Identification of a novel plasmin(ogen)-binding motif in surface displayed α-enolase of Streptococcus pneumoniae
Identification of a novel plasmin(ogen)-binding motif in surface displayed α-enolase of Streptococcus pneumoniae
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DOI：
10.1046/j.1365-2958.2003.03557.x
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发表时间：
2003-07-01
影响因子：
3.6
通讯作者：
Hammerschmidt, S
中科院分区：
文献类型：
--
作者：
Bergmann, S;Wild, D;Hammerschmidt, S
肺炎链球菌与人纤溶酶（原）的相互作用代表了通过捕获感染宿主中表面相关的蛋白水解活性来增强细菌毒力的机制。纤溶酶原与表面展示的肺炎球菌α-烯醇化酶（Eno）结合，随后被宿主来源的组织纤溶酶原激活物（tPA）或尿激酶（uPA）激活为丝氨酸蛋白酶纤溶酶。Eno的C-末端赖氨酰残基在位置433和434处被鉴定为含有赖氨酸结合位点的纤溶酶（原）的kringle基序的结合位点。在这份报告中，我们已经确定了一个新的内部纤溶蛋白（原）结合位点的伊诺通过研究蛋白质-蛋白质相互作用。在结合测定以及表面等离子体共振研究中使用的C-末端突变的Eno蛋白的纤溶酶（原）结合活性表明Eno的另外的结合基序参与Eno-纤溶酶（原）复合物的形成。对代表Eno序列的斑点合成的合成肽的分析鉴定了位于氨基酸248-256之间的9个氨基酸的肽作为介导纤溶酶原与Eno结合的最小第二结合表位。放射性标记的纤溶酶原与活肺炎球菌的结合被代表Eno的新型内部纤溶酶（原）结合基序的合成肽FYDKERKVYD竞争性抑制。相反，在通过系统突变分析鉴定的内部结合基序的关键位置具有氨基酸取代的合成肽不抑制纤溶酶原与肺炎球菌的结合。表达α-烯醇化酶的肺炎球菌突变体在内部结合基序中具有氨基酸取代，显示出显著降低的纤溶酶原结合活性。这些突变体的毒力也减弱，在小鼠模型中的鼻内感染，表明新的纤溶酶原结合基序在肺炎球菌疾病的发病机制的意义。
The interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surface-associated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal a-enolase (Eno) and is subsequently activated to the serine protease plasmin by host-derived tissue plasminogen activator (tPA) or urokinase (uPA). The C-terminal lysyl residues of Eno at position 433 and 434 were identified as a binding site for the kringle motifs of plasmin(ogen) which contain lysine binding sites. In this report we have identified a novel internal plamin(ogen)-binding site of Eno by investigating the protein-protein interaction. Plasmin(ogen)-binding activity of C-terminal mutated Eno proteins used in binding assays as well as surface plasmon resonance studies suggested that an additional binding motif of Eno is involved in the Eno-plasmin(ogen) complex formation. The analysis of spot synthesized synthetic peptides representing Eno sequences identified a peptide of nine amino acids located between amino acids 248-256 as the minimal second binding epitope mediating binding of plasminogen to Eno. Binding of radiolabelled plasminogen to viable pneumococci was competitively inhibited by a synthetic peptide FYDKERKVYD representing the novel internal plasmin(ogen)-binding motif of Eno. In contrast, a synthetic peptide with amino acid substitutions at critical positions in the internal binding motif identified by systematic mutational analysis did not inhibit binding of plasminogen to pneumococci. Pneumococcal mutants expressing alpha-enolase with amino acid substitutions in the internal binding motif showed a substantially reduced plasminogen-binding activity. The virulence of these mutants was also attenuated in a mouse model of intranasal infection indicating the significance of the novel plasminogen-binding motif in the pathogenesis of pneumococcal diseases.