Fig 1.
The Cek1 and Mkc1 MAP kinase signaling cascades in C. albicans are involved in cell wall biogenesis.
The Cek1 and Mkc1 MAP kinase cascades, and their respective upstream activator signaling proteins are shown. Rho1 activates protein kinase C (Pkc1), which activates the Mkc1 MAP kinase cascade. Cdc42 activates the PAK kinase Cst20 which activates the Cek1 MAP kinase cascade.
Fig 2.
Cek1 and Mkc1 MAPKs exhibit increased activation in cho1Δ/Δ cells compared to wild-type.
(A) Proteins from yeast-form cells growing in log phase in YPD media were extracted and Western blotting was performed with anti-phospho-p44/42 antibody to detect Phospho-Mkc1 and Phospho-Cek1. Anti-Mkc1 was used for total Mkc1, anti-Cek1 for total Cek1, and anti-tubulin as a loading control. Graphs of quantification by Image J of the phosphorylated forms of each kinase are expressed as a percent of the wild-type control after being normalized to the total kinase blot for each respective MAP kinase and the tubulin loading controls for each gel. Quantification is based on three biological replicates. The statistical analysis was performed by using One-way ANOVA *, p = 0.0308. ***, p = 0.0005. (B) Western blotting was performed on extracts from cells grown as hyphae in RPMI media and probed with antibodies detecting Phospho-Cek1 and Phospho-Mkc1 as well as the tubulin loading control.
Fig 3.
Hyperactive Ste11 (STE11ΔN467) causes significant increases in β (1,3)-glucan exposure and TNF-α secretion.
(A) The Cek1 MAPK is hyper-activated by transforming wild-type cells with a hyperactive allele of STE11 (PMAL-STE11ΔN467), which is induced by adding maltose as a carbon source. (B) Both wild-type and STE11ΔN467 expressing cells were cultured overnight (16hrs) in YPD or YPM individually and then were doubly stained. The Y-axis represents staining with soluble Dectin-1-Fc (sDectin-1-Fc) that binds to exposed β (1,3)-glucan, and the X-axis represents anti-Als3 antibody, which binds to the hyphal-specific protein Als3. Secondary antibodies were used for fluorescence as described in the Methods. Flow cytometry was performed to quantify β (1,3)-glucan exposure from the yeast-form population (Q1: sDectin-1 single positive staining; Q2: sDectin-1 & Als3 double positive staining; Q3: Als3 single positive staining; Q4: double negative staining). Gates were established with an unstained control where 97% of unstained cells gated within Q4. Gating strategies are further described in the Methods. (C) Left graph: Comparison of β (1,3)-glucan exposure from the yeast-form population of STE11ΔN467 versus that of wild-type, both of which are cultured in YPM. Data were compared by unpaired t-test *, p = 0.0289. Right graph: β (1,3)-glucan exposure was compared between these two strains when grown in YPD medium. (D) Expression of STE11ΔN467 significantly induces TNF-α secretion after growing in YPM overnight. RAW264.7 macrophages were challenged with various C. albicans stains. C. albicans strains were grown in YPD or YPM, washed, UV-inactivated, and then add to the macrophages for 4hrs. The macrophage supernatant was collected and assayed by ELISA to quantify TNF-α production. **, P = 0.0030; ***, P = 0.0002; ****, p<0.0001.
Fig 4.
Cdc42 activity is upregulated in cho1Δ/Δ compared to wild-type.
(A) GTP-Cdc42 was pulled-down with beads conjugated with GST-CRIB, which specifically binds with active GTP-Cdc42/Rac1. Cdc42 that was pulled down was then detected via Western blotting with anti-Cdc42 antibody. The amount of total Cdc42 in the extract was also probed as a control. The GTP-Cdc42/Total-Cdc42 ratio is expressed as a percentage of wild-type. (B) CaCRIB-GFP localization is not altered upon CHO1 deletion. The CaCRIB-GFP probe was transformed into Candida strains to investigate the active GTP-Cdc42 localization. The scale bar represents 10μm. (C) Cells from Fig 4B were analyzed by microscopy for the number that exhibited CaCRIB-GFP localization to buds. Quantification is of three biological replicates, and each replicate has at least 50 cells.
Fig 5.
Cho1 is essential for GFP-Cdc42 polarization at the plasma membrane.
(A) GFP-Cdc42 localization is examined for each strain by microscopy. The red arrows indicate the fluorescence concentrated at the bud tips or bud necks. The scale bar represents 5μm. (B) Quantification of the degree of polarization of GFP-Cdc42 in Candida cells. A minimum of 50 cells was counted for each strain and the imaging experiment was repeated three times. The statistical analysis was carried out by One-way ANOVA. ****, p<0.0001.
Fig 6.
Deletion of MKC1 in cho1Δ/Δ did not diminish β (1,3)-glucan exposure.
(A) Cells were stained with primary anti-β (1,3)-glucan antibody and Cy3-conjugated secondary antibody, and imaged by epi-fluorescent microscopy. The scale bar indicates 10μm. (B) Flow cytometry was carried out to quantify β (1,3)-glucan exposure. Cells were incubated with primary anti-β (1,3)-glucan antibody and PE-conjugated secondary antibody. The statistical analysis was carried out by doing One-way ANOVA analysis. *, P = 0.0485; **, P = 0.0024.
Fig 7.
Active GTP-Rho1 is de-localized in cho1Δ/Δ.
(A-C) GFP-RID localization was used as proxy for active (GTP-bound) Rho1 in cells, and was analyzed by epifluorescent microscopy. (D) Quantification of the degree of polarization of GTP-Rho1 in Candida cells. A minimum of 50 cells were counted for each strain and this repeated three times. The statistical analysis was done by One-way ANOVA. *, p<0.019.
Fig 8.
Hyper-activated Rho1 causes β (1,3)-glucan exposure.
(A-B) Candida cells were stained for β (1,3)-glucan exposure as described in Fig 6. The scale bar represents 10μm. ***, p = 0.0003. (C) Western blotting was performed to examine the effect of expressing hyperactive RHO1Q67L on the regulation of downstream MAPK activities. Phospho-p44/42 antibody was used to detect Phospho-Cek1 and Phospho-Mkc1 and anti-tubulin, anti-Mkc1, and anti-Cek1 antibodies were used as controls.