title: Cryptococcus neoformans induces alterations in the cytoskeleton of human brain microvascular endothelial cells.
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.
authors.affiliation: Divisions 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.
authors.forename: Steven H M
authors.initials: SHM
authors.lastname: Chen
authors.affiliation: Divisions 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.
authors.forename: Monique F
authors.initials: MF
authors.lastname: Stins
authors.affiliation: Divisions 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.
authors.forename: Sheng-He
authors.initials: SH
authors.lastname: Huang
authors.affiliation: Divisions 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.
authors.forename: Yu Hua
authors.initials: YH
authors.lastname: Chen
authors.affiliation: Divisions 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.
authors.forename: K J
authors.initials: KJ
authors.lastname: Kwon-Chung
authors.affiliation: Divisions 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.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chang
authors.affiliation: Divisions 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.
authors.forename: Kwang Sik
authors.initials: KS
authors.lastname: Kim
authors.affiliation: Divisions 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.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Suzuki
authors.affiliation: Divisions 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.
authors.forename: Ambrose Y
authors.initials: AY
authors.lastname: Jong
mesh_terms: D051339:Actin Depolymerizing Factors; D001422:Bacterial Adhesion; D001921:Brain / Q000098:blood supply*; D002478:Cells, Cultured; D003455:Cryptococcus neoformans / Q000472:pathogenicity*; D003599:Cytoskeleton / Q000648:ultrastructure*; D042783:Endothelial Cells / Q000648:ultrastructure*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D008833:Microcirculation / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D062793:Occludin; D010766:Phosphorylation; D012995:Solubility
chemical_list: D051339:Actin Depolymerizing Factors; D008565:Membrane Proteins; D008840:Microfilament Proteins; C568985:OCLN protein, human; D062793:Occludin