Engineered Fluorescent Strains of Cryptococcus neoformans: a Versatile Toolbox for Studies of Host-Pathogen Interactions and Fungal Biology, Including the Viable but Nonculturable State

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hyg^r] or neomycin resistance [Neo^r]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans. IMPORTANCE Cryptococcus neoformans is a human-pathogenic yeast that can undergo a dormant state and is responsible for over 180,000 deaths annually worldwide. We engineered a set of fluorescent transformants to aid in research on C. neoformans. A mutant with GFP-tagged Pab1 improved fluorescence-based techniques used in host interaction studies. Moreover, this mutant induced a viable but nonculturable phenotype and uncovered posttranscriptional mechanisms associated with dormant C. neoformans. The experimental use of fluorescent mutants may shed light on C. neoformans-host interactions and fungal biology, including dormant phenotypes.

Keywords: Cryptococcus neoformans; GFP; H99; VBNC; dormancy; fluorescently tagged strains; histone H3; mCherry; poly(A) binding protein; stress granules.

FIG 1

Engineered fluorescent strains exhibit a nuclear or cytoplasmic fluorescence distribution. (A) GFP or mCherry (mC) transformant strains were stained with the NucBlue (NB) nucleus probe and observed under a fluorescence microscope for assessment of the intracellular distribution of fluorophores (BF, bright field; Mrg, merge) (bars = 5 μm). (B and C) Fluorescence intensity histograms (B) and median fluorescence intensities (MFI) (C) of the transformant strains (normalized to the values for the WT) evaluated by flow cytometry. The results are presented as the means ± SD of data obtained from three independent experiments. Representative fluorescence micrographs were taken using automatic exposure, and histograms are adjusted to highlight the subcellular localization of the fluorescent fusion proteins, whereas the relative quantification of fluorescence signals was done by flow cytometry.

FIG 2

The fluorescent strains preserved the production of mating filaments. The transformant strains were mixed with an equal amount (10 μL of a suspension of 1 × 10⁷ cells/mL) of KN99 cells (MATα) and cocultured on agar filament plates for 7 days at room temperature. Hyphal fragments were assessed by fluorescence photomicroscopy at a ×200 magnification using automatic exposure, and histograms are adjusted to highlight the fluorescent fusion proteins. The edges of mating colonies were photographed with a stereomicroscope under a ×2.5 magnification.

FIG 3

Expression of virulence-associated phenotypes. (A) Transformant strains were evaluated for laccase-dependent melanization (Niger seed agar), urease (Christiansen’s urea agar) and phospholipase (PLase) (egg yolk agar) activities, and capsule formation (growth in liquid minimal medium and Indian ink preparation). (B) Measurement of capsule thickness was performed based on the formation of the ink exclusion zone observed under a light microscope. Black bars represent the means ± SD of data pooled from two experiments. *, P < 0.05 (compared to the WT).

FIG 4

The fluorescent strains are virulent in a wax moth model and survive after phagocytosis by activated murine macrophages. (A) G. mellonella caterpillars (n = 14 to 17) were inoculated with either PBS alone (inoculation trauma control) or 5 × 10⁴ yeast cells from each transformant strain. (B) After 5 days of infection, hemolymph was recovered from individual larvae for analysis of yeast by fluorescence microscopy. (C) Fungal burdens in G. mellonella caterpillars (n = 5) at the LT₅₀ upon infection with the transformant strains. (D) Primary murine macrophages were activated for 3 h with LPS (1 μg/mL) and IFN-γ (20 ng/mL) and inoculated (MOI of 2) with the previously opsonized transformant strains. After 2 and 24 h of interaction, the cell monolayer was washed and lysed, and the resulting yeast suspension was diluted and seeded onto SDA plates for CFU counting. The percentage of fungal survival was calculated based on the 24-h/2-h CFU ratio multiplied by 100. The results are representative of data from two independent experiments. (E and F) For phagocytosis analysis, nonactivated primary murine macrophages were infected at an MOI of 5. After 2 h of interaction, the cell monolayer was stained with a panoptic stain for analysis of the phagocytic index (yeast cells/macrophages) and the percentage of phagocytosis (percentage of macrophages containing yeast) by light microscopy. The results are presented as the means ± SD and are representative of data from two independent experiments. *, P < 0.05; **, P < 0.01 (compared to the WT).

FIG 5

The transformant strains show no difference from the parental strain in phagocytosis by murine macrophages by fluorescence-based analysis. (A and B) Primary murine macrophages were cocultured at an MOI of 5 with the WT, FITC-labeled WT, or PAB-GFP strain previously opsonized with mAb 18B7. After 2 h of interaction, the phagocytic index (yeast cells/macrophages) and percentage of phagocytosis (percentage of macrophages containing yeast) were evaluated by fluorescence microscopy. (C) For analysis of the percentage of phagocytosis by flow cytometry, the cell monolayer was washed, detached, and stained with anti-CD11b antibody conjugated to APC for the identification of macrophages. (D) Flow cytometry dot plots of CD11b-APC versus FITC/GFP. (E) Fluorescence micrographs of the interaction between macrophages and yeast cells under a magnification of ×400. The results are presented as the means ± SD and are representative of data from two independent experiments.

FIG 6

C. neoformans PAB-GFP grown under nutrient starvation and hypoxia exhibits the viable but nonculturable cell phenotype and stable GFP expression by flow cytometry. (A and B) WT or PAB-GFP yeast cells in stationary phase (STAT) or incubated for 7 days under hypoxia (HYPOx) were inoculated onto SDA plates for CFU counting, which was used to determine the percentage of culturable cells (A), or inoculated into fresh liquid medium for analysis of growth latency (B). (C and D) Cells were labeled with LIVE/DEAD violet dye (LVD) for cell viability analysis by flow cytometry. (E and F) GFP expression in viable or heat-killed (HK) cells was evaluated. In panel E, filled histograms denote cultures of STAT yeast cells, while empty histograms represent HYPOx cells. FSC-A, forward scatter area. The results are presented as the means ± SD from at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

C. neoformans PAB-GFP VBNC cells display GFP expression and a specific cell distribution pattern by fluorescence microscopy. WT or PAB-GFP yeast cells in stationary phase (STAT) or in a hypoxic atmosphere (HYPOx) were heat killed (HK) or not, subjected to LIVE/DEAD Violet Dye (LVD) staining, and analyzed for cell viability and GFP expression by fluorescence microscopy. Dead cells appear blue. The arrowheads indicate stress granules. The results are representative of data from two independent experiments.

FIG 8

C. neoformans PAB-GFP VBNC cells exhibit accumulation of Pab1 in stress granules and the nucleus. WT or PAB-GFP yeast cells were grown until stationary phase (STAT) and then incubated for 7 days in a hypoxic atmosphere (HYPOx). Nuclei (blue) were stained with NucBlue. The results are representative of data from two independent experiments.