Calcineurin controls hyphal growth, virulence, and drug tolerance of Candida tropicalis

Abstract

Candida tropicalis, a species closely related to Candida albicans, is an emerging fungal pathogen associated with high mortality rates of 40 to 70%. Like C. albicans and Candida dubliniensis, C. tropicalis is able to form germ tubes, pseudohyphae, and hyphae, but the genes involved in hyphal growth machinery and virulence remain unclear in C. tropicalis. Recently, echinocandin- and azole-resistant C. tropicalis isolates have frequently been isolated from various patients around the world, making treatment difficult. However, studies of the C. tropicalis genes involved in drug tolerance are limited. Here, we investigated the roles of calcineurin and its potential target, Crz1, for core stress responses and pathogenesis in C. tropicalis. We demonstrate that calcineurin and Crz1 are required for hyphal growth, micafungin tolerance, and virulence in a murine systemic infection model, while calcineurin but not Crz1 is essential for tolerance of azoles, caspofungin, anidulafungin, and cell wall-perturbing agents, suggesting that calcineurin has both Crz1-dependent and -independent functions in C. tropicalis. In addition, we found that calcineurin and Crz1 have opposite roles in controlling calcium tolerance. Calcineurin serves as a negative regulator, while Crz1 plays a positive role for calcium tolerance in C. tropicalis.

FIG 1

Calcineurin is required for hyphal growth in C. tropicalis. (A) Hyphal growth of C. tropicalis wild-type (WT) and mutant strains on filament-inducing agar plates. Cells were grown overnight, washed twice with dH₂O, and serially diluted to 10³ cells/ml (based on an OD₆₀₀ of 1 being equal to ∼4 × 10⁷ cells/ml). One hundred microliters containing ∼100 cells was spread on a variety of filament-inducing media and incubated at 37°C for the number of days indicated. The experiments were repeated at least three times, and one representative image is shown. Bar = 0.1 mm. (B) Hyphal growth of C. tropicalis wild-type and mutant strains in liquid bovine calf serum (100%). Cell preparations were as described above with minor modifications. Two microliters of cells at an OD₆₀₀ of 1/ml were added to microtiter wells prefilled with 98 μl of 100% bovine calf serum, resulting in an OD₆₀₀ of 0.00004 (∼1.6 × 10³ cells) in each well. Cultures in the 96-well polystyrene plates were incubated at 37°C without shaking for 24 h. Bar = 40 μm.

FIG 2

Scanning electron microscopy images of C. tropicalis on filament-inducing media. Calcineurin mutants (cnb1/cnb1) display yeast or pseudohyphal growth, while wild-type and crz1/crz1 mutants exhibit hyphal growth. Cells grown on 50% serum agar medium for 48 h at 37°C were processed for scanning electron microscopy and imaged (see Materials and Methods). Magnification = ×2,000. Bars = 10 μm.

FIG 3

C. tropicalis calcineurin and crz1/crz1 mutants are compromised for virulence in a murine systemic infection model. (A) The survival of mice following intravenous challenge with 5 × 10⁶ C. tropicalis yeast cells was monitored for up to 42 days. Ten mice per strain were used for all strains, except 9 mice were used for the wild type (1 animal died during the experimental procedures). (B) The fungal burden in the kidneys and spleens was determined on day 10 after C. tropicalis infection. Five mice per strain were used for all strains.

FIG 4

Histopathological sections of kidneys dissected from mice infected with wild-type or calcineurin or crz1/crz1 mutant strains. Mice were infected with 10⁶ yeast cells and sacrificed at day 10. GMS and H&E stains were used to observe C. tropicalis colonization and tissue necrosis, respectively. Bars = 100 μm.

FIG 5

C. tropicalis calcineurin mutants are attenuated in a murine ocular infection model. (A) Clinical photographs of corneas of immunosuppressed (cyclophosphamide-treated) ICR mice 8 days after inoculation with 10⁸ yeast cells. Fungal keratitis (red arrows) was observed only in animals infected with the C. tropicalis (Ct) wild type, crz1/crz1 mutants, and C. albicans (Ca) type strain SC5314 and not in animals infected with C. tropicalis calcineurin mutants. (B) Each cornea of an immunosuppressed mouse was inoculated with 10⁸ yeast cells of each strain, and the disease severity was scored for 8 days. C. albicans type strain SC5314 served as a reference control. Mice infected with C. tropicalis calcineurin mutants or the PBS control exhibited normal corneas. The disease scores of mice infected with the C. tropicalis wild type or crz1/crz1 mutants and C. albicans strain SC5314 exhibiting visible signs of keratitis were plotted.

FIG 6

Calcineurin is required for cell wall integrity, drug tolerance, and cation homeostasis in C. tropicalis. (A) Calcineurin mutants are sensitive to cell wall integrity-damaging agents (SDS and Congo red) and an endoplasmic reticulum stress-inducing chemical (tunicamycin). Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, spotted onto YPD medium containing SDS, Congo red, or tunicamycin at the concentrations indicated, and then incubated at 30°C for 48 h and photographed. (B) Calcineurin mutants are sensitive to echinocandins. Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto YPD medium containing caspofungin, micafungin, or anidulafungin at the concentrations indicated, and then incubated at 30°C for 48 h and photographed. (C) Calcineurin mutants did not exhibit sensitivity to triazoles. Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto YPD medium containing fluconazole (FLC), posaconazole (PSC), or voriconazole (VRC) at the concentrations indicated, and then incubated at 30°C for 48 h and photographed. (D) Roles of calcineurin and Crz1 in controlling cation homeostasis in C. tropicalis. Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto YPD medium containing CaCl₂ (with or without FK506), MnSO₄, LiCl, or NaCl at the concentrations indicated. The plates were incubated at 30°C for 36 h.

FIG 7

Proposed roles of calcineurin and Crz1 in hyphal growth, virulence, and drug tolerance in C. tropicalis. C. tropicalis calcineurin controls hyphal growth (in response to carbon source starvation), virulence, and micafungin tolerance in a Crz1-dependent fashion. Meanwhile, C. tropicalis calcineurin governs Crz1-independent functions, such as hyphal growth induced by serum or SLAD medium, cell wall integrity, caspofungin/anidulafungin tolerance, azole tolerance, or Mn²⁺ tolerance. Interestingly, Crz1 plays a positive role for Ca²⁺ tolerance functions, while calcineurin serves a negative role. MCF, micafungin; CSF, caspofungin; ANF, anidulafungin.