The dipeptide repeat region of the fibrinogen-binding protein (clumping factor) is required for functional expression of the fibrinogen-binding domain on the Staphylococcus aureus cell surface
The dipeptide repeat region of the fibrinogen-binding protein (clumping factor) is required for functional expression of the fibrinogen-binding domain on the Staphylococcus aureus cell surface
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DOI：
10.1046/j.1365-2958.1997.5291896.x
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发表时间：
1997-09-01
影响因子：
3.6
通讯作者：
Foster, TJ
中科院分区：
文献类型：
--
作者：
Hartford, O;Francois, P;Foster, TJ
金黄色葡萄球菌聚集因子是一种位于细菌细胞表面的纤维蛋白原结合蛋白。该蛋白有一个不寻常的重复结构域(区域R)，主要由二肽、天冬氨酸和丝氨酸组成。为了确定区域R是否在纤维蛋白原结合区A结构域的表面显示中起作用，产生了缺少区域R编码区的clfA基因。为了确定野生型ClfA表达所需的最小R区长度，构建了截短R区结构域的变体。对表达突变clfA基因的金黄色葡萄球菌细胞进行了检测：(1)溶葡萄球菌酶处理后释放的蛋白与A区特异性抗血清反应；(2)聚集在可溶性纤维蛋白原中；(3)结合固定化纤维蛋白原；(4)荧光激活细胞分选分析检测细胞表面ClfA抗原的表达。每个构建物都表达三种主要的免疫反应蛋白，其中两种可能是N-末端降解产物。A区和W区(A区和LPDTG分选信号之间的72个残基)之间的R区残基需要大于40个残基，才能在纤维蛋白原中形成野生型水平的聚集。当A区与LPDTG之间的距离从72个残基减少到4个残基时，凝集滴度逐渐下降。类似地，在表达40个或更少R区的ClfA的细胞中，观察到抗ClfA血清的结合和与纤维蛋白原涂层塑料表面的结合减少。然而，在完全缺乏R区的ClfA衍生物中，与纤维蛋白原的粘附率和与抗ClfA血清的结合水平较低。这表明，一小部分ClfA分子连接到距离细胞表面非常近的肽链上，但需要超过72个残基才能使足够的ClfA分子跨越整个细胞壁，并以一种能够完全参与纤维蛋白原结合的形式展示具有生物活性的A结构域。
Clumping factor of Staphylococcus aureus is a fibrinogen-binding protein that is located on the bacterial cell surface. The protein has an unusual repeat domain (region R) comprising mainly the dipeptide aspartate and serine. To determine if region R has a role in the surface display of the fibrinogen-binding region A domain, deletions lacking the region R encoding region of the clfA gene were generated. To determine the minimum length of region R required for wild-type levels of ClfA expression, variants with truncated region R domains were constructed. S. aureus cells expressing mutated clfA genes were tested for (i) proteins released by lysostaphin treatment that reacted with antisera specific for region A, (ii) clumping in soluble fibrinogen, (iii) adherence to immobilized fibrinogen and (iv) expression of the ClfA antigen on the cell surface by fluorescent activated cell sorting analysis. Each construct expressed three major immunoreactive proteins, two of which were putative N-terminal degradation products. Region R residues greater than 40 were required between region A and W (72 residues between region A and the LPDTG sorting signal) for wild-type levels of clumping in fibrinogen. A stepwise decrease in clumping titre was observed as the distance between region A and LPDTG was decreased from 72 to 4 residues. Similarly, a decrease in binding of anti-ClfA serum and in binding to fibrinogen-coated plastic surfaces was observed with cells expressing ClfA with 40 region R residues or less. Nevertheless, low levels of adherence to fibrinogen and binding to anti-ClfA serum occurred with ClfA derivatives that lacked region R altogether. This indicates that a small proportion of the ClfA molecules are linked to peptidoglycan very close to the cell surface but that residues greater than 72 are needed to allow sufficient ClfA molecules to span the entire cell wall and to display the biologically active A domain in a form that can participate fully in fibrinogen binding.