An Enterococcus faecium secreted antigen, SagA, exhibits broad-spectrum binding to extracellular matrix proteins and a appears essential for E-faecium growth
An Enterococcus faecium secreted antigen, SagA, exhibits broad-spectrum binding to extracellular matrix proteins and a appears essential for E-faecium growth
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DOI：
10.1128/iai.71.9.5033-5041.2003
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发表时间：
2003-09-01
影响因子：
3.1
通讯作者：
Murray, BE
中科院分区：
文献类型：
--
作者：
Teng, F;Kawalec, M;Murray, BE
通过用屎肠球菌相关心内膜炎患者的血清筛选屎肠球菌基因组表达文库，在屎肠球菌中鉴定出编码主要分泌抗原 SagA 的基因。重组 SagA 蛋白显示出与细胞外基质 (ECM) 蛋白的广谱结合，包括纤维蛋白原、1 型胶原蛋白、IV 型胶原蛋白、纤连蛋白和层粘连蛋白。从屎肠球菌临床分离株的培养上清液中纯化出的纤维蛋白原结合蛋白与预测的 SagA 蛋白的 N 端序列相匹配，并与抗 SagA 抗体发生反应，证实它就是 SagA 蛋白；该蛋白质在蛋白质印迹上显示为 80 至 90 kDa 涂片，对蛋白酶 K 敏感，并且对高碘酸盐处理和糖蛋白染色具有抗性。当在屎肠球菌和大肠杆菌中过表达时，天然和重组SagA蛋白形成稳定的寡聚体，显然是通过它们的C-末端结构域。 SagA 蛋白由三个结构域组成：(i) 假定的卷曲螺旋 N 端结构域，与变形链球菌 SagA 蛋白的 N 端结构域具有同源性（42% 相似性），之前显示其参与细胞壁完整性和细胞形状维持，并且与单核细胞增生李斯特氏菌的 P45 蛋白具有同源性（41% 相似性）； (ii) 包含直接重复序列的中心结构域； (iii) 与多种蛋白质中发现的相似的 C 末端结构域，包括单核细胞增生李斯特菌的 P45（50% 相似性）和 P60（52% 相似性）。 P45和P60蛋白均具有细胞壁水解酶活性，并且后者也被证明与毒力有关，而SagA蛋白未检测到细胞壁水解酶活性。与变形链球菌同源物一样，屎肠球菌 sagA 基因位于一组编码似乎参与细胞壁代谢的蛋白质的基因中，除非首先反式互补，否则不会被破坏，这表明 sag,4 基因对于屎肠球菌生长至关重要，并且可能参与细胞壁代谢。总之，胞外屎肠球菌 SagA 蛋白显然对于生长至关重要，显示出与 ECM 蛋白的广谱结合，形成寡聚体，并且在感染期间具有抗原性。
A gene encoding a major secreted antigen, SagA, was identified in Enterococcus faecium by screening an E. faecium genomic expression library with sera from patients with E. faecium-associated endocarditis. Recombinant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type 1, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of an E. faecium clinical isolate, was found to match the N-terminal sequence of the predicted SagA protein and to react with the anti-SagA antibody, confirming that it was the SagA protein; this protein appeared as an 80- to 90-kDa smear on a Western blot that was sensitive to proteinase K and resistant to periodate treatment and glycoprotein staining. When overexpressed in E. faecium and Escherichia coli, the native and recombinant SagA proteins formed stable oligomers, apparently via their C-terminall domains. The SagA protein is composed of three domains: (i) a putative coiled-coil N-terminal domain that shows homology to the N-terminal domain of Streptococcus mutans SagA protein (42% similarity), previously shown to be involved in cell wall integrity and cell shape maintenance, and to the P45 protein of Listeria monocytogenes (41% similarity); (ii) a central domain containing direct repeats; and (iii) a C-terminal domain that is similar to that found in various proteins, including P45 (50% similarity) and P60 (52% similarity) of L. monocytogenes. The P45 and P60 proteins both have cell wall hydrolase activity, and the latter has also been shown to be involved in virulence, whereas cell wall hydrolase activity was not detected for SagA protein. The E. faecium sagA gene, like the S. mutans homologue, is located in a cluster of genes encoding proteins that appear to be involved in cell wall metabolism and could not be disrupted unless it was first transcomplemented, suggesting that the sag,4 gene is essential for E. faecium growth and may be involved in cell wall metabolism. In conclusion, the extracelluar E. faecium SagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.