Institute of Hygiene and Microbiology, University of Würzburg, Würzburg, Germany.
PLoS One. 2012;7(6):e39613. doi: 10.1371/journal.pone.0039613. Epub 2012 Jun 29.
Entry of Neisseria meningitidis (the meningococcus) into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK), which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK) blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells.
脑膜炎奈瑟菌（脑膜炎球菌）进入人脑微血管内皮细胞（HBMEC）是通过纤维连接蛋白或玻连蛋白与表面蛋白 Opc 结合形成桥接至相应整合素来介导的。这种相互作用导致细胞骨架重排和脑膜炎奈瑟菌的摄取。在这项研究中，我们确定了与整合素直接相关的粘着斑激酶（FAK）参与了 HBMEC 中脑膜炎奈瑟菌的整合素介导的内化。通过特异性 FAK 抑制剂 PF 573882 抑制 FAK 活性可使 Opc 介导的 HBMEC 侵袭减少 90%以上。此外，表达激酶活性受损或不能自动磷酸化或过表达 FAK （FRNK）的显性负性形式的 FAK 突变体可阻断整合素介导的脑膜炎奈瑟菌内化。重要的是，FAK 缺陷型成纤维细胞被脑膜炎奈瑟菌侵袭的程度明显降低。此外，脑膜炎奈瑟菌诱导了包括 FAK/Src 复合物底物 cortactin 在内的几种宿主蛋白的酪氨酸磷酸化。通过 siRNA 沉默抑制 cortactin 表达和突变 cortactin 内的关键氨基酸残基（包括 Arp2/3 结合和 dynamin 结合），显著降低了脑膜炎奈瑟菌进入真核细胞的侵袭能力，表明这两个结构域对于脑膜炎奈瑟菌进入真核细胞的有效摄取都是至关重要的。总之，这些结果表明，脑膜炎奈瑟菌利用整合素信号通路进入宿主细胞，FAK 介导激活整合素向细胞骨架传递信号。然后，FAK、Src 和 cortactin 之间的协同作用使脑膜炎奈瑟菌能够进入宿主细胞进行内吞作用。
