C. albicans flocculation in response to CASP. (A) C. albicans wild-type strain (SC5314) growing in the exponential growth phase at 30°C in YPD or RPMI 1640 medium was treated for 3 h with CASP at 10 or 100 ng/ml or left untreated. Cultures were photographed 2 min after vortex mixing of the culture tubes. ctrl, control. (B) The cultures described for panel A were used for measuring the OD₆₀₀ after 1 and 3 h of treatment. The optical density of the cultures was measured directly after vortex mixing. Some 15 min later, the same tubes were measured again without vortex mixing. Values shown represent the results of the second measurement relative to those of the first measurement. All treatments and measurements were done in triplicate; standard deviations are shown as error bars. *;, P < 0.05; *;*;, P < 0.01 (Student's t test) relative to the untreated control results. (C) High sugar concentrations inhibit CASP-induced flocculation. Wild-type strain SC5314 was grown to the exponential growth phase in YPD medium or YPD medium supplemented with the indicated concentrations of mannose (Man). CASP was added at a final concentration of 100 ng/ml. After 3 h of CASP treatment, the optical density of the cultures was measured as described for panel B. *;*;*;, P < 0.001 (Student's t test).

High sugar concentrations improve survival in the presence of CASP. (A) Wild-type SC5314 cells were grown to the exponential growth phase in YPD medium, YPD-10% mannose, or YPD plus 0.3 M NaCl before treatment with CASP at 100 ng/ml. After 3 h, cells were stained with FUN1 and Calcofluor White (CFW) and microscopy pictures were taken. BF, bright field. (B) Wild-type SC5314 cells were grown to the exponential growth phase in YPD medium or YPD medium supplemented with the indicated concentrations of glucose (Glu), NaCl, or KCl. Cultures were split, and one half was treated with CASP at 100 ng/ml whereas the other remained untreated. After 3 h of incubation at 30°C, cells were harvested, washed, and reinoculated into fresh YPD medium. After 6 h of recovery at 30°C, the optical density was determined and the OD value was used for calculating the number of cells surviving CASP treatment. Data are presented relative to untreated control results. *;*;, P < 0.01; *;*;*;, P < 0.001 (Student's t test) relative to cells treated in YPD medium only. (C) Logarithmically grown wild-type strain SC5314 was diluted to an OD₆₀₀ of 0.1 and spotted on a YPD medium plus CASP (350 ng/ml) agar plate containing a gradient of 2% to 12% glucose. The plate was inspected and scanned after 3 days of incubation at 30°C. (D) Logarithmically growing strains (mkc1 mutant strain and wild-type CA-IF100 strain) were spotted in serial dilutions at an OD₆₀₀ ranging from 0.1 to 10⁻⁴ onto YPD medium and YPD medium plus 10% glucose plates with or without the indicated concentrations of CASP. Plates were scanned after 2 days of incubation at 30°C.

ALS1 expression is induced by CASP and required for flocculation. (A) A reporter strain expressing GFP under the control of the ALS1 promoter was grown to the mid-logarithmic growth phase before treatment with CASP at 50 ng/ml. Microscopy pictures of the treated and untreated control cultures were taken after 3 h of incubation with CASP at 30°C. (B) Reporter strains expressing GFP under the control of different ALS promoters were grown to the exponential growth phase and treated with CASP at 10 or 50 ng/ml for 3 h. Microscopy pictures were taken, and the GFP signal intensity of cells was determined by measuring the average value for the gray regions of interest selected in the pictures captured with the GFP filter set. For each strain and set of conditions, a minimum of 100 cells were measured for quantification. (C) Wild-type (CAI4), als1Δ/Δ, and ALS1-reconstituted strains were grown to the exponential growth phase at 30°C in glass tubes before CASP at 100 ng/ml was added for 3 h. Cultures were photographed after vortex-mixing (upper panel). To quantify flocculation, the optical density of cultures was measured directly after vortex-mixing. The same tubes were measured again without prior mixing at 5 min, 10 min, 15 min, and 20 min after the first measurement. Treatments and measurements were performed in triplicate; standard deviations are included as error bars (lower panel). *;, P < 0.05 (Student's t test). (D) A reporter strain expressing GFP under the control of the ALS1 promoter was grown to the exponential growth phase and treated with CASP at 50 ng/ml for 3 h in the presence or absence of high glucose concentrations. Microscopy pictures were taken, and the GFP signal intensity of cells was determined as described for panel B.

Efg1 is required for CASP tolerance and flocculation. (A) Northern analysis of ALS1 expression. Wild-type SC5314 and efg1Δ/Δ strains were grown to the exponential growth phase followed by treatment with CASP at 10 ng/ml. Samples were taken at indicated time points. Total RNA was isolated, and mRNA levels of ALS1 were detected by Northern blotting. CHR11 served as a control for an Efg1-independent CASP-responsive gene and ACT1 as a loading control. (B) Wild-type SC5314 and efg1Δ/Δ cells in the exponential growth phase at 30°C in glass tubes were treated with CASP at 80 ng/ml for 3 h. Cultures were photographed 2 min after vortex-mixing (upper panel). To quantify flocculation, the OD₆₀₀ of cultures was measured directly after vortex-mixing. The same tubes were measured again without prior mixing at 5 min, 10 min, 15 min, and 20 min after the first measurement. Treatments and measurements were done in triplicate; standard deviations are indicated as error bars (lower panel). *;*;, P < 0.01; *;*;*;, < 0.001 (Student's t test). (C) Identical volumes of 10-fold serial dilutions of two independent efg1 mutant strains were spotted onto YPD plates containing CASP at 250 ng/ml along with their parental strains (SC5314 and CAI-4). Colony growth was inspected after 48 h of incubation at 30°C. (D) Strains CAYF5 (P_ADH1-ALS1) and CADS1 (P_ADH1-ALS1-efg1Δ/Δ) and the corresponding wild type were grown to the exponential growth phase at 30°C in synthetic medium before treatment with CASP at 10 or 50 ng/ml for 3 h. The optical density of the cultures was measured directly after vortex-mixing. Some 15 min later, the same tubes were measured again without vortex-mixing. Values shown represent the results of the second measurement relative to the results of the first measurement. All treatments and measurements were done in triplicate; standard deviations are shown as error bars. *;, P < 0.05; *;*;, < 0.01 (Student's t test) relative to the untreated control results.

CASP treatment induces agar invasion. (A) Logarithmically growing wild-type cells (SC5314) were spotted in serial dilutions at an of OD₆₀₀ ranging from 0.1 to 10⁻⁴ onto YPD plates containing the indicated concentrations of CASP. Cells were scanned from the bottom after 2 days of incubation at 30°C. Plates were then washed and scanned again. (B) Logarithmically growing wild-type and efg1Δ/Δ mutant strains were spotted in serial dilutions onto YPD plates containing the indicated concentrations of CASP. Plates were treated as described for panel A. (C) Logarithmically growing wild-type and efg1Δ/Δ mutant strains were spotted in serial dilutions onto YPD plates without CASP or YPD plates containing CASP at 10 and 50 ng/ml. Plates were incubated at 30°C for 4 days. Cells were washed from the surface of the plates, and the agar was cut through the center of colonies; pictures of cross-sections were then taken. Since the efg1Δ/Δ mutant failed to grow on plates with CASP at 50 ng/ml, no cross-section pictures were taken.

Model for CASP-activated signaling response in C. albicans. CASP-induced cell wall damage activates Mkc1- and Efg1-dependent signaling pathways, each driving expression of a distinct subset of target genes implicated in cell wall remodeling and repair. ALS1 is a major target of the Efg1-dependent CASP response. Mkc1 regulates expression of genes implicated in cell wall remodeling but most likely also of genes involved in invasive growth in the presence of CASP, whereas Efg1 normally acts as an inhibitor of invasive growth.