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Published on: July 14, 2020
Cryptococcus neoformans induces alterations in the cytoskeleton of human brain microvascular endothelial cells
Steven H M Chen1, Monique F Stins1, Sheng-He Huang1
1Divisions of Hematology-Oncology1 and Infectious Diseases3, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA 2The Johns Hopkins University, Pediatric Infectious Diseases, 600 N. Wolfe St, Park 256, Baltimore, MD 21287, USA 4Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA 5National Institute of Health Sciences, Tokyo, Japan.
Abstract:
The fungal pathogen Cryptococcus neoformans has a predilection for the central nervous system (CNS), resulting in devastating meningoencephalitis. At present, it is unclear how C. neoformans traverses the blood-brain barrier (BBB) and causes CNS infection. The present study has examined and characterized the interaction of C. neoformans with human brain microvascular endothelial cells (HBMEC), which constitute the BBB. Adhesion of and transcytosis of HBMEC by C. neoformans was inoculum- and time-dependent and occurred with both encapsulated and acapsulated strains. C. neoformans induced marked morphological changes in HBMEC, for example membrane ruffling, irregular nuclear morphology and swelling of the mitochondria and the ER. These findings suggest that C. neoformans induced actin cytoskeletal reorganization of the host cells. In addition, it was observed that the dephosphorylated form of cofilin was increased during cryptococcal adherence to HBMEC, concomitant with the actin rearrangement. Cryptococcal binding to HBMEC was increased in the presence of Y27632, a Rho kinase (ROCK)-specific inhibitor. Since ROCK activates LIM kinase (LIMK), which phosphorylates cofilin (inactive form), this suggests the involvement of the ROCK-->LIMK-->cofilin pathway. In contrast, the phosphatase inhibitor sodium orthovanadate decreased adherence of Cryptococcus to HBMEC, concomitant with the increase of phosphorylation of cofilin. Furthermore, the tight junction marker protein occludin became Triton-extractable, indicating alteration of tight junctions in brain endothelial cells. This is the first demonstration that C. neoformans is able to adhere to and transcytose across the HBMEC monolayer and alter the cytoskeleton morphology in HBMEC. Further characterization of the interactions between C. neoformans and HBMEC should help the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.
Insights
Cryptococcus neoformans invades the central nervous system by crossing the blood-brain barrier. This study reveals how the fungus interacts with brain endothelial cells, altering their structure and facilitating entry.
Area of Science:
- Mycology
- Infectious Diseases
- Neuroscience
- Cell Biology
Background:
- Cryptococcus neoformans is a fungal pathogen causing meningoencephalitis.
- The mechanism of C. neoformans crossing the blood-brain barrier (BBB) into the central nervous system (CNS) remains unclear.
- Human brain microvascular endothelial cells (HBMEC) form the BBB.
Purpose of the Study:
- To characterize the interaction between C. neoformans and HBMEC.
- To elucidate the cellular mechanisms underlying C. neoformans' ability to breach the BBB.
Main Methods:
- In vitro study using HBMEC and C. neoformans.
- Assessment of fungal adhesion and transcytosis across HBMEC monolayers.
- Microscopic analysis of HBMEC morphology and cytoskeletal changes.
- Investigation of the Rho kinase (ROCK) signaling pathway and cofilin phosphorylation.
Main Results:
- C. neoformans adheres to and transcytoses across HBMEC in a dose- and time-dependent manner.
- Fungal interaction induces significant HBMEC morphological changes, including actin cytoskeletal reorganization.
- The ROCK-LIMK-cofilin pathway is implicated in cryptococcal adherence, with altered cofilin phosphorylation observed.
- Tight junctions in HBMEC are disrupted, indicated by Triton-extractable occludin.
Conclusions:
- C. neoformans actively interacts with HBMEC, adhering, transcytosing, and disrupting endothelial cell structure.
- The study identifies the ROCK-LIMK-cofilin pathway and tight junction alterations as key mechanisms in BBB penetration.
- Understanding these interactions may lead to novel strategies for preventing cryptococcal meningitis.
