Fig 1.
Spores result in higher fungal burdens in the brain than yeast in a mouse intranasal model of infection.
(A) Representative survival plot of mice (8 per group) infected intranasally with 1x105 BT63 (a) yeast alone (triangles), 1x105 H99 (α) yeast alone (circles), 1x105 yeast as a 1:1 mixture of both strains (BT63 + H99; diamonds), or 1x105 spores derived from a cross between strains (BT63 x H99; squares). (B) Fungal burdens (as colony-forming units per gram of tissue) at endpoint in the lungs (gray bars) and brains (striped bars) of mice infected with 1x105 yeast in a 1:1 mixture of both parent strains (BT63 + H99) or with 1x105 spores derived from a cross between strains (BT63 x H99) (3 mice per group). Each circle represents an individual mouse. *p = 0.011 for brain comparisons; for lung and all other tissues, p ≥ 0.10 (using a Student’s t-test). Data are representative of multiple biological replicates (see Materials and Methods).
Fig 2.
Spores produced by avirulent yeast strains cause uniformly fatal cryptococcal meningoencephalitis in a mouse intranasal model of infection.
(A) Survival plot of mice (8 mice per group) infected intranasally with 2.5x105 B-3502 yeast (triangles) or 2.5x105 B-3501 yeast (circles), 2.5x105 yeast as a 1:1 mixture of B-3502 and B-3501 (B-3502 + B-3501; diamonds), or 2.5x105 spores derived from a cross between B-3502 and B-3501 (B-3502 x B-3501; squares). (B) Fungal burdens (as colony-forming units per gram of tissue plus 1) in the lung (dark gray bars), kidney (light gray bars), and brain (striped bars) at 3, 6, 14, 21, and 100 days after intranasal infection of mice with 2.5x105 yeast as a 1:1 mixture of B-3502 and B-3501. Circles represent individual mice (3 mice per time point). (C) Fungal burdens (as colony-forming units per gram of tissue plus 1) in the lung (dark gray bars), kidney (light gray bars) and brain (striped bars) at 3, 6, 14, 21 days and at endpoint after intranasal infection of mice with 2.5x105 spores derived from a cross between B-3502 and B-3501. Circles represent individual mice (3 mice per time-point). (D) Survival curves of 6 mice infected by intratracheal instillation with 1x105 spores derived from a B-3502 x B-3501 cross (squares) or 5 mice infected with 1x105 recombinant yeast (generated by germination of spores derived from a B-3502 x B-3501 cross into yeast; triangles). (E) Survival plots of mice infected by tail-vein injection with 1x106 yeast in a 1:1 mixture (B-3502 + B-3501; triangles) or 1x106 spores derived from a B-3502 x B-3501 cross (squares) (5 mice per group). Data in A, B, and C are representative of independent biological replicates (see Materials and Methods), and data in D and E are derived from single experiments.
Fig 3.
Yeast and spores of the same genetic background do not elicit differences in overall lung immune response.
(A) Quantification of lung myeloid cell (monocytes, neutrophils, or eosinophils) recruitment elicited by intranasal infection with 2.5x105 yeast of a 1:1 mixture of B-3502 + B-3501 (dark gray), 2.5x105 spores from a B-3502 x B-3501 cross (gray), or 1x105 H99 yeast (light gray) at 11 days post infection (4 mice per group). (B) Quantification of lung CD44+ T-cell cytokine production (interferon gamma (IFNg), interleukin-5 (IL-5), and interleukin-13 (IL-13)) following intranasal infection with Cryptococcus yeast or spores as described in (A). In all panels, the Y-axis indicates total number of host cells, error bars represent the mean ± standard error of the mean (SEM). ns = not significantly different (p > 0.1 in a Student's t-test). Circles, squares, and triangles represent individual mice. Data are derived from a single experiment.
Fig 4.
Cryptococcus disseminates to the draining lymph node very early in spore-mediated infections.
(A) Fungal burden (colony forming units per gram of tissue) in the lungs of mice (3 per group) infected intranasally with 1x106 yeast in a 1:1 mixture of JEC20 +J EC21 (dark gray) or 1x106 spores derived from a JEC20 x JEC21 cross (light gray) after 1, 5, and 7 days of infection. (B) Fungal burden (total colony forming units per organ) in the lymph nodes of mice infected intranasally with 1x106 yeast in a 1:1 mixture of JEC20 + JEC21 (dark gray) or 1x106 spores derived from a JEC20 x JEC21 cross (light gray) after 1, 5, and 7 days of infection. Data are representative of multiple biological replicates (see S6 Fig).
Fig 5.
Spore phagocytosis and lymph node dissemination are dependent on intrinsic spore properties.
(A) Phagocytosis of formaldehyde-fixed spores by RAW 264.7 macrophages at various points in germination (germination progression indicated by increasingly darker shading). Data are representative of multiple, independent (3) biological replicates. (B) Phagocytosis of spores and/or yeast expressing GFP or mCherry (incubated individually or together) by RAW 264.7 macrophages in vitro. Results are illustrated as stacked bar graphs showing the mean±SEM percent of macrophages containing mCherry-positive fungal cells (red), GFP-positive fungal cells (green) or both (black) (n = 3) after 4 hours of co-incubation. Compositions of the populations incubated with macrophages are represented below the plots (G = GFP, m = mCherry). (C) Fungal burden quantity and composition in the lungs 3 days post-infection in mice infected with 5x106 mCherry-spores, 5x106 GFP-yeast, mCherry-yeast and GFP-yeast (5x106 of each), or mCherry-spores and GFP-yeast (5x106 of each). The results are expressed in jitter plots of pie-charts with the position of each pie chart indicating the fungal burden identified in a single mouse (y-axis). The contents of each pie-graph indicate the composition of the fungal burden in that same mouse (in 50 randomly-selected isolates per mouse). A horizontal line identifies the mean fungal burden of each group, and the mean composition of the fungi of all mice per group is represented by pie-graphs along the x-axis (n = 5–8). Compositions of the initial inocula are represented below the plot (G = GFP, m = mCherry). (D) Fungal burden quantity and composition in the lymph nodes (L.N.) 3 days post-infection in mice infected with mCherry-spores, GFP-yeast, mCherry-yeast and GFP-yeast, or mCherry-spores and GFP-yeast. Data are from lymph nodes from the same mice shown in (C) and are represented in the same manner. Empty pie charts represent mice in which no fungal colonies were recovered from the lymph nodes. For the difference in the ratios of mCherry+:GFP+ colonies in lung vs. lymph node p = 1.73x10-8 (by a Student's t-test).
Fig 6.
CD11c+ cells display more associated spores than yeast at 6 hours post-infection in the mouse lung.
(A) The total number of each lung cell type (CD11c+, AMs = alveolar macrophages, DCs = dendritic cells) recovered at 6-hours post-infection in mice infected with PBS (“naïve," black bars, 2 mice), 5x106 mCherry-spores (light gray bars, 5 mice), or 5x106 mCherry yeast (dark gray bars, 5 mice). The results are expressed as bar plots illustrating the average ± SEM. For the number of DCs in spore- vs. yeast-infected mice p = 0.14. (B) Percent of each cell type (as identified in panel A) with associated mCherry+ Cryptococcus cells. The results are expressed as bar plots illustrating the average ± SEM. For the percentage of CD11c+ phagocytes associated with spores vs. yeast p = 0.019. (C) Cryptococcal cells per macrophage at 6 hours post-infection. Alveolar macrophages were recovered by bronchoalveolar lavage 6 hours post-infection in mice infected with 5x106 mCherry-spores (light gray bars, 3 mice), or 5x106 mCherry yeast (dark gray bars, 3 mice). The number of mCherry+ cryptococcal cells in 100 occupied macrophages was counted for each infected mouse. The results are expressed as the average occupancy of the three mice for each group ± SEM. For the number of spores vs. yeast per macrophage p = 0.0002 (by a Student's t-test).
Fig 7.
Dissemination of Cryptococcus spores to the lymph node is dependent on CD11c+ lung phagocytes.
(A) Schematic illustrating the effects of administering diphtheria toxin to wild type (WT) mice or mice expressing the diphtheria toxin receptor under the control of a CD11c promoter (CD11c-DTR). AMs = Alveolar Macrophages, DCs = Dendritic Cells, DTR = Diphtheria Toxin Receptor. (B) Fungal burden (total colony forming units per organ) 2 days post-infection in the lungs of mice infected with 1x107 spores derived from JEC20 x JEC21 crosses. Burdens are from wild type mice on the left and from CD11c-DTR mice on the right, each in the absence (gray bars) or presence (bars with red outline) of diphtheria toxin administration. (C) Fungal burden (total colony forming units per organ) 2 days post-infection in the lymph nodes of mice infected with 1x107 spores derived from JEC20 x JEC21 crosses. Burdens are from wild type mice on the left and from CD11c-DTR mice on the right, each in the absence (gray bars) or presence (bars with red outline) of diphtheria toxin administration.
Fig 8.
Model of cryptococcal spore-mediated disseminated disease.
Graphical representation of our model of spore-mediated lung escape and disseminated disease: (1) spores are inhaled from the environment into the lungs where they are taken up by resident lung CD11c-positive phagocytes, likely alveolar macrophages (2). Inside phagocytes, spores are able to (3) germinate and efficiently (4) traffic to the mediastinal lymph nodes (the order of events 3 and 4 is unknown). From the lymph node, (5) Cryptococcus gains access to the blood stream, which can be used to disseminate to and colonize peripheral organs, eventually resulting in fatal CNS disease.