Cryptococcus neoformans induces alterations in the cytoskeleton of human brain microvascular endothelial cells.
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.
Chen
Steven H M
SHM
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.
Stins
Monique F
MF
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.
Huang
Sheng-He
SH
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.
Chen
Yu Hua
YH
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.
Kwon-Chung
K J
KJ
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.
Chang
Yun
Y
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.
Kim
Kwang Sik
KS
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.
Suzuki
Kazuhiro
K
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.
Jong
Ambrose Y
AY
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.
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
D051339:Actin Depolymerizing Factors; D008565:Membrane Proteins; D008840:Microfilament Proteins; C568985:OCLN protein, human; D062793:Occludin