Cryptococcus neoformans host adaptation: toward biological evidence of dormancy

Abstract

Cryptococcosis is an opportunistic infection due to the ubiquitous yeast Cryptococcus neoformans. This yeast interacts closely with innate immune cells, leading to various fates, including fungal persistence within cells, making possible the dissemination of the yeast cells with monocytes via a Trojan horse strategy. In humans, the natural history of the infection begins with primoinfection during childhood, which is followed by dormancy and, in some individuals, reactivation upon immunosuppression. To address the question of dormancy, we studied C. neoformans infection at the macrophage level (in vitro H99-macrophage interaction) and at the organ level in a murine model of cryptococcosis. We analyzed the diversity of yeast adaptation to the host by characterizing several C. neoformans populations with new assays based on flow cytometry (quantitative flow cytometry, multispectral imaging flow cytometry, sorting), microscopy (dynamic imaging), and gene expression analysis. On the basis of parameters of multiplication and stress response, various populations of yeast cells were observed over time in vivo and in vitro. Cell sorting allowed the identification of a subpopulation that was less prone to grow under standard conditions than the other populations, with growth enhanced by the addition of serum. Gene expression analysis revealed that this population had specific metabolic characteristics that could reflect dormancy. Our data suggest that dormant yeast cells could exist in vitro and in vivo. C. neoformans exhibits a huge plasticity and adaptation to hosts that deserves further study. In vitro generation of dormant cells is now the main challenge to overcome the limited number of yeast cells recovered in our models.

Importance: Cryptococcus neoformans is a sugar-coated unicellular fungus that interacts closely with various cells and organisms, including amoebas, nematodes, and immune cells of mammals. This yeast is able to proliferate and survive in the intracellular environment. C. neoformans causes cryptococcosis, and yeast dormancy in humans has been suggested on the basis of epidemiological evidence obtained years ago. By studying an in vitro model of yeast-macrophage interaction and murine models of cryptococcosis, we observed that yeast cells evolve in heterogeneous populations during infection on the basis of global metabolic activity. We compared the growth ability and gene expression of yeast cells belonging to various populations in those two models. We eventually found a population of yeast cells with low metabolism that fit some of the criteria for dormant cells. This paves the way for further characterization of dormancy in C. neoformans.

FIG 1

Quantification of C. neoformans (H99) multiplication, stress response, and viability with fluorescent dyes and flow cytometry. (A) C. neoformans multiplication was evaluated after Calcofluor (CALCO) staining of yeast cells prior to incubation into YPD medium, interaction with murine macrophages, or inoculation to outbred OF1 mice. The yeast cells were recovered from different environments (YPD culture, J774 cells, and lungs) at different time points and compared to unstained yeast cells by flow cytometry (MacsQuant analyzer, Miltenyi Biotec). A population with lower CALCO fluorescence intensity (daughter cells) appeared over time under the three conditions. Gates allowing segregation of CALCO^high and CALCO^med+low cells are depicted for each condition (YPD culture, J774 cells, and lungs). Of note, the decrease in CALCO fluorescence intensity observed in yeast cells recovered from lungs as soon as 30 min after inoculation can be attributed to quenching and not to multiplication. (B) In vitro C. neoformans stress response evaluated on the basis of CMFDA staining. Yeast cells were stained with CMFDA (10 µM) for 30 min at 37°C. Various stresses (H₂O, H₂O₂, fresh medium [DMEM–10% FCS–1% penicillin/streptomycin] and heat killing [HK]) were applied to stationary-phase (H0) yeast cells for 2 and 24 h (H24). CMFDA fluorescence increased over time, except at H24 after H₂O₂ incubation, where it became negative. (C) Viability of C. neoformans populations was assessed with Topro-3 iodide (TOPRO) with dead yeast cells (HEAT) with a higher fluorescence intensity (TOPRO^high) than viable yeast cells harvested in stationary phase upon standard culture (YPD culture, TOPRO^low).

FIG 2

Evolution of C. neoformans multiplication and stress response in two host environments. The CALCO and the CMFDA fluorescence intensities were analyzed after interaction with macrophages (A and B) and in lungs of infected mice (C and D). After macrophage lysis or organ grinding, the pellet of yeast cells was stained with CMFDA (10 µM) and TOPRO (10 µM) for 30 min at 37°C (CMFDA/TOPRO assay). Analysis of the fluorescence was performed by flow cytometry after exclusion of the TOPRO^high population (dead yeast cells). The generation of daughter cells (CALCO^med+low) was noticed upon 2 h of macrophage interaction (A) and 15 h after mouse inoculation (C) and increased over time (B and D). An increased stress response (higher CMFDA fluorescence) was already seen at 2 h (H2) in macrophages (A) and 7 h (H7) in the lungs (B). A population with a lower CMFDA fluorescence level (CMFDA^med+low) (arrows) was observed at 24 h (H24) in macrophages (A and B) and at 30 h (H30) in lung cells (C and D) that affected first mother cells (24 h [H24]) and then daughter cells (48 h [H48]) in macrophages.

FIG 3

C. neoformans stress response and multiplication depends on individuals (outbred OF1 mice) and tissues. Seven days after inoculation with 10⁵ CALCO-stained yeast cells (H99), mice were sacrificed and their organs (brains, lungs, and spleens) were ground. Yeast cells were stained for stress response and viability by CMFDA/TOPRO assay. Flow cytometry analysis was performed after exclusion of the TOPRO^high population (dead yeast cells) (MacsQuant analyzer; Miltenyi Biotec). The C. neoformans brain and spleen profile was homogeneous in terms of multiplication and stress response, whereas two profiles were observed in lungs. A well-defined CALCO^high/CMFDA^high population (black gate) was observed in some mice (M1 and M2, M3, and M5) and not in others (M4 and M6).

FIG 4

Multispectral imaging flow cytometry confirms the heterogeneity of the C. neoformans populations in the lungs of outbred OF1 mice. C. neoformans from the pooled lungs of 14 mice (a total of 2,635 yeast cells) were analyzed for multiplication, stress response, and viability. (A) Nine H99 populations (1 to 9) were delineated by multispectral imaging flow cytometry (Imagestream^X). The nine gates were adjusted on the basis of optical control of the fluorescence intensity of the corresponding pictured events. One representative of two independent experiments is shown. (B) The distribution of the CALCO populations (high, medium, and low) in the different CMFDA population is shown. Bars represent the means ± the standard deviations of the nine populations, with numbers corresponding to those shown in panel A. The CALCO^low population always represented <20%. In the CMFDA^high population, the CALCO^high population was predominant, whereas the CALCO^med population was predominant in the CMFDA^med and CMFDA^low populations. (C) Heat map generated from the BF channel pictures on the basis of the geometric mean of 54 different algorithms. Five groups of yeast cells composed mainly of CALCO^low, CALCO^high CMFDA^high, CMFDA^med, CMFDA^low, and eight groups (a to h) of algorithms clustered. Cluster h, which includes all of the size and most of the location algorithms, delineated the CALCO^high (yellow, red outline) and CALCO^low (blue, pink outline) populations, indicating that the CALCO^high cells consisted mostly of big yeast cells and the CALCO^low cells consisted mostly of small yeast cells. Cluster b distinguished CALCO^low cells from the other four populations as having a specific signal strength and texture (higher value of the corresponding algorithms, red outline). Cluster c distinguished CMFDA^low cells from the other four populations on the basis of some shape and location algorithms (higher value of the corresponding algorithms, red outline).

FIG 5

Morphological and fluorescence features of the Calco^high/CMFDA^high H99 population in the lungs of outbred OF1 mice. (A) Multispectral imaging flow cytometry (Imagestream^X; Amnis) was used to picture each event of the CALCO^high/CMFDA^high population in five channels: BF (transmitted light), yellow (Cy3, capsule), green (CMFDA, stress response), blue (CALCO, multiplication), and red (TOPRO, viability). Different patterns of morphology and CMFDA fluorescence were observed. (B) The CALCO^high CMFDA^high population of yeast cells from lung homogenates was also observed by classical fluorescence microscopy. CMFDA fluorescence was observed surrounding the cell wall CMFDA^sur (white dotted arrows in panel A), or within the yeast cell cytoplasm (CMFDA^intra, white arrows in panel A). (C) Multispectral imaging flow cytometry based on the modulation algorithm (texture) and the area (size) of the yeast cells easily discriminated CMFDA^intra and CMFDA^sur cells. The CMFDA^sur yeast cells were composed of regular and drop C. neoformans cells.

FIG 6

Morphological features and metabolic activity revealed that the drop C. neoformans (Drop Cn) cells in the lungs of outbred OF1 mice were dead yeast cells. Yeast cells from lung homogenates were observed by interference contrast microscopy. (A) Examples of cells composing a subpopulation of yeast cells with a typical morphology (small size [5.80 ± 0.80 µm, n = 12], thick cell wall, and one well-defined round refringent vesicle) are shown. (B) Nucleus morphology (DAPI, blue), RNA content (SYTO85, orange), and mitochondrial activity (Mitotracker, orange) were assessed in yeast cells previously stained with CMFDA (green). Drop C. neoformans cells were devoid of nucleus (regular shape and regular DAPI fluorescence), RNA, and mitochondrial activities, in contrast to regular C. neoformans (Reg Cn) cells. (C) Lipid membrane layers (MDY64, green) were assessed in unstained yeast cells. Drop C. neoformans cells harbored a complete retraction of the cytoplasm around the central refringent vesicle.

FIG 7

Analysis of the growth curves of different CMFDA populations after sorting. Yeast cells recovered from macrophage lysis (MP) after 24 h of interaction (A and B) or lung homogenates (MO) from infected outbred mice (C and D) were stained and then sorted (FACSAria II; BD). Stationary-phase yeast cells stained at the same time (sSTAT, red) were used as a control. Yeast cells (10⁴/ml) of each population were allowed to grow in YPDps (dashed lines) or YPDps plus 10% FCS (+FCS, solid lines) in the wells of a 96-well plate at 30°C with agitation (800 rpm). The OD₆₀₀ was recorded over time. (A) Four CMFDA populations (CMFDA/CALCO dot plot) were studied: CMFDA^high CALCO^high (yellow), CMFDA^med CALCO^high (orange) CMFDA^low CALCO^high (blue) and CMFDA^high CALCO^med+low (green). (B) The exponential growth phase of the CMFDA^low population (blue) was delayed compared to that of the other three populations and sSTAT. Onset of the exponential phase occurred earlier with the addition of FCS. (C) Three CMFDA populations (CMFDA SSC dot plot) of yeast cells recovered from pooled lung homogenates were analyzed: CMFDA^high (yellow), CMFDA^med (orange), and CMFDA^low (blue). (D) The CMFDA^low population (blue) failed to grow in YPD even after 48 h of incubation. Addition of FCS restored growth capacities even though onset was delayed by ≥10 h compared to that of the other populations and sSTAT. In addition, growth of CMFDA^high and CMFDA^med populations was similar and delayed compared to that of sSTAT.

FIG 8

Heat map of gene expression analysis (n = 37) of different CMFDA populations by real-time quantitative PCR. CMFDA populations of yeast cells recovered from macrophages (MP) or murine lungs (MO) were studied. After lyophilization, yeast cells were lysed and primer-specific reverse transcription and quantitative PCR were performed. The targets selected (n = 37) were genes involved in growth, stationary phase, resistance to oxidative stress, autophagy, adaptation to the lung environment, and capsule and chitin formation. For each gene, the fold change compared to the ACT1 and GAPDH genes was normalized between 0 and 1. The genes for which amplification failed are depicted in grey. MO_CMFDA^med and MO_CMFDA^high from mice clustered together with each other and apart from the other populations. All of the macrophage populations except MP_CMFDA^low clustered together. The pattern of expression of MP_CMFDA^low and MO_CMFDA^low was different from that of the other populations and between them, except for PCK1 and COX1 (red arrows). Four clusters of genes can be differentiated, with C1 specifically composed of genes involved in lung adaptation during infection (21) and C4 composed of genes expressed more in the MP_CMFDA^low population.