Calcineurin governs thermotolerance and virulence of Cryptococcus gattii

Abstract

The pathogenic yeast Cryptococcus gattii, which is causing an outbreak in the Pacific Northwest region of North America, causes life-threatening pulmonary infections and meningoencephalitis in healthy individuals, unlike Cryptococcus neoformans, which commonly infects immunocompromised patients. In addition to a greater predilection for C. gattii to infect healthy hosts, the C. gattii genome sequence project revealed extensive chromosomal rearrangements compared with C. neoformans, showing genomic differences between the two Cryptococcus species. We investigated the roles of C. gattii calcineurin in three molecular types: VGIIa (R265), VGIIb (R272), and VGI (WM276). We found that calcineurin exhibits a differential requirement for growth on solid medium at 37°, as calcineurin mutants generated from R265 were more thermotolerant than mutants from R272 and WM276. We demonstrated that tolerance to calcineurin inhibitors (FK506, CsA) at 37° is linked with the VGIIa molecular type. The calcineurin mutants from the R272 background showed the most extensive growth and morphological defects (multivesicle and larger ring-like cells), as well as increased fluconazole susceptibility. Our cellular architecture examination showed that C. gattii and C. neoformans calcineurin mutants exhibit plasma membrane disruptions. Calcineurin in the C. gattii VGII molecular type plays a greater role in controlling cation homeostasis compared with that in C. gattii VGI and C. neoformans H99. Importantly, we demonstrate that C. gattii calcineurin is essential for virulence in a murine inhalation model, supporting C. gattii calcineurin as an attractive antifungal drug target.

Keywords: FK506; capsule; cation homeostasis; cyclosporin A; fluconazole tolerance; melanin; pathogenicity; temperature sensitivity.

Figure 1

Calcineurin is critical for thermotolerance in C. gattii. (A and B) Inhibition of calcineurin via calcineurin inhibitors (FK506, CsA). Cells were grown overnight in YPD at 24°, fivefold serially diluted, spotted onto YPD medium containing FK506 (A) or cyclosporin A (CsA) (B), and incubated at the indicated temperatures for 48 hr. The plate incubated at 37° was transferred to 24° for 48-hr incubation to test viability of the strains after FK506 or CsA inhibition at 37°. (C) Deletion of C. gattii calcineurin catalytic A subunit results in temperature sensitivity. Cells were grown overnight in YPD at 24°, fivefold serially diluted, spotted onto YPD medium, and incubated at the indicated temperatures for 48 hr. The plate incubated at 37° was transferred to 24° for 48-hr incubation to test whether the temperature-sensitive calcineurin mutants survived exposure to the nonpermissive temperature.

Figure 2

Molecular-type VGIIa isolates are more tolerant to calcineurin inhibitors at 37°. Thirty-six randomly selected C. gattii isolates covering four molecular types [VGI, VGII(a,b,c), VGIII, and VGIV] were grown overnight in YPD at 30°, fivefold serially diluted, spotted onto YPD medium in the absence or presence of FK506 (1 µg/mL) and CsA (100 µg/mL), and incubated for 48 hr at 24° and 37°. FK506 and CsA have no obvious effects on cryptococcal growth at 24°, and thus data are not shown. The calcineurin inhibitor tolerance of R265 was set as a control scale with three pluses (+++). The isolates that were hypersensitive to calcineurin inhibitor compared with R265 were labeled with ++ or + symbol, whereas isolates that were extremely sensitive to FK506 or CsA were given a – symbol, indicating that calcineurin is essential in the isolate. NA indicates not available due to the wild-type strain being hypersensitive to growth at 37° in the absence of either calcineurin inhibitor.

Figure 3

Growth kinetics of wild-type and calcineurin mutants in response to thermal stress in C. gattii and C. neoformans. Cells were grown overnight at 24°, washed twice with dH₂O, diluted to 0.2 OD₆₀₀/mL in fresh YPD medium, and incubated at 24° (left) and 37° (right) with shaking. The OD₆₀₀ of cultures was measured at 0, 4, 8, 24, 48, 72, and 96 hr. The experiments were performed in triplicate, and data were plotted using Prism 5.03.

Figure 4

C. gattii calcineurin mutants are compromised for virulence in a murine inhalation infection model. (A) Groups of 10 A/Jcr mice were each infected via intranasal instillation with an infectious inocula of 5 × 10⁴ cells (in 50 µL) of the wild-type and two independent cna1Δ mutants. Survival percentage was monitored for 8 wk postinfection. (B) The fungal burden in the lungs was determined at 14 d postinfection (5 × 10⁴ cells per mouse). Five mice per strain were used. P values were determined by ANOVA and Dunnett’s multiple comparison test. (C) Histopathological sections of lungs dissected from mice infected with wild-type and mutant from R265, R272, and WM276 strains. The mice were infected with 5 × 10⁴ yeast cells and sacrificed at day 14. Mucicarmine and H&E stains were used to observe C. gattii colonization and tissue necrosis, respectively. Scale bar = 50 µm.

Figure 5

Calcineurin mutants exhibit multivesicular phenotypes at 37°. Strains were grown in liquid YPD medium overnight at 24°. Cells were washed twice, diluted into two tubes of 0.4 OD₆₀₀/mL, and incubated for 5 hr at 24° and 37° to reach log phase. The cells were spun and washed twice with PBS buffer, then resuspended in 5 mL of fixative buffer [1:1 ratio of 0.2 M Sodium cacodylate (pH = 6.8) and 6% glutaraldehyde in dH₂O]. The cna1Δ mutants tested were YL136 (from R265), YL165 (from R272), YL277 (from WM276), and KK1 (from H99). Cells were viewed by light microscopy at 1000× magnification and photographed. Scale bar = 5 µm.

Figure 6

Calcineurin mutants exhibit plasma membrane disruptions at 37° (indicated by red arrows). Cell growth and fixation conditions are described in Materials and Methods. Transmission electron microscopy was used to observe intracellular architecture. Only cells grown at 37° are shown. Images were taken at 25,000× magnification. Scale bar = 200 nm.

Figure 7

Divergent roles of calcineurin in cell wall integrity, ER stress response, and cation homeostasis. (A) Cells were grown overnight in YPD at 24°, fivefold serially diluted, and spotted onto YPD medium containing SDS, calcofluor white (CFW), Congo red (CR), and tunicamycin (TM) at the indicated concentrations and incubated at 24° for 48 hr. (B) In C. gattii calcineurin plays a larger role than in C. neoformans in controlling cation homeostasis. Cells were grown overnight in YPD at 24°; fivefold serially diluted; spotted onto YPD medium containing CaCl₂, LiCl, or NaCl at the indicated concentrations; and incubated at 24°C for 48 hr.

Figure 8

Divergent roles of calcineurin in fluconazole tolerance between C. gattii and C. neoformans. (A) Cells were grown overnight in YPD medium at 24°, fivefold serially diluted, and spotted onto YPD medium in the absence or presence of fluconazole (5 µg/mL). The plates were incubated at 24° for 48 hr. (B) Disk diffusion halo assays were used to determine fluconazole susceptibility of wild-type and mutant strains. Cells were grown overnight at 24°, and 100 µL of 0.1 OD₆₀₀/mL was spread on the surface of YPD medium. A disk was placed on the surface of the medium, and fluconazole (20 µg) was added to each disk. The plates were incubated at 24° for 48 hr and photographed. Scale bar = 6 mm.