Cloning and expression of the Escherichia coli K1 outer membrane protein A receptor, a gp96 homologue
Cloning and expression of the Escherichia coli K1 outer membrane protein A receptor, a gp96 homologue
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DOI：
10.1128/iai.71.4.1680-1688.2003
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发表时间：
2003-04-01
影响因子：
3.1
通讯作者：
Sukumaran, SK
中科院分区：
文献类型：
--
作者：
Prasadarao, NV;Srivastava, PK;Sukumaran, SK
大肠杆菌是引起新生儿脑膜炎的最常见的革兰氏阴性菌之一。我们前期的研究表明，大肠杆菌外膜蛋白A（OmpA）在大肠杆菌中的表达量明显高于大肠杆菌外膜蛋白A。大肠杆菌与95-kDa的人脑微血管内皮细胞（HBMEC）糖蛋白Ecgp相互作用，用于侵袭。在这里，我们报告的识别基因，编码Ecgp的筛选HBMEC cDNA表达文库，以及通过5'快速扩增cDNA末端。Ecgp基因的序列表明，它与肿瘤排斥抗原-1 gp 96高度相似，并含有内质网滞留信号KDEL。ECgp或gp 96在HBMEC和CHO细胞中的过表达增加E. coli结合和侵袭。我们进一步表明，尽管存在KDEL基序，但转染Ecgp基因的HBMEC在细胞表面表达Ecgp。北方印迹分析表明，ECgp在HBMEC中有显著表达。重组His标记的Ecgp阻断E.通过直接与细菌结合来有效地侵入大肠杆菌。这些结果表明，E. coli K1与HBMEC上的gp 96样分子相互作用以进行侵袭。
Escherichia coli is one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) of E. coli interacts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5' rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a tumor rejection antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Overexpression of either Ecgp or gp96 in both HBMECs and CHO cells increases E. coli binding and invasion. We further show that Ecgp gene-transfected HBMECs express Ecgp on the cell surface despite the presence of the KDEL motif. Northern blot analysis of total RNA from various eukaryotic cells indicates that Ecgp is significantly expressed in HBMECs. Recombinant His-tagged Ecgp blocked E. coli invasion efficiently by binding directly to the bacteria. These results suggest that OmpA of E. coli K1 interacts with a gp96-like molecule on HBMECs for invasion.