Fowler Trent, Johansson Staffan, Wary Kishore K, Höök Magnus
Department of Biochemistry and Biophysics, Institute of Biosciences and Technology, Texas A and M University System Health Science Center, Houston, TX, USA.
Cell Microbiol. 2003 Jun;5(6):417-26. doi: 10.1046/j.1462-5822.2003.00290.x.
Traditionally recognized as an extracellular pathogen, the Gram-positive bacterium Staphylococcus aureus can also be internalized by a variety of cell types in vitro. Internalization is known to involve binding of the host extracellular protein fibronectin to the bacterium, recognition of the fibronectin-coated bacterium by the fibronectin-binding integrin alpha5beta1 on the host cell surface, and integrin-mediated internalization. Here we examine elements of mammalian cell signalling pathways involved in S. aureus internalization. The mouse fibroblast cell line GD25, in which the gene encoding the beta1 integrin subunit is inactivated, has been complemented with a beta1 integrin cDNA encoding a tyrosine (Y) to phenylalanine (F) mutation in each of the two beta1 integrin intracellular NPXY motifs. This cell line, GD25beta1 A Y783/795F, is defective in migration on fibronectin coated surfaces and intracellular signalling activities involving the tyrosine kinase Src. GD25beta1 A Y783/795F cells have a decreased ability to internalize S. aureus compared to GD25beta1 A cells expressing wild-type beta1 integrins. Furthermore, using mouse embryo fibroblasts in which different members of the Src family kinases are genetically inactivated, we demonstrate that optimal internalization is dependent on expression of Src kinase. Interferon, which has been implicated in repression of the effects of the viral homologue of Src inhibits internalization of S. aureus indicating that internalization may be blocked by inhibitors of Src kinase function. We then demonstrate that Src family kinase specific inhibitors effectively block S. aureus internalization into HeLa cells leading to the conclusion that a function unique to Src is required for optimal internalization of S. aureus in vitro.
革兰氏阳性菌金黄色葡萄球菌传统上被认为是一种胞外病原体，但在体外它也能被多种细胞类型内化。已知内化过程涉及宿主细胞外蛋白纤连蛋白与细菌的结合、宿主细胞表面纤连蛋白结合整合素α5β1对纤连蛋白包被细菌的识别以及整合素介导的内化。在此，我们研究了参与金黄色葡萄球菌内化的哺乳动物细胞信号通路的相关成分。小鼠成纤维细胞系GD25中编码β1整合素亚基的基因已失活，该细胞系已用一种β1整合素cDNA进行了补充，该cDNA在两个β1整合素细胞内NPXY基序中的每一个中编码酪氨酸（Y）到苯丙氨酸（F）的突变。这个细胞系，GD25β1 A Y783/795F，在纤连蛋白包被表面上的迁移以及涉及酪氨酸激酶Src的细胞内信号传导活动方面存在缺陷。与表达野生型β1整合素的GD25β1 A细胞相比，GD25β1 A Y783/795F细胞内化金黄色葡萄球菌的能力有所下降。此外，利用Src家族激酶的不同成员在基因上失活的小鼠胚胎成纤维细胞，我们证明最佳内化依赖于Src激酶的表达。干扰素与Src病毒同源物作用的抑制有关，它抑制金黄色葡萄球菌的内化，这表明内化可能被Src激酶功能抑制剂阻断。然后我们证明Src家族激酶特异性抑制剂能有效阻断金黄色葡萄球菌内化到HeLa细胞中，从而得出结论：Src的独特功能是体外金黄色葡萄球菌最佳内化所必需的。
