Beckmann Christiane, Waggoner Joshua D, Harris Theresa O, Tamura Glen S, Rubens Craig E
Division of Infectious Disease, Children's Hospital and Regional Medical Center and University of Washington, Seattle 98105, USA.
Infect Immun. 2002 Jun;70(6):2869-76. doi: 10.1128/IAI.70.6.2869-2876.2002.
Group B streptococci (GBS) are a major cause of pneumonia, sepsis, and meningitis in newborns and infants. GBS initiate infection of the lung by colonizing mucosal surfaces of the respiratory tract; adherence of the bacteria to host cells is presumed to be the initial step in and prerequisite for successful colonization (G. S. Tamura, J. M. Kuypers, S. Smith, H. Raff, and C. E. Rubens, Infect. Immun. 62:2450-2458, 1994). We have performed a genome-wide screen to identify novel genes of GBS that mediate adherence to fibronectin. A shotgun phage display library was constructed from chromosomal DNA of a serotype Ia GBS strain and affinity selected on immobilized fibronectin. DNA sequence analysis of different clones identified 19 genes with homology to known bacterial adhesin genes, virulence genes, genes involved in transport or metabolic processes, and genes with yet-unknown function. One of the isolated phagemid clones showed significant homology to the gene (scpB) for the GBS C5a peptidase, a surface-associated serine protease that specifically cleaves the complement component C5a, a chemotaxin for polymorphonuclear leukocytes. In this work we have demonstrated that affinity-purified recombinant ScpB and a peptide ScpB fragment (ScpB-PDF), similar to the peptide identified in the phagemid, bound fibronectin in a concentration-dependent manner. Adherence assays to fibronectin were performed, comparing an isogenic scpB mutant to the wild-type strain. Approximately 50% less binding was observed with the mutant than with the wild-type strain. The mutant phenotype could be fully restored by in trans complementation of the mutant with the cloned wild-type scpB gene, providing further evidence for the role of ScpB in fibronectin adherence. Our results suggest that C5a peptidase is a bifunctional protein, which enzymatically cleaves C5a and mediates adherence to fibronectin. Since binding of fibronectin has been implicated in attachment and invasion of eukaryotic cells by streptococci, our results may imply a second important role for this surface protein in the pathogenesis of GBS infections.
B族链球菌（GBS）是新生儿和婴儿肺炎、败血症及脑膜炎的主要病因。GBS通过在呼吸道黏膜表面定植引发肺部感染；细菌与宿主细胞的黏附被认为是成功定植的起始步骤及前提条件（G. S. 田村、J. M. 凯珀斯、S. 史密斯、H. 拉夫和C. E. 鲁本斯，《感染与免疫》62:2450 - 2458，1994年）。我们进行了全基因组筛选，以鉴定GBS中介导与纤连蛋白黏附的新基因。从一株血清型Ia GBS菌株的染色体DNA构建了一个鸟枪法噬菌体展示文库，并在固定化的纤连蛋白上进行亲和筛选。对不同克隆的DNA序列分析鉴定出19个与已知细菌黏附素基因、毒力基因、参与转运或代谢过程的基因以及功能未知的基因具有同源性的基因。其中一个分离的噬菌粒克隆与GBS C5a肽酶的基因（scpB）具有显著同源性，C5a肽酶是一种表面相关的丝氨酸蛋白酶，可特异性切割补体成分C5a，C5a是多形核白细胞的趋化因子。在这项研究中，我们证明亲和纯化的重组ScpB和一个肽段ScpB片段（ScpB - PDF），类似于在噬菌粒中鉴定出的肽段，以浓度依赖的方式结合纤连蛋白。进行了与纤连蛋白的黏附试验，将同基因的scpB突变体与野生型菌株进行比较。观察到突变体的结合比野生型菌株减少了约50%。通过用克隆的野生型scpB基因对突变体进行反式互补，可完全恢复突变体表型，为ScpB在纤连蛋白黏附中的作用提供了进一步证据。我们的结果表明，C5a肽酶是一种双功能蛋白，它能酶切C5a并介导与纤连蛋白的黏附。由于纤连蛋白的结合与链球菌对真核细胞的附着和侵袭有关，我们的结果可能意味着这种表面蛋白在GBS感染发病机制中具有第二个重要作用。
