Real-time imaging of trapping and urease-dependent transmigration of Cryptococcus neoformans in mouse brain

Abstract

Infectious meningitis and encephalitis is caused by invasion of circulating pathogens into the brain. It is unknown how the circulating pathogens dynamically interact with brain endothelium under shear stress, leading to invasion into the brain. Here, using intravital microscopy, we have shown that Cryptococcus neoformans, a yeast pathogen that causes meningoencephalitis, stops suddenly in mouse brain capillaries of a similar or smaller diameter than the organism, in the same manner and with the same kinetics as polystyrene microspheres, without rolling and tethering to the endothelial surface. Trapping of the yeast pathogen in the mouse brain was not affected by viability or known virulence factors. After stopping in the brain, C. neoformans was seen to cross the capillary wall in real time. In contrast to trapping, viability, but not replication, was essential for the organism to cross the brain microvasculature. Using a knockout strain of C. neoformans, we demonstrated that transmigration into the mouse brain is urease dependent. To determine whether this could be amenable to therapy, we used the urease inhibitor flurofamide. Flurofamide ameliorated infection of the mouse brain by reducing transmigration into the brain. Together, these results suggest that C. neoformans is mechanically trapped in the brain capillary, which may not be amenable to pharmacotherapy, but actively transmigrates to the brain parenchyma with contributions from urease, suggesting that a therapeutic strategy aimed at inhibiting this enzyme could help prevent meningitis and encephalitis caused by C. neoformans infection.

Figure 1. C. neoformans does not roll and adhere to endothelial cells under flow conditions in vitro or to postcapillary venules in vivo.

Monolayers of HUVECs on glass coverslips were treated with 10 ng/ml LPS. Leukocytes or C. neoformans (10⁶/ml) were perfused over the monolayers of the endothelial cells at 2 dyn/cm². (A–C) A series of images taken by phase contrast microscopy showing the rolling leukocytes (white arrows) and stationary leukocytes (black arrows). (D–F) A series of images taken by phase contrast microscopy, with the imaging optimized to show the movement of the yeast cells (white arrows). Time in seconds is shown. Mice were anesthetized, and the brain microcirculation was visualized by IVM. C. neoformans (100 × 10⁶ strain H99) labeled with FITC were suspended in 100 μl saline and injected via the tail vein. (G) An image of a postcapillary venule (white arrow) before injection of C. neoformans. (H–J) A series of images of the same capillary taken 5 minutes after injection; time in minutes and seconds. Black arrows indicate the same moving yeast cell. Scale bars: 20 μm. (K) Numbers of C. neoformans passing a fixed point in a postcapillary venule (with a diameter between 30 and 70 μm) at various times after injection. Data are expressed as mean ± SEM of 2 independent experiments (n = 8 mice).

Figure 2. C. neoformans stops suddenly in the brain capillaries.

C. neoformans (strain H99) labeled with FITC was injected via the tail vein. (A) An image of the brain capillary bed before injection of C. neoformans. (B and C) Images taken by IVM showing the same field of view 5 minutes after injection. Time in minutes and seconds is shown. (D) An image was taken by IVM showing the same field of view 60 minutes after injection. Arrows indicate the same yeast immediately following arrest and when stationary. (E) The number of stationary C. neoformans in a field of view at various time points after injection. Data are expressed as mean ± SEM of 2 independent experiments (n = 8 mice). (F) Tie-2 GFP mice (which allowed visualization of the capillaries in vivo) were anesthetized, and spinning disk confocal microscopy of the brain microcirculation was performed. C. neoformans (100 × 10⁶ strain H99) labeled with TRITC (arrowhead) in 100 μl saline was injected via tail vein. (G) Mice were injected with 20 × 10⁶ C. neoformans (strain H99) via the tail vein. Sixty minutes later, the mice were anesthetized, and vascular perfusion was performed. The brain was removed, and immunohistochemistry was performed using anticryptococcal antibody (red, arrows) and anti–collagen IV (green). The images in H–K are 3D reconstructive images of the image in G. Scale bars: 20 μm.

Figure 3. Polystyrene microspheres stop in the brain with the same kinetics as C. neoformans.

Fluorescent polystyrene microspheres that are similar in size to C. neoformans (strain H99) in 100 μl saline were injected via tail vein. (A) An image of the brain capillary bed before injection of microspheres. (B and C) A series of images taken by IVM showing the same field of view 8 minutes after injection. The arrow indicates a suddenly arrested microsphere. (D) An image was taken by IVM showing the same field of view 60 minutes after injection. The arrow indicates the same microsphere after suddenly trapping shown in C. Time in minutes and seconds is shown. (E) Numbers of stationary C. neoformans in the capillary bed 60 minutes after injection of microspheres or C. neoformans (H99). Data are expressed as mean ± SEM of 2 independent experiments (n = 8). (F) C57BL/6 mice were injected with 20 × 10⁶ microspheres (red, arrow) via the tail vein. Sixty minutes later, the mice were anesthetized and perfusion was performed. The brain was removed for frozen sections as described in Methods. Immunohistochemistry was performed to label vessels (green). Scale bars: 20 μm.

Figure 4. Real-time visualization of C. neoformans transmigration in the capillary of a living host.

Mice were injected with 20 × 10⁶ C. neoformans (strain H99) via the tail vein. Six hours later, the mice were anesthetized and perfused. The brain was removed, and immunohistochemistry was performed to label C. neoformans (red) and vessels (green). (A) C. neoformans was found partially within (arrow) or outside (asterisk) the vessels. (B and C) Relationship between C. neoformans (arrow) and the vessel. (C) 3D reconstructive image of B. (D–F) C. neoformans (arrowhead) crossing the capillary wall in a living host. Tie-2 GFP BALB/c mice were injected with 100 × 10⁶ H99 via the tail vein. The mice were anesthetized, and spinning disk confocal microscopy of the brain microcirculation was performed as described in Methods. Time-lapse video was taken 3 hours after injection of C. neoformans. A series of single z-stack images is shown. (D) At the start of image acquisition, C. neoformans was present within the capillaries (arrowhead). C. neoformans (arrowheads) moving out from the capillaries across the vessel wall at 50 minutes (E) and 1 hour and 55 minutes (F). Squares represent areas enlarged in insets. Scale bars: 20 μm.

Figure 5. Viability is required for C. neoformans transmigration in the brain.

Mice were injected with 20 × 10⁶ FITC-labeled live strain H99 (A, vessel appears red; C. neoformans appears green [arrow]), heat-killed strain H99 (B, vessel, red; C. neoformans, green [arrow]), fluorescence-labeled polystyrene microspheres (C; vessel, green; microspheres, red [arrows]). Twenty-four hours later, the mice were anesthetized, and perfusion was performed. The brain was removed, and immunohistochemistry was performed. Scale bars: 20 μm. (D) The percentage of the yeasts and polystyrene microspheres located in parenchyma of the brain. Data are presented as mean ± SEM (n = 5). Data are representative of 2 independent experiments. ND, none detected.

Figure 6. Transmigration of temperature-sensitive strains of C. neoformans from capillaries to the brain.

Mice were injected with 10 × 10⁶ FITC-labeled C. neoformans strains ras1 mutant, cdc24 mutant, reconstituted ras1 + RAS1, or reconstituted cdc24 + CDC24 via the tail vein. Twenty-four hours later, the mice were anesthetized, perfusion was performed, and immunohistochemistry was performed. (A) Injection of ras1 + RAS1 reconstituted strain. (B) Injection of ras1 mutant strain. (C) The percentage of the yeasts in the parenchyma. Data are expressed as mean ± SEM of 2 independent experiments (n = 6). (D) Injection of cdc24 + CDC24 reconstituted strain. (E) Injection of cdc24 mutant strain C. neoformans. (F) The percentage of the yeasts in the parenchyma. Data are expressed as mean ± SEM of 2 independent experiments (n = 6). C. neoformans (arrows), green; vessels, red. Scale bars: 20 μm.

Figure 7. Urease contributes to the transmigration of C. neoformans in brain.

(A) Survival of mice injected i.v. with 20 × 10⁶ C. neoformans H99, ure1, or ure1+URE1-1. (B) Brain CFU of mice 3 and 72 hours after i.v. injection of 20 × 10⁶ of C. neoformans H99, ure1, or ure1+URE1-1. (C) An image taken by spinning disk confocal microscopy 2 hours after i.v. injection of a mixture of equal numbers (50 × 10⁶) of FITC-labeled ure1 and TRITC-labeled ure1+URE1-1 showing equal distribution of the two yeasts in the capillary bed. (D) The number of yeasts at each transmigration site, 72 hours after i.v. injection of 20 × 10⁶ C. neoformans H99, ure1, or ure1+URE1-1. (E) An immunofluorescence image from the brain 24 hours after injection of ure1 showing yeasts (green) located inside vessels (red). (F) An immunofluorescence image of the brain taken 24 hours after injection of ure1+URE1-1 showing yeasts (green) localized in the parenchyma of the brain as well as inside the vessels (red). (G) Percentage of colonies within the parenchyma of the brain (transmigrated) as a percentage of the total colonies, 24 and 72 hours after i.v. injection of 20 × 10⁶ C. neoformans H99, ure1, or ure1+URE1-1. Data are presented as mean ± SEM (n = 4). Data are representative of 2 or 3 independent experiments. *P < 0.05, **P < 0.01. Scale bars: 20 μm.

Figure 8. Flurofamide ameliorates brain infection.

(A) Survival of the mice treated with flurofamide and receiving 20 × 10⁶ flurofamide-treated C. neoformans strain H99 compared with DMSO treatment, which served as a control. (B) Brain CFU of mice treated with flurofamide and receiving 20 × 10⁶ flurofamide-treated C. neoformans strain H99 compared with DMSO treatment. (C) Percentage of colonies within the parenchyma of the brain as a percentage of the total colonies at 72 hours in mice treated with flurofamide and receiving 20 × 10⁶ flurofamide-treated C. neoformans strain H99. Data are presented as mean ± SEM (n = 4). Data are representative of 2 or 3 independent experiments. *P < 0.05.