Hartford Orla, McDevitt Damien, Foster Timothy J
Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland1.
Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, USA2.
Microbiology (Reading). 1999 Sep;145 ( Pt 9):2497-2505. doi: 10.1099/00221287-145-9-2497.
The fibrinogen-binding protein ClfA and the collagen-binding protein Cna are surface-associated adhesins of Staphylococcus aureus. ClfA has a dipeptide repeat region R composed mainly of serine and aspartate residues, more than 40 of which are required along with the 28-residue region W, the LPXTG motif and region M to display the ligand-binding region A on the cell surface in a functional form. Cna has a 61-residue region W and at least one 187-residue region B linking the collagen-binding region A to peptidoglycan. A cna mutant of S. aureus lacking region B was shown to bind collagen at the same level as wild-type Cna+ cells, indicating that region B is not necessary for ligand binding. Furthermore, altering the number of B repeats did not influence the level of collagen binding. In order to study the ability of C-terminal domains of Cna and ClfA to support functional ligand-binding activity of different adhesins, chimeric proteins were constructed and expressed in S. aureus. Surprisingly, the presence of a single Cna B domain and a nonapeptide linker located between ClfA region A and Cna region WM failed to support fibrinogen binding by S. aureus cells, despite the fact that ClfA region A was detected on the bacterial surface by immunoblotting. In contrast, the ClfA region A-Cna region B hybrid expressed as a recombinant protein in Escherichia coli did bind fibrinogen in Western ligand blots and in an ELISA-type assay. It is concluded that Cna region B cannot support functional display of ClfA region A on the bacterial cell surface. However, the ClfA dipeptide repeat region R and region WM did promote functional surface expression of the Cna collagen-binding domain in a hybrid Cna-ClfA protein.
纤维蛋白原结合蛋白ClfA和胶原蛋白结合蛋白Cna是金黄色葡萄球菌的表面相关黏附素。ClfA有一个主要由丝氨酸和天冬氨酸残基组成的二肽重复区域R，其中40多个这样的残基与28个残基的区域W、LPXTG基序和区域M一起，才能使配体结合区域A以功能形式展示在细胞表面。Cna有一个61个残基的区域W和至少一个将胶原蛋白结合区域A与肽聚糖相连的187个残基的区域B。一个缺乏区域B的金黄色葡萄球菌cna突变体被证明与野生型Cna+细胞结合胶原蛋白的水平相同，这表明区域B对于配体结合不是必需的。此外，改变B重复序列的数量并不影响胶原蛋白结合水平。为了研究Cna和ClfA的C末端结构域支持不同黏附素功能配体结合活性的能力，构建了嵌合蛋白并在金黄色葡萄球菌中表达。令人惊讶的是，尽管通过免疫印迹在细菌表面检测到了ClfA区域A，但位于ClfA区域A和Cna区域WM之间的单个Cna B结构域和一个九肽接头的存在并不能支持金黄色葡萄球菌细胞结合纤维蛋白原。相反，在大肠杆菌中作为重组蛋白表达的ClfA区域A - Cna区域B杂合体在Western配体印迹和ELISA型试验中确实能结合纤维蛋白原。得出的结论是，Cna区域B不能支持ClfA区域A在细菌细胞表面的功能展示。然而，ClfA二肽重复区域R和区域WM确实促进了杂合Cna - ClfA蛋白中Cna胶原蛋白结合结构域在表面的功能表达。
