Calcineurin Is Required for Pseudohyphal Growth, Virulence, and Drug Resistance in <em>Candida lusitaniae</em>

<div><p><em>Candida lusitaniae</em> is an emerging fungal pathogen that infects immunocompromised patients including HIV/AIDS, cancer, and neonatal pediatric patients. Though less prevalent than other <em>Candida</em> species, <em>C. lusitaniae</em> is unique in its ability to develop resistance to amphotericin B. We investigated the role of the calcium-activated protein phosphatase calcineurin in several virulence attributes of <em>C. lusitaniae</em> including pseudohyphal growth, serum survival, and growth at 37°C. We found that calcineurin and Crz1, a <em>C. albicans</em> Crz1 homolog acting as a downstream target of calcineurin, are required for <em>C. lusitaniae</em> pseudohyphal growth, a process for which the underlying mechanism remains largely unknown in <em>C. lusitaniae</em> but hyphal growth is fundamental to <em>C. albicans</em> virulence. We demonstrate that calcineurin is required for cell wall integrity, ER stress response, optimal growth in serum, virulence in a murine systemic infection model, and antifungal drug tolerance in <em>C. lusitaniae</em>. To further examine the potential of targeting the calcineurin signaling cascade for antifungal drug development, we examined the activity of a calcineurin inhibitor FK506 in combination with caspofungin against echinocandin resistant <em>C. lusitaniae</em> clinical isolates. Broth microdilution and drug disk diffusion assays demonstrate that FK506 has synergistic fungicidal activity with caspofungin against echinocandin resistant isolates. Our findings reveal that pseudohyphal growth is controlled by the calcineurin signaling cascade, and highlight the potential use of calcineurin inhibitors and caspofungin for emerging drug-resistant <em>C. lusitaniae</em> infections.</p> </div

Calcineurin inhibitor FK506 exhibits synergistic antifungal activity with caspofungin against echinocandin resistant strains.

#<p>Strains were grown overnight with shaking at 30°C and washed twice in dH₂O. The OD₆₀₀ was taken of the cultures with a spectrophotometer and diluted to 0.01 OD₆₀₀/ml in RPMI-1640 medium [Sigma R1383 (8.4g) and MOPS (34.5g) in 1L dH₂O buffered to pH7 with NaOH]. Minimum inhibitory concentrations (MIC) of each drug alone and fractional inhibitory concentrations of the drugs in combination were determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) protocol M27-A3. Final concentrations of caspofungin (CAS) ranged from 16 to 0.0312 µg/ml. FK506 concentrations ranged from 4.0 to 0.063 µg/ml.</p>&<p>FIC index = (MIC_combined drug 1/MIC _alone drug 1) + (MIC_combined drug 2/MIC_alone drug 2) FIC ≤0.5 (synergy), >0.5 but <1.0 (additive), >1.0 but ≤2.0 (no interaction), >2.0 (antagonism).</p>*<p><i>C. lusitaniae</i> clinical echinocandin resistant isolates with the Ser645Phe mutation in the Fks1 protein.</p

Calcineurin is required for drug tolerance in <i>C. lusitaniae</i>.

#<p>Cells were grown overnight at 30°C and washed twice with dH₂O. Then 0.5 OD (in 500 µl) of cells was spread on RPMI 1640 media (Remel; R04067). After 20 min, the E-test strips (bioMérieux Corp.) were transferred to the surface of the media. The minimum inhibitory concentrations (MIC) were read after 24 h incubation at 35°C according to the manufacturer’s instructions.</p

Optimal growth in serum is controlled by calcineurin in <i>C. lusitaniae</i>.

<p>(<b>A</b>) 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>B</b>) The growth kinetics of <i>C. lusitaniae</i> 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), <i>cnb1</i> mutants (YC198 and YC202) and <i>crz1</i> mutants (YC187 and YC467). (<b>C</b>) Doubling time of wild-type and calcineurin pathway mutants in 100% serum. * <i>P</i><0.01, ** <i>P</i><0.0001.</p

Calcineurin and Crz1 control colony pseudohyphal growth of <i>C. lusitaniae</i>.

<p>(<b>A</b>) 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>B</b>) Scanning electron microscopy (SEM) images of <i>C. lusitaniae</i> 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), <i>cnb1</i> mutant (YC198), and <i>crz1</i> mutant (YC187).</p

PCR primers used in this study.

<p>Sequences complementary to the <i>SAT1</i> marker are underlined.</p

Transcription factor Crz1 plays a greater role than calcineurin in controlling Ca²⁺ ion homeostasis in <i>C. lusitaniae</i>.

<p>(<b>A</b>) 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>B</b>) The growth kinetics of <i>C. lusitaniae</i> 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), <i>cnb1</i> mutants (YC198 and YC202), and <i>crz1</i> mutants (YC187 and YC467). (<b>C</b>) Doubling time of wild-type and calcineurin pathway mutants in 1 M CaCl₂. *<i>P</i> = 0.0002, **<i>P</i><0.0001.</p

Calcineurin contributes to kidney tissue colonization in a murine systemic infection model.

<p>(<b>A</b>) 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), <i>cnb1</i> mutants (YC198 and YC202), and <i>crz1</i> mutants (YC187 and YC467). The <i>P</i> value (ANOVA, Dunnett’s Multiple Comparison) between wild-type and mutants is shown. (<b>B</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 <i>P</i> value (ANOVA, Dunnett’s Multiple Comparison) between wild-type and mutants is shown. (<b>C</b>) Histopathological sections of kidneys dissected from immunocompromised mice infected with wild-type, <i>cnb1</i>, or <i>crz1</i> mutant strains. The mice were challenged with 10⁷ cells and sacrificed at day 7. Gomori Methenamine Silver stain was used to observe <i>C. lusitaniae</i> colonization (black dots). Bar = 50 µm.</p

Calcineurin inhibitor exhibits synergistic antifungal activity with caspofungin against <i>C. lusitaniae</i> wild-type and echinocandin-resistant strains.

<p>Disk diffusion assays were used to determine synergistic antifungal activity with caspofungin against clinical echinocandin-resistant <i>C. lusitaniae</i> 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.</p

Proposed roles of calcineurin and Crz1 in core stress responses in <i>C. lusitaniae</i>.

<p><i>C. lusitaniae</i> 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).</p