Dziewanowska K, Carson A R, Patti J M, Deobald C F, Bayles K W, Bohach G A
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844, USA.
Infect Immun. 2000 Nov;68(11):6321-8. doi: 10.1128/IAI.68.11.6321-6328.2000.
We reported previously that internalization of Staphylococcus aureus by nonprofessional phagocytes involves an interaction between fibronectin (Fn) binding protein (FnBP) and the host cell, resulting in signal transduction, tyrosine kinase activity, and cytoskeletal rearrangement (K. Dziewanowska, J. M. Patti, C. F. Deobald, K. W. Bayles, W. R. Trumble, and G. A. Bohach, Infect. Immun. 67:4673-4678, 1999). The goal of the present study was to identify the host molecules responsible for uptake of the organism through an interaction with FnBP. First, Fn was required for internalization. Addition of small amounts of exogenous Fn stimulated the uptake of S. aureus by HEp-2 cells, which are deficient in Fn synthesis. Fn antibodies blocked internalization of the organism by MAC-T cell monolayers, a bovine epithelial cell line which expresses Fn. Second, a monoclonal antibody (MAb) specific for beta(1) integrins dramatically reduced S. aureus invasion, suggesting that the formation of a Fn bridge linking the host cell beta(1) integrin and FnBP precedes internalization. However, ligand blotting of cell membrane proteins with a functional fragment of FnBP consistently identified an additional approximately 55-kDa receptor on both human and bovine epithelial cells. This protein was purified and identified by N-terminal microsequencing as heat shock protein 60 (Hsp60). The interaction between FnBP and Hsp60 also occurred when the whole cells were used. Cell membrane localization of Hsp60 was confirmed by biotinylation with an agent nonpermeable to the cell membrane. Pretreatment of epithelial cells with a MAb specific for eukaryotic Hsp60 significantly reduced internalization of S. aureus. Combined, these results suggest that the FnBP binds directly to both Hsp60 and Fn and is linked to beta(1) integrins through a Fn bridge. The simultaneous involvement of Fn and two host cell ligands, beta(1) integrins and Hsp60, suggests that FnBP is a multifunctional adhesin that mediates internalization in a manner similar to that proposed for OpaA, the Neisseria gonorrhoeae FnBP homolog (J. P. M. van Putten, T. D. Duensing, and R. L. Cole, Mol. Microbiol. 29:369-379, 1998).
我们之前报道过，非专职吞噬细胞对金黄色葡萄球菌的内化作用涉及纤连蛋白（Fn）结合蛋白（FnBP）与宿主细胞之间的相互作用，从而导致信号转导、酪氨酸激酶活性以及细胞骨架重排（K. Dziewanowska、J. M. Patti、C. F. Deobald、K. W. Bayles、W. R. Trumble和G. A. Bohach，《感染与免疫》67:4673 - 4678，1999年）。本研究的目的是通过与FnBP的相互作用来鉴定负责摄取该病原体的宿主分子。首先，内化作用需要Fn。添加少量外源性Fn可刺激缺乏Fn合成的HEp - 2细胞摄取金黄色葡萄球菌。Fn抗体可阻断MAC - T细胞单层（一种表达Fn的牛上皮细胞系）对该病原体的内化作用。其次，一种针对β(1)整合素的单克隆抗体（MAb）显著降低了金黄色葡萄球菌的侵袭，这表明在内化作用之前，连接宿主细胞β(1)整合素和FnBP的Fn桥的形成。然而，用FnBP的功能片段对细胞膜蛋白进行配体印迹分析，始终在人和牛上皮细胞上鉴定出另一种约55 kDa的受体。该蛋白经纯化并通过N端微测序鉴定为热休克蛋白60（Hsp60）。当使用完整细胞时，FnBP与Hsp60之间也会发生相互作用。通过用一种不能透过细胞膜的试剂进行生物素化，证实了Hsp60在细胞膜上的定位。用一种针对真核Hsp60的MAb预处理上皮细胞，可显著降低金黄色葡萄球菌的内化作用。综合这些结果表明，FnBP直接与Hsp60和Fn结合，并通过Fn桥与β(1)整合素相连。Fn以及两种宿主细胞配体β(1)整合素和Hsp6０的同时参与表明，FnBP是一种多功能黏附素，其介导内化作用的方式类似于淋病奈瑟菌FnBP同源物OpaA所提出的方式（J. P. M. van Putten、T. D. Duensing和R. L. Cole，《分子微生物学》29:３69 - 379，1998年）。
