Teng Fang, Kawalec Magdalena, Weinstock George M, Hryniewicz Waleria, Murray Barbara E
Division of Infectious Disease, Department of Internal Medicine and Center for the Study of Emerging and Reemerging Pathogens, University of Texas Houston Medical School, Houston, Texas 77030, USA.
Infect Immun. 2003 Sep;71(9):5033-41. doi: 10.1128/IAI.71.9.5033-5041.2003.
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 I, 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-terminal 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 sagA 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.
通过用粪肠球菌相关性心内膜炎患者的血清筛选粪肠球菌基因组表达文库，在粪肠球菌中鉴定出一个编码主要分泌抗原SagA的基因。重组SagA蛋白与细胞外基质（ECM）蛋白具有广谱结合性，这些蛋白包括纤维蛋白原、I型胶原、IV型胶原、纤连蛋白和层粘连蛋白。从一株粪肠球菌临床分离株的培养上清中纯化得到的一种纤维蛋白原结合蛋白，被发现与预测的SagA蛋白的N端序列匹配，并能与抗SagA抗体发生反应，证实它就是SagA蛋白；该蛋白在蛋白质印迹上呈现为一条80至90 kDa的条带，对蛋白酶K敏感，对高碘酸盐处理和糖蛋白染色具有抗性。当在粪肠球菌和大肠杆菌中过表达时，天然和重组的SagA蛋白明显通过其C端结构域形成稳定的寡聚体。SagA蛋白由三个结构域组成：（i）一个假定的卷曲螺旋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（相似性为42%），先前已证明该结构域参与细胞壁完整性和细胞形态维持，并且与单核细胞增生李斯特菌的P45蛋白具有同源性（相似性为41%）；（ii）一个包含直接重复序列的中央结构域；（iii）一个C端结构域，与多种蛋白质中的结构域相似，包括单核细胞增生李斯特菌的P45（相似性为50%）和P60（相似性为52%）。P45和P6I蛋白都具有细胞壁水解酶活性，并且后者也已被证明与毒力有关，而未检测到SagA蛋白具有细胞壁水解酶活性。粪肠球菌sagA基因与变形链球菌的同源基因一样，位于一组编码似乎参与细胞壁代谢的蛋白质的基因簇中，并且除非首先进行反式互补，否则无法被破坏，这表明sagA基因对粪肠球菌的生长至关重要，并且可能参与细胞壁代谢。总之，粪肠球菌细胞外SagA蛋白显然对生长至关重要，与ECM蛋白具有广谱结合性，形成寡聚体，并且在感染期间具有抗原性。
