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

Jong-Chul Kim,Benjamin Crary,Kee J. Kim,Yun C. Chang,Kyung J. Kwon-Chung

doi:10.1074/jbc.m112.389676

Abstract

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.

Highlights

Cryptococcus neoformans traverses across the blood-brain barrier (BBB) to penetrate into the brain
Our findings demonstrate that C. neoformans activates RhoGTPases and subsequently focal adhesion kinase (FAK), ezrin, and PKC␣ to promote their traversal across the human brain microvascular endothelial cells (HBMEC) monolayer, which is the critical step for cryptococcal brain infection and development of meningitis
Our results demonstrate that C. neoformans induces activation of RhoGTPases followed by phosphorylation of FAK, PKC␣, and ezrin of HBMEC, all of which lead to fungal transmigration across the BBB

Summary

Background

Cryptococcus neoformans traverses across the BBB to penetrate into the brain. Results: C. neoformans activates RhoGTPases and subsequently PKC␣, focal adhesion kinase, and ezrin in HBMEC. CD44, the hyaluronic acid receptor, in lipid rafts has been identified as a host receptor, and its binding to Cryptococcus is involved in the activation of protein kinase C␣ (PKC␣), which is required for fungal invasion and transmigration [13,14,15] These findings strongly indicate the role of actin cytoskeleton reorganization during Cryptococcus-HBMEC interaction. Our results demonstrate that C. neoformans induces activation of RhoGTPases followed by phosphorylation of FAK, PKC␣, and ezrin of HBMEC, all of which lead to fungal transmigration across the BBB This is the first report demonstrating the role of host RhoGTPases and other signaling proteins related to actin cytoskeleton rearrangements in the traversal of C. neoformans across the BBB, which is the critical step in disease development

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION