Cryptococcus neoformans Activates RhoGTPase Proteins Followed by Protein Kinase C, Focal Adhesion Kinase, and Ezrin to Promote Traversal across the Blood-Brain Barrier

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However,...

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Bibliographic Details
Published in:The Journal of biological chemistry 2012-10, Vol.287 (43), p.36147-36157
Main Authors: Kim, Jong-Chul, Crary, Benjamin, Chang, Yun C., Kwon-Chung, Kyung J., Kim, Kee J.
Format: Article
Language:English
Subjects:
Blood-Brain Barrier
Blood-Brain Barrier - metabolism
Blood-Brain Barrier - microbiology
Blood-Brain Barrier - pathology
Brain
Cells, Cultured
Cryptococcosis - metabolism
Cryptococcosis - pathology
Cryptococcus Neoformans
Cytoskeletal Proteins - metabolism
Endothelial Cell
Endothelial Cells - metabolism
Endothelial Cells - pathology
Enzyme Activation
Focal Adhesion Kinase 1 - metabolism
Fungi
Humans
Meningoencephalitis - metabolism
Meningoencephalitis - pathology
Microbial Pathogenesis
Microbiology
Phosphorylation
Protein Kinase C-alpha - metabolism
Protein Phosphorylation
rho GTP-Binding Proteins - metabolism
Rho GTPases
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Summary:Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis. Background:Cryptococcus neoformans traverses across the BBB to penetrate into the brain. Results:C. neoformans activates RhoGTPases and subsequently PKCα, focal adhesion kinase, and ezrin in HBMEC. Conclusion: Activation of host signaling proteins promotes Cryptococcus crossing the BBB. Significance: Elucidation of the host signaling events that contributes to cryptococcal BBB traversal is important for a better understanding of its pathogenesis.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M112.389676