Real-time imaging of trapping and urease-dependent transmigration of Cryptococcus neoformans in mouse brain
Meiqing Shi, … , Paul Kubes, Christopher H. Mody
Meiqing Shi, … , Paul Kubes, Christopher H. Mody
Published April 26, 2010
Citation Information: J Clin Invest. 2010;120(5):1683-1693. https://doi.org/10.1172/JCI41963.
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Research Article

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.

Authors

Meiqing Shi, Shu Shun Li, Chunfu Zheng, Gareth J. Jones, Kwang Sik Kim, Hong Zhou, Paul Kubes, Christopher H. Mody

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Figure 1

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

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C. neoformans does not roll and adhere to endothelial cells under flow ...
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).