Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Thu, 01 Aug 2019 19:34:51 Journal of Medical Microbiology (2003), 52, 961–970 DOI 10.1099/jmm.0.05230-0 05230 & 2003 SGM Printed in Great Britain 961 Correspondence Ambrose Y. Jong ajong@chla.usc.edu Received 20 February 2003 Accepted 3 July 2003 Cryptococcus neoformans induces alterations in the cytoskeleton of human brain microvascular endothelial cells Steven H. M. Chen, 1 Monique F. Stins, 2 Sheng-He Huang, 3 Yu Hua Chen, 3 K. J. Kwon-Chung, 4 Yun Chang, 4 Kwang Sik Kim, 2 Kazuhiro Suzuki 5 and Ambrose Y. Jong 1 1,3 Divisions of Hematology–Oncology 1 and Infectious Diseases 3 , Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA 2 The Johns Hopkins University, Pediatric Infectious Diseases, 600 N. Wolfe St, Park 256, Baltimore, MD 21287, USA 4 Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA 5 National Institute of Health Sciences, Tokyo, Japan 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. INTRODUCTION Infection by Cryptococcus neoformans, an encapsulated fun- gal organism, has increased considerably over recent years (Mitchell & Perfect, 1995; Gottfredsson & Perfect, 2000; Kwon-Chung et al., 2000). The expanded use of immuno- suppressive drugs and the onset of AIDS have contributed to this epidemic. Patients with T-cell deficiencies, including AIDS, lymphoma, corticosteroid therapy and idiopathic CD4 lymphocytopenia, are the most susceptible to C. neofor- mans infection. Dehydrated haploid yeast or basidiospores of C. neoformans enter the host via the respiratory system. They are then likely to spread haematogeneously to extrapulmon- ary tissues, including the brain, where meningoencephalitis develops. Untreated cryptococcal meningoencephalitis is fatal in normal hosts. Even when treated with the most Abbreviations: BBB, blood–brain barrier; CNS, central nervous system; HBMEC, human brain microvascular endothelial cell; HUVEC, human umbilical vein endothelial cell; LIMK, LIM kinase; ROCK, Rho kinase; TEER, trans-endothelial electrical resistance.