Elucidating the pathogenesis of spores from the human fungal pathogen Cryptococcus neoformans

Abstract

Cryptococcus neoformans was first described as a human fungal pathogen more than a century ago. One aspect of the C. neoformans infectious life cycle that has been the subject of earnest debate is whether the spores are pathogenic. Despite much speculation, no direct evidence has been presented to resolve this outstanding question. We present evidence that C. neoformans spores are pathogenic in a mouse intranasal inhalation model of infection. In addition, we provide mechanistic insights into spore-host interactions. We found that C. neoformans spores were phagocytosed by alveolar macrophages via interactions between fungal beta-(1,3)-glucan and the host receptors Dectin-1 and CD11b. Moreover, we discovered an important link between spore survival and macrophage activation state: intracellular spores were susceptible to reactive oxygen-nitrogen species. We anticipate these results will serve as the basis for a model to further investigate the pathogenic implications of infections caused by fungal spores.

FIG. 1.

C. neoformans var. grubii spores are pathogenic in a murine model of cryptococcosis. Groups of four or eight AJ/Cr (A) or C57BL/6 (B) mice were infected with C. neoformans var. grubii spores (10⁵) or yeast (10⁵) via intranasal inhalation. All mice succumbed to infection and died by day 30 postinfection. There was no significant difference in the median time to death of AJ/Cr or C57BL/6 mice infected with C. neoformans var. grubii spores or yeast (P > 0.05). Median times of death for AJ/Cr mice infected with C. neoformans var. grubii spores or yeast were 24 and 25 days, respectively. Median times of death for C57BL/6 mice infected with C. neoformans var. grubii spores or yeast were 28 and 27 days, respectively.

FIG. 2.

Mice infected with C. neoformans var. grubii spores have low lung CFU early in infection. Groups of four C57BL/6 mice were infected with C. neoformans var. grubii spores (10⁵) or yeast (10⁵) via intranasal inhalation. Mice were sacrificed 3 and 6 days postinfection. Pulmonary CFU were determined by homogenizing whole lungs in PBS and plating dilutions of homogenates on YPD plates. The CFU/g of lung tissue were calculated. Bars represent mean ± the standard error.

FIG. 3.

C. neoformans var. neoformans spores are phagocytosed by alveolar macrophages. C. neoformans var. neoformans spores (A) and yeast (B) were coincubated with adherent mouse alveolar macrophages at a multiplicity of infection of 4:1. Fluorescence microscopy images of alveolar macrophages (differential interference contrast) incubated with calcofluor stained (blue) spores and yeast. Only spores are phagocytosed by alveolar macrophages. Flow cytometric analysis of alveolar macrophages incubated with calcofluor-stained spores and yeast confirmed that spores were phagocytosed. An AF488-labeled anti-CD11c antibody was used to detect alveolar macrophages, spores and yeast were labeled with calcofluor white, and an AF594-labeled anti-GXM antibody was used to discern between extracellular and intracellular spores or yeast.

FIG. 4.

β-(1,3)-Glucan is accessible on the surface of C. neoformans var. neoformans spores. (A) Fc-Dectin chimera protein was used to assay for β-(1,3)-glucan on the surface of C. neoformans var. neoformans spores and yeast. Fluorescence microscopy of Fc-Dectin binding showed that β-(1,3)-glucan was on the surface of spores but not yeast. Bar, 5 μm. (B) Flow cytometric analysis of anti-β-(1,3)-glucan IgG antibody binding to C. neoformans var. neoformans spores and yeast confirmed that β-(1,3)-glucan was accessible on the surface of spores but not yeast. No binding was observed with the secondary antibody alone control.

FIG. 5.

Intracellular survival of C. neoformans var. neoformans spores is dependent on alveolar macrophage activation state. (A) C. neoformans var. neoformans spores were cocultured with untreated alveolar macrophages, and intracellular germination was assessed 24 h postinfection. Numerous budding yeast (white arrowheads) enveloped in capsule (black arrowheads) were observed inside alveolar macrophages. (B) Within 24 h, intracellular C. neoformans var. neoformans spores germinated into yeast (white arrowheads). Confocal microscopy of calcofluor labeled yeast inside actin stained alveolar macrophages showed yeast exiting intact alveolar macrophages (yellow arrow). (C) Three-dimensional reconstruction of a deconvolved confocal image of calcofluor labeled yeast inside actin-stained alveolar macrophages. The three-dimensional image confirmed the intracellular localization of yeast (white arrowheads) and exit of yeast from the alveolar macrophages (yellow arrowhead). The images are representative of three independent experiments. (D) C. neoformans var. neoformans spores were cocultured with alveolar macrophages (AM) treated with LPS and IFN-γ (Stim.) or left untreated as a control. After 4 h of coculture the wells were washed to remove extracellular spores and yeast, and cultures were allowed to continue for 24 h. Phagocytosis (time point [TP] 0 h) and inhibitory activity (TP 24 h) were assessed by CFU analysis. The results demonstrate that activation state affects alveolar macrophage (AM) antifungal activity. (E) An identical experiment was performed with spores and yeast opsonized with anti-GXM IgG. Activated macrophages inhibited the growth of spores, but not yeast. The bars represent means plus the standard errors.

FIG. 6.

Alveolar macrophage (AM) inhibitory activity is mediated by reactive oxygen-nitrogen species. (A) C. neoformans var. neoformans spores were cocultured with mouse alveolar macrophages from iNOS^−/− gp47^phox−/−double-knockout mice for 4 h. Phagocytosis (time point [TP] 0 h) and inhibitory activity (TP 24 h) were assessed by CFU analysis. Stimulated macrophages from knockout mice did not inhibit spore germination. (B) Groups of four iNOS^−/− gp47^phox−/− or C57BL/6 mice were infected with 10⁵ C. neoformans var. grubii spores via intranasal inhalation. There was a statistically significant difference in the median time to death (P < 0.004) of iNOS^−/− gp47^phox−/− mice infected mice compared to or C57BL/6 wild-type mice. (C) In contrast, there was no significant difference in the median time to death of groups of four iNOS^−/− gp47^phox−/− or C57BL/6 mice infected with C. neoformans var. grubii strain H99 yeast (10⁵) via intranasal inhalation.