Figure 1.
Calcineurin mutation confers cell wall integrity defects in C. lusitaniae.
(A) Calcineurin mutants are sensitive to cell wall integrity-damaging agents and ER stress chemicals. Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto YPD medium containing sodium dodecyl sulfate (SDS), calcofluor white (CFW), congo red (CR), tunicamycin (TM), or dithiothreitol (DTT) and incubated at 30°C for 48 h. (B) The growth kinetics of C. lusitaniae wild-type and mutant strains at 30°C. Cells were grown overnight at 30°C, washed twice with dH2O, diluted to 0.2 OD600/ml in fresh liquid YPD medium, and incubated at 30°C with shaking at 250 rpm. The OD600 of cultures was measured at 0, 3, 6, 9, 12, 15, 18, 21, and 24 h. The experiments were performed in triplicate, and data was plotted using Prism 5.03. Strains tested were wild-type (ATCC42720), cnb1 mutants (YC198 and YC202), and crz1 mutants (YC187 and YC467).
Figure 2.
Calcineurin and Crz1 control colony pseudohyphal growth of C. lusitaniae.
(A) Cells were grown overnight and washed twice with dH2O. Cells were diluted to 500 cells/ml. One hundred microliters containing ∼50 cells were spread on a variety of filament-inducing media lacking or containing FK506 (1 µg/ml), and incubated at 37°C for the number of days indicated. FA, Filament Agar; PDA, Potato Dextrose Agar. The experiments were repeated at least three times and one representative image is shown. Scale bar = 0.5 mm. (B) Scanning electron microscopy (SEM) images of C. lusitaniae on filament-inducing media. Cells grown on V8 (pH = 7) media for 7 days at 37°C were processed for SEM, and imaged (see Materials and Methods). Scale bars for upper panel (1000x) and lower panel (5000x) images represent 10 µm and 2 µm, respectively. WT (ATCC42720), cnb1 mutant (YC198), and crz1 mutant (YC187).
Figure 3.
Optimal growth in serum is controlled by calcineurin in C. lusitaniae.
(A) Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto agar water medium containing 10% or 50% fetal bovine serum, and incubated at 37°C for 48 h. (B) The growth kinetics of C. lusitaniae wild-type and mutant strains on 100% serum at 37°C. Cells were grown overnight at 30°C, washed twice with dH2O, diluted to 0.2 OD600/ml in 100% serum, and incubated at 37°C with shaking at 250 rpm. The OD600 of cultures was measured at 0, 3, 6, 9, 24, 30, 48, 72, and 96 h (upper panel). The lower panel shows the growth kinetics between 0 and 9 h extracted from the upper panel. The experiments were performed in triplicate, and data was plotted using Prism 5.03. Strains tested were wild-type (ATCC42720), cnb1 mutants (YC198 and YC202) and crz1 mutants (YC187 and YC467). (C) Doubling time of wild-type and calcineurin pathway mutants in 100% serum. * P<0.01, ** P<0.0001.
Figure 4.
Transcription factor Crz1 plays a greater role than calcineurin in controlling Ca2+ ion homeostasis in C. lusitaniae.
(A) Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto YPD medium with or without CaCl2 at the concentrations indicated, and incubated at 37°C for 48 h. (B) The growth kinetics of C. lusitaniae wild-type and mutant strains on YPD containing 1 M CaCl2 at 37°C. Cells were grown overnight at 30°C, washed twice with dH2O, diluted to 0.2 OD600/ml in fresh liquid YPD medium, and incubated at 37°C with shaking at 250 rpm. The OD600 of cultures was measured at 0, 3, 6, 9, 12, 15, 18, 21, 24, 48, 72, 96, and 120 h (upper panel). The lower panel shows the growth kinetics between 0 and 24 h extracted from the upper panel. The experiments were performed in triplicate, and data was plotted using Prism 5.03. Strains tested were wild-type (ATCC42720), cnb1 mutants (YC198 and YC202), and crz1 mutants (YC187 and YC467). (C) Doubling time of wild-type and calcineurin pathway mutants in 1 M CaCl2. *P = 0.0002, **P<0.0001.
Figure 5.
Calcineurin contributes to kidney tissue colonization in a murine systemic infection model.
(A) The fungal burden in the kidneys and spleen of immunocompetent mice was determined at day 14 after challenge with 107 yeast cells via lateral tail vein injection. Strains tested were wild-type (ATCC42720), cnb1 mutants (YC198 and YC202), and crz1 mutants (YC187 and YC467). The P value (ANOVA, Dunnett’s Multiple Comparison) between wild-type and mutants is shown. (B) The fungal burden in the kidneys and spleen of immunocompromised mice (cyclophosphamide-treated) was determined at day 7 after challenge with 107 yeast cells via lateral tail vein injection. The P value (ANOVA, Dunnett’s Multiple Comparison) between wild-type and mutants is shown. (C) Histopathological sections of kidneys dissected from immunocompromised mice infected with wild-type, cnb1, or crz1 mutant strains. The mice were challenged with 107 cells and sacrificed at day 7. Gomori Methenamine Silver stain was used to observe C. lusitaniae colonization (black dots). Bar = 50 µm.
Figure 6.
Calcineurin controls antifungal drug tolerance in C. lusitaniae.
Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto YPD medium containing echinocandins (A) or azoles (B) at the indicated concentrations, and incubated at 30°C for 48 h.
Table 1.
Calcineurin is required for drug tolerance in C. lusitaniae.
Table 2.
Calcineurin inhibitor FK506 exhibits synergistic antifungal activity with caspofungin against echinocandin resistant strains.
Figure 7.
Calcineurin inhibitor exhibits synergistic antifungal activity with caspofungin against C. lusitaniae wild-type and echinocandin-resistant strains.
Disk diffusion assays were used to determine synergistic antifungal activity with caspofungin against clinical echinocandin-resistant C. lusitaniae strains. Cells were grown overnight at 30°C, and 0.1 OD600 (in 100 µl) was spread on the surface of RPMI media lacking or containing FK506 (1 µg/ml). A disk was placed on the surface of the medium and 12.5 µg caspofungin (5 µl of 2.5 mg/ml) was added to each disk. The plates were incubated at 30°C for 48 h and photographed. S = caspofungin-sensitive; R = caspofungin-resistant (less susceptible). Scale bar = 6 mm.
Figure 8.
Proposed roles of calcineurin and Crz1 in core stress responses in C. lusitaniae.
C. lusitaniae core stress responses including pseudohyphal growth, drug tolerance, virulence, serum growth, cell membrane and wall integrity, ER stress, and Ca2+ homeostasis are controlled by either calcineurin-dependent or -independent signaling cascades. The pseudohyphal development, serum growth, and virulence are controlled by Crz1-mediated calcineurin signaling, while cell wall integrity and echinocandin tolerance are governed by Crz1-independent calcineurin signaling (green shading). Crz1 also exhibits calcineurin-independent functions to: 1) negatively regulate cell membrane integrity, ER stress, and azole tolerance; 2) positively regulate Ca2+ tolerance (red shading).
Table 3.
Strains used in this study.
Table 4.
PCR primers used in this study.