Cryptococcus neoformans induces alterations in the cytoskeleton of human brain microvascular endothelial cells
Cryptococcus neoformans induces alterations in the cytoskeleton of human brain microvascular endothelial cells
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DOI：
10.1099/jmm.0.05230-0
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发表时间：
2003-11-01
影响因子：
3
通讯作者：
Jong, AY
中科院分区：
文献类型：
--
作者：
Chen, SHM;Stins, MF;Jong, AY
真菌病原体新型隐球菌偏爱中枢神经系统（CNS），导致破坏性脑膜脑炎。目前，尚不清楚新生C.如何穿过血脑屏障（BBB）并引起中枢神经系统感染。本研究研究了新生弓形虫与构成血脑屏障的人脑微血管内皮细胞（HBMEC）的相互作用。新生C.菌对HBMEC的粘附和胞吞作用是接种量和时间依赖性的，并且在包被和包被菌株中都发生。新生弓形虫引起HBMEC明显的形态学改变，如膜褶皱、核形态不规则、线粒体和内质网肿胀。这些发现表明，新生C.诱导了宿主细胞的肌动蛋白细胞骨架重组。此外，观察到在隐球菌粘附HBMEC时，伴随着肌动蛋白重排，cofilin的去磷酸化形式增加。在Rho激酶（ROCK）特异性抑制剂Y27632的存在下，隐球菌与HBMEC的结合增加。由于ROCK激活LIM激酶（LIMK），使cofilin磷酸化（无活性形式），这表明ROCK- > LIMK- >cofilin通路参与其中。相比之下，磷酸酶抑制剂原钒酸钠降低隐球菌对HBMEC的粘附，同时增加cofilin的磷酸化。此外，紧密连接标记蛋白occludin成为triton可提取的，表明脑内皮细胞紧密连接的改变。这是首次证明C. neoformans能够粘附并跨HBMEC单层转糖，并改变HBMEC的细胞骨架形态。进一步表征新生隐球菌与HBMEC之间的相互作用，将有助于制定预防隐球菌脑膜炎及其相关发病率的新策略。
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.