Kingsley Robert A, Keestra A Marijke, de Zoete Marcel R, Bäumler Andreas J
Department of Medical Microbiology and Immunology, College of Medicine, Texas A and M University System Health Science Center, 407 Reynolds Medical Building, College Station, TX77843-1114, USA.
Mol Microbiol. 2004 Apr;52(2):345-55. doi: 10.1111/j.1365-2958.2004.03995.x.
Introduction of Salmonella enterica serotype Typhimurium into food products results from its ability to persist in the intestine of healthy livestock by mechanisms that are poorly understood. The non-fimbrial adhesin ShdA is a fibronectin binding protein required for persistent intestinal carriage of S. Typhimurium. We further investigated the molecular mechanism of ShdA-mediated intestinal persistence by determining the binding-site of this receptor in fibronectin. Analysis of ShdA binding to fibronectin proteolytic fragments and to recombinant fibronectin fusion proteins identified the (13)FnIII repeat module of the Hep-2 domain as the primary binding site for this adhesin. The (13)FnIII repeat module of fibronectin contains a cationic cradle formed by six basic residues (R6, R7, R9, R23, K25 and R54) that is a high affinity heparin-binding site conserved among fibronectin sequences from frogs to man. Binding of ShdA to the (13)FnIII repeat module of fibronectin and to a second extracellular matrix protein, Collagen I, could be inhibited by heparin. Furthermore, binding of ShdA to the Hep-2 domain was sensitive to the ionic buffer strength, suggesting that binding involved ionic interactions. We therefore determined whether amino acid substitutions of basic residues in the cationic cradle of the Hep-2 domain that inhibit heparin binding also abrogate binding of ShdA. Combined substitution of R6S and R7S strongly reduced ShdA binding to (13)FnIII. These data suggest that ShdA binds the Hep-2 domain of fibronectin by a mechanism that may mimic binding of the host polysaccharide heparin.
肠炎沙门氏菌鼠伤寒血清型进入食品是由于其能够通过尚不清楚的机制在健康家畜肠道中持续存在。非菌毛黏附素ShdA是鼠伤寒沙门氏菌肠道持续携带所需的一种纤连蛋白结合蛋白。我们通过确定该受体在纤连蛋白中的结合位点，进一步研究了ShdA介导的肠道持续存在的分子机制。对ShdA与纤连蛋白蛋白水解片段以及重组纤连蛋白融合蛋白的结合分析确定，Hep-2结构域的（13）FnIII重复模块是该黏附素的主要结合位点。纤连蛋白的（13）FnIII重复模块包含一个由六个碱性残基（R6、R7、R9、R23、K25和R54）形成的阳离子支架，这是一个在从青蛙到人类的纤连蛋白序列中保守的高亲和力肝素结合位点。肝素可抑制ShdA与纤连蛋白的（13）FnIII重复模块以及第二种细胞外基质蛋白胶原蛋白I的结合。此外，ShdA与Hep-2结构域的结合对离子缓冲强度敏感，表明结合涉及离子相互作用。因此，我们确定抑制肝素结合的Hep-2结构域阳离子支架中碱性残基的氨基酸取代是否也会消除ShdA的结合。R6S和R7S的联合取代强烈降低了ShdA与（13）FnIII的结合。这些数据表明，ShdA通过一种可能模拟宿主多糖肝素结合的机制与纤连蛋白的Hep-2结构域结合。
