Snippet: Each strain was spotted (3 μl) on Niger seed medium (70 g Niger seed and 20 g Bacto agar per liter), dopamine medium, or epinephrine agar medium (1 g l -asparagine, 3 g KH 2 PO 4 , 250 mg MgSO 4 , 1 mg thiamine, 5 μg biotin, and 100 mg l -DOPA or epinephrine hydrochloride per liter) with a limited glucose concentration (0.1% or 0.2%).

Techniques: Mutagenesis, Staining, Fluorescence, Microscopy

Journal: mBio

Article Title: Unraveling Melanin Biosynthesis and Signaling Networks in Cryptococcus neoformans

doi: 10.1128/mBio.02267-19

Figure Lengend Snippet: The role of the Sks1 kinase downstream of Usv101, Hob1, and Mbs1 in melanin production. (A) Fold change in SKS1 expression upon shifting from nutrient-rich conditions (YPD) to nutrient-starved conditions (YNB without glucose) from the RNA-seq data of the C. neoformans WT and indicated mutant strains . Double and triple asterisks indicate P values of <0.01 and <0.001, respectively. NS, not significant. (B) The expression level of SKS1 was measured in WT and mutant strains under nutrient-rich (R; YPD) or nutrient-starved (S; YNB without glucose) conditions. Three biologically independent experiments were performed with three technical replicates each. Error bars indicate SEM, and statistical differences among gene expression levels were calculated by one-way ANOVA multiple comparisons with Bonferroni’s correction (*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant). (C) C. neoformans WT and mutant strains on Niger seed, dopamine, and epinephrine medium. Darker cultures had more effective melanin synthesis than those with lighter colors.

Article Snippet: Each strain was spotted (3 μl) on Niger seed medium (70 g Niger seed and 20 g Bacto agar per liter), dopamine medium, or epinephrine agar medium (1 g l -asparagine, 3 g KH 2 PO 4 , 250 mg MgSO 4 , 1 mg thiamine, 5 μg biotin, and 100 mg l -DOPA or epinephrine hydrochloride per liter) with a limited glucose concentration (0.1% or 0.2%).

Techniques: Expressing, RNA Sequencing, Mutagenesis, Gene Expression