Isolation and Characterization of Senescent Cryptococcus neoformans and Implications for Phenotypic Switching and Pathogenesis in Chronic Cryptococcosis

C. neoformans and C. albicans strains.RC-2 is a variant of the serotype D strain 24067 generated in our laboratory (3). Strain 24067 was originally obtained from the American Type Culture Collection (Manassas, VA). The RC-2 strain can produce two colony morphologies on Sabouraud dextrose agar (SDA) plates known as SM and MC, both of which are characteristic of C. neoformans colonies. C. albicans strain WO-1 switches between white and opaque colony morphologies as described previously (27). C. neoformans was grown in standard Sabouraud dextrose (SDB) and synthetic dextrose (SD) media (6.7 g Bacto yeast nitrogen base without amino acids and 20 g glucose per liter). Also, SD+ medium [SD medium supplemented with 0.4% ethanol, 5 g (NH₄)₂SO₄ per liter, and 3.3 g NaCl per liter] was used because it inhibits capsule expression. WO-1 was grown in Lee's medium (13), and switching was assessed on agar plates supplemented with 5 mg/liter phloxine B as described previously (31).

Isolation of senescent C. neoformans cells.A method to isolate C. neoformans cells with advanced generational age was developed on the basis of previously published protocols for S. cerevisiae (12, 25). C. neoformans RC-2 cells were grown overnight in SD medium. Because larger polysaccharide capsules interfere with the isolation of senescent cells, the overnight culture was diluted 1:50 and grown for approximately 6 h in SD+ medium. Under these growth conditions, the mean thickness of the polysaccharide capsule was less than 1 μm. Counterflow centrifugal elutriation was performed to isolate newly budded cells (<5 μm in diameter). This method isolates fractions of different-sized cells on the basis of their sedimentation coefficient, which is a function of cell volume and density. A cell suspension (1 × 10⁸ to 5 × 10⁸ yeast cells) was loaded at 14 ml/min into a rotating elutriator rotor (Beckman JE-5.0 in a Beckman J-6B centrifuge; Beckman Instruments Inc., Palo Alto, CA) while the rotor speed was kept constant at 3,500 rpm. Cells were collected in 100-ml fractions at increasing flow rates with a peristaltic pump. The flow rates were 16 ml/min, 20 ml/min, and 25 ml/min for fractions 1 to 3, respectively. The first two fractions contained small C. neoformans cells (<5 μm; generation 0). Generation 0 cells were washed one time, suspended in phosphate-buffered saline (PBS) at 5 × 10⁷/ml, and labeled at room temperature with biotin (Sulfo-NHS-LC-biotin; Pierce, Rockford, IL) at a concentration of 4 mg/ml in PBS. These cells constituted the starting population from which we derived old cells of advanced generational age. This label is unable to permeate the cell membrane and thus labels only surface proteins and can be visualized by staining with fluorescein streptavidin conjugate (Molecular Probes Inc., Eugene, OR). Biotin-labeled cells retain the label and exhibit normal function and a normal growth rate. Cells were washed five times with PBS, and positive staining was confirmed in a small fraction before cells were grown overnight in SDB. After approximately five or six doubling times, they were washed in PBS, resuspended at 10⁷/90 μl, and incubated with 10 μl streptavidin-conjugated magnetic microbeads (Miltenyi Biotec, Auburn, CA) for 15 min at 6 to 10°C. Magnetic-bead-labeled cells were washed, resuspended at 10⁸/500 μl PBS, and isolated via a LS magnetic column (Miltenyi Biotec). The retained bead-positive cell fraction was released by disrupting the magnetic field. The purity of the positive cells was confirmed by staining, and the cells were reintroduced into overnight culture as described above. The procedure was repeated four or five times to isolate senescent (18 to 30 generations old) cells. With increasing isolation, a significant percentage of the cells (>50%) was lost. Notably, restaining and relabeling of the positive cells after two rounds (10 generations) could increase the yield. For C. albicans, the same protocol was applied. The initial elutriation step was not required for Candida cells, as they lack a capsule. To verify the generational age of isolated cells, the input and output cells were carefully counted and the doubling times and generations were mathematically inferred. Fluorescent staining of total populations was used as an independent confirmation of the result (percentage of green fluorescent cells).

Switching frequency.Phenotypic switching was defined as the spontaneous generation of colony variants with altered colony morphology (6). For calculation of switching frequencies, 200 to 300 cells with the SM “parent” phenotype were plated on 100 SDA plates and 10⁴ colonies were scored for the respective MC “switch variant” phenotype. The percentage of the total number of CFU with MC morphology relative to the percentage with SM morphology was determined. For WO-1 cells, the white cells were plated on phloxine B-supplemented agar plates. The ratio of pink (opaque) to white (white) cells was calculated (29).

Phenotypic characterization of senescent cells and their detection in vivo. (i) Cell morphology and capsule size measurements.For capsule size measurements, senescent and young yeast cells were suspended in India ink (Becton Dickinson, Franklin Lakes, NJ) and visualized at ×1,000 magnification with an Olympus AX70 microscope. Images were captured with a QImaging Retiga 1300 digital camera with the QCapture Suite V2.46 software (QImaging, Burnaby, British Columbia, Canada). Capsule measurements were made on 25 to 40 randomly chosen cells of each strain with Adobe Photoshop 7.0 for Windows, and capsule thickness was calculated with the conversion of 15 pixels/μm, as described previously (33). Capsule of senescent cells was induced in vitro by incubating the cells at 37°C for 48 h in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (33). Bud scars were stained with fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (WGA) lectin in accordance with the manufacturer's protocol (Sigma-Aldrich). As a control, senescent S. cerevisiae cells (approximately 15 generations old) were stained with WGA.

(ii) In vivo detection.For in vivo staining, mice were sacrificed at day 14 and day 21 post i.t. infection with 10⁶ cells (overnight culture biotin labeled). Single-cell suspensions were obtained from lung homogenates and stained with FITC-conjugated streptavidin to identify biotin-labeled cells. As a negative control, cells from mice infected with unlabeled cells were used. For a rough estimate of percentage of biotin-labeled cells, we counted all of the fungal cells under phase-contrast microscopy and then the FITC-positive cells under fluorescence microscopy.

(iii) Phagocytosis assay.For in vitro phagocytosis assays, J774.16 cells were grown in a 96-well tissue culture plate and young or old C. neoformans cells were added at a fungus-to-macrophage ratio of 1:1 in the presence of capsule-specific monoclonal antibody 18B7 (1 μg/ml). After 2 h, the cell layers were washed, fixed, and stained with Giemsa and the phagocytosis index (number of yeast cells in macrophages/number of macrophages) was determined. The assay was performed in triplicate, and a total of 500 macrophages per group were counted to calculate the phagocytosis index.

(iv) Karyotype analysis of senescent cells.Senescent cells (24 generations old) of both the SM parent (O) and MC switch (O*) phenotypes were regrown briefly for two or three generations to produce enough cells (>10⁸) for the isolation of chromosomal DNA. Also, plugs were made from young cells (overnight culture). Plugs were prepared from cultures as described before (4). After washing, the plugs were inserted into a 1% pulsed-field-certified agarose gel (Bio-Rad, Richmond, CA) and electrophoresis was done in a CHEF DRIII variable-angle pulsed-field gel electrophoresis system (Bio-Rad) in 0.5% Tris-borate-EDTA at 12°C. The electrophoresis conditions were programmed in two sequential blocks (first a switch time of 90 s for 9 h and then switch times of 120 s and 360 s for 63 h). Both blocks were run at 3.5 V/cm at an angle of 115°. Gels were stained with ethidium bromide and photographed.

Killing assay.The in vitro killing efficiency of increasing doses of fluconazole, amphotericin B, and caspofungin was determined for young and old cells. Briefly, young and old cells were isolated as described above and diluted to 1 × 10⁴/ml in RPMI 1640 medium (morpholinepropanesulfonic acid [MOPS] buffered to pH 7). Fluconazole, amphotericin B, or caspofungin was serially diluted in 100-μl volumes at concentrations of 128 to 1,000 μg/ml, 0.125 to 2 μg/ml, and 4 to 512 μg/ml, respectively, in RPMI medium buffered with MOPS in a 96-well plate. Volumes of 100 μl of young and old cells were added to achieve final concentrations of 5 × 10³ cells/ml, 64 to 500 μg/ml fluconazole, 0.06 to 1 μg/ml amphotericin B, and 2 to 256 μg/ml caspofungin. Wells with no antifungal agent were included in the plate as controls. The plate was incubated at 37°C with shaking for 2.5 h (C. neoformans) and 4 h (C. albicans), and 100 μl of each well was plated on SDA plates and incubated at 37°C for 48 h. All experiments were performed in triplicate, and the killing percentage was calculated by comparison of antifungal-treated CFU count to the CFU count of the respective control (without antifungal treatment).

Animal studies.BALB/c mice (male, 6 to 12 weeks old) were obtained from the National Cancer Institute (Bethesda, MD). Groups of anesthetized mice (10 per group) were infected by i.t. inoculation of 10⁵ old and young C. neoformans cells in 50 μl sterile, nonpyrogenic PBS with a 26-gauge needle as previously described (9). Dilutions of the infecting suspension were plated onto SDA plates to ensure that comparable numbers of viable yeast cells were injected. Mice were observed daily for signs of disease. Mice were killed by cervical dislocation after anesthesia, and organ fungal burdens were determined by homogenizing lung and brain tissues in 10 ml of PBS and plating 100-μl volumes of different dilutions of the homogenate on SDA (Difco Laboratories, Detroit, MI). Colonies were counted after 48 h (1 colony = 1 CFU). To compare in vivo crossing of the blood-brain barrier by young and old cells, intravenous (i.v.) infection was performed as described previously (11). Briefly, young and old cells were isolated as described above and BALB/c mice (five per group) were injected in the tail vein with 6 × 10⁵ cells. The inoculum size was verified by back plating onto SDA plates. After 3 h, the mice were anesthetized and perfused with PBS and the brains were removed and homogenized in 2 ml of sterile PBS and plated on SDA plates to determine the number of CFU per brain.

Mathematical modeling.The time-dependent growth or (decrease) of biological populations can be mathematically represented by first-order differential equations (19). We devised a simple mathematical model for the growth of two classes of cells, A (SM) and B (MC), whose in vivo growth is influenced by the immune response as follows: dA/dt = α1 × A(t) × H(t) for SM cells and dB/dt = β1 × B(t) × h(t) for MC cells. A(t) and B(t) represent the (unknown) number of cells at time t. dA/dt represents the change in the population size (A) at any time t [same for B(t)]. H(t) and h(t) are immune factors which modify the growth of A(t) or B(t). These factors are assumed to be a function of time t but might also be dependent on other parameters, such as the number of A or B cells (in this case, the equation depends on the square of A, or B would become nonlinear). α1 and β1 are constants representing the growth rates. These differential equations can be solved numerically, yielding solutions A(t) and B(t). If H is constant (i.e., a number), the differential equations describe an exponential increase or decrease in the number of A or B cells in time (depending on the sign of α1). We have assumed for H = H(t) that it is time dependent.

Statistical analysis.Standard statistical analyses, including the Kaplan-Meier test, log rank regression analysis, and the t test, were performed with the programs SPSS version 7.5.1 and Microsoft EXCEL version 11.5.3.

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FIG. 1.

Phenotypic characterization of senescent C. neoformans and C. albicans cells. (a) The larger size of 24-generation-old C. neoformans cells involves increases in both the capsule and the cell body. The increase is progressive over generations, as shown in FITC-stained, biotin-labeled cells. Bar size, 2 μm. (b) Similar size increases were seen in senescent C. albicans cells. Bar size, 2 μm. Notably, the biotin label faded more over time. (c) Staining with WGA lectin cannot be used to adequately determine the generation. Opposed to S. cerevisiae (right panel), the bud scars of C. neoformans appear to heal over or become undetectable with time. Bar size, 3 μm.

We successfully adapted published protocols for S. cerevisiae and developed a method to isolate senescent C. neoformans cells that were more than 30 generations old. In contrast to C. neoformans cells, we were not able to isolate senescent Candida cells that were older than 14 generations because C. albicans cells consistently transformed into a hyphal phenotype as they grew older. Hyphal cells could not be isolated by this strategy because the hyphae promoted clumping, which clotted the magnetic column and prevented flowthrough. The doubling time increased with generational age, and for C. neoformans cells older than 25 generations, it was about twice as long. Old cells of both C. neoformans and C. albicans were significantly larger than young cells (P < 0.001 by t test and analysis of variance). The size of C. neoformans biotin-positive cells was 9.1 ± 1.2 μm (24 generations), and that of biotin-negative cells was 7.3 ± 0.71 μm (overnight logarithmic culture) (Fig. 1a). The size of biotin-positive C. albicans cells was 6.2 ± 0.8 μm at 6 generations and 7.6 ± 1.7 μm at 12 generations, and that of biotin-negative cells from an overnight culture was 4.3 ± 0.4 μm (Fig. 1b). Notably, the biotin label faded much more rapidly in C. albicans than in C. neoformans (Fig. 1b). Also, the increase in C. neoformans cell size was proportional for the cell body and the capsule. Bud scar staining was performed with WGA, a lectin that preferably binds to bud scars. In contrast to S. cerevisiae, we detected no more than five bud scars per C. neoformans cell (Fig. 1c).

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FIG. 2.

Phenotypic switching rate is augmented in senescent RC-2 cells. (a) Karyotype analysis of young (Y) and old (O = old parent SM, O* = old switched MC) C. neoformans cells indicates changes in the chromosome 2 of old (O, O*) cells but no karyotype changes in old cells compared to young (Y) cells. (b, c, d) The rate of switching from SM to MC (arrowhead) increased with age, and many sectored colonies (arrow) were detected. MW, molecular weight markers.

Phenotypic switching rates were compared between young and old C. neoformans strain RC-2 cells. Prior to plating, we confirmed the 95% purity of senescent cells by staining with FITC-conjugated streptavidin. A dramatic increase in the switching rate between young and old cells was seen and confirmed in repeat experiments. The switching rate increased with age and was 6- and 11-fold greater in cells 27 and 31 generations old, respectively (Fig. 2b to d). Cells that were 15 generations old exhibited no significant increase in the switching rate. An effect of senescence on the switching of C. albicans strain WO-1 (30) could not be accurately determined because only cells up to 14 generations old could be isolated and the switching rate, which was determined by the color of colonies, was noted to be highly variable between experiments (data not shown). Comparison of the karyotype patterns of old C. neoformans cells with either SM colony morphology (O) or switched MC colony morphology (O*) demonstrated no detectable rearrangements in the karyotype pattern. However, changes in the second largest chromosome were seen in the karyotype patterns of both old cell types (O, O*). Their karyotype pattern appeared to have a wider or extra band in that chromosome compared to the karyotype pattern of young cells (Fig. 2a).

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FIG. 3.

In vivo detection, survival, and migration of senescent cells. (a) Biotin-positive cells in lung homogenates from mice infected for 14 days. Note that biotin-labeled, actively budding cells were observed in these lung homogenates (left). (b) No survival difference was documented in mice infected i.t with young (overnight culture) or old (24 generations) cells. (c) After i.v. infection, old (18 generations) and young C. neoformans cells crossed the blood-brain barrier equally.

Criteria to reliably identify senescent cells within a pathogen population are not available. Therefore, we took several indirect approaches to investigate these cells in vivo. First, we determined if the persistence of old cells occurs during chronic murine infection. For these experiments, mice were infected with biotin-labeled C. neoformans cells (overnight culture) and sacrificed at 14 and 21 days postinfection. At this time, the lung CFU count is highest in infected mice. Lung homogenates were stained with FITC-conjugated streptavidin to identify biotin-labeled cells. These cells would represent cells from the original inoculum that could have theoretically undergone up to 80 doubling times (assuming a 4-h doubling time) by day 14. Biotin-positive cells were detected in lung homogenates from mice infected for 14 and 21 days (Fig. 3a) in low numbers (approximately 1 to 10% of the extracellular yeast cells). In these lung homogenates, actively budding biotin-positive cells were also seen (Fig. 3a). Next, we compared the survival times of mice injected with young and old (24 generations) C. neoformans cells. This experiment documented comparable median survival times, specifically, 53 days versus 56 days in mice that were infected with young and old cells (P = 0.6) (Fig. 3b). Lastly, we compared the transmigration of young and old C. neoformans cells (18 generations) across the blood-brain barrier in mice infected i.v. These experiments documented that both old and young C. neoformans cells crossed the blood-brain barrier equally (Fig. 3c).

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FIG. 4.

Increased resistance of senescent C. neoformans and C. albicans cells in time-kill assays with antifungals. (a) Old (18 generations) C. neoformans cells were more resistant to killing by therapeutic doses of amphotericin B and (b) higher fluconazole doses. (c) Old (10 generations) C. albicans cells were also more resistant to killing by caspofungin (* represents P values of <0.02; experiments were done in triplicate).

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FIG. 5.

Enhanced resistance of senescent C. neoformans cells to phagocytosis. Antibody-mediated phagocytosis of old (18 generations) C. neoformans cells was significantly (* represents P < 0.05; experiments were done in triplicate) decreased compared to that of young cells (grown overnight). In vitro capsule induction was also more pronounced in senescent C. neoformans cells. Bar size, 5 μm. Arrowheads indicate phagocytosis of C. neoformans cells by macrophages. Left, uptake of many young, small C. neoformans cells. Right, uptake of less old, big C. neoformans cells.

To further investigate mechanisms that could facilitate the persistence and selection of senescent cells, we examined their resistance to killing by antifungal agents and to antibody-mediated phagocytosis by macrophages. Both assays are not dependent on the growth of C. neoformans. Senescent C. neoformans cells were more resistant to killing with therapeutic doses of amphotericin B, a lethal antifungal agent. At higher-than-therapeutic levels of fluconazole, a static antifungal agent, enhanced resistance to killing was also observed. For C. albicans, enhanced resistance to killing by caspofungin in therapeutic dose ranges was established. Furthermore, antibody-mediated phagocytosis of senescent C. neoformans cells was significantly (P = 0.04) decreased in comparison to the phagocytosis of young cells grown overnight. Capsule induction in vitro was also more pronounced in senescent C. neoformans cells (Fig. 5) but, relative to the enlarged cell body, comparable for old and young cells.

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FIG. 6.

Mathematical modeling of the emergence of MC variants in the setting of altered switching rates. The emergence of MC cells (CFU count in the lung, y axis) in mice infected with a “switching” SM strain (blue and red lines) is modeled mathematically based on experimental data. Note that, according to this model, emergence would be greatly affected by a 10-fold higher switching rate. Gray represents decreasing CFU counts of SM cells in infected mice that mount a host response. Green represents increasing CFU counts in MC cell-infected mice that cannot clear the infection.

The emergence of MC colonies was modeled according to biological data derived from two large murine infection experiments (5, 6) in which >100 mice were infected i.t. with 10⁴ SM or MC CFU (5, 6). These experiments had demonstrated that mice infected i.t with 10⁴ SM cells exhibited a lung CFU count of log 6.25 at day 14. These mice cleared the infection by day 140. In contrast, mice infected i.t with 10⁴ MC cells exhibited a comparable lung CFU count of log 6.0 at day 14. MC cell-infected mice did not clear the infection (median survival time of 33 days and a lung CFU count of more than log 7). A simple mathematical model for the growth of A (SM) and B (MC) cells whose in vivo growth was influenced by the immune response was devised as outlined in Materials and Methods. The emergence of MC cells derived from switching of SM to MC at rates of 1:20,000 and 1:2,000 was modeled in parallel (Fig. 6), demonstrating that changes in switching rate without changes in host response would greatly affect the emergence of MC cells in vivo.