Champion C I, Blanco D R, Skare J T, Haake D A, Giladi M, Foley D, Miller J N, Lovett M A
Department of Microbiology & Immunology, UCLA School of Medicine 90024.
Infect Immun. 1994 Jul;62(7):2653-61. doi: 10.1128/iai.62.7.2653-2661.1994.
In this study, we report the cloning, sequencing, and molecular analysis of a gene located on a 9.0-kbp circular plasmid of virulent Borrelia burgdorferi B31 designated eppA (exported plasmid protein A). This gene encodes a precursor protein of 174 amino acids including a signal peptide of 20 amino acids and a type I signal peptidase cleavage site. The mature EppA protein of 154 amino acids has a calculated molecular weight of 17,972. Several lines of evidence suggest that eppA is not expressed by B. burgdorferi B31 during in vitro cultivation. Immunoblot analysis using hyperimmune rabbit antiserum to recombinant EppA (rEppA) did not detect the presence of EppA in B. burgdorferi B31 cultivated in vitro. Northern blot analysis using total RNA isolated from in vitro-cultivated virulent B. burgdorferi B31 failed to detect an eppA transcript. EppA was not detected in culture supernatants of virulent B. burgdorferi B31 in a sensitive antigen-capture enzyme-linked immunosorbent assay. In contrast, evidence for expression of eppA during infection was based on the observation that patients with Lyme disease as well as rabbits experimentally infected with B. burgdorferi B31 produced antibodies that recognized rEppA. Because the cellular location of EppA in B. burgdorferi cannot be determined in vivo because of very small numbers of organisms present in vertebrate infection, we examined the cellular location of rEppA expressed in Escherichia coli. In E. coli, rEppA is targeted to the outer membrane. In addition, purified E. coli outer membranes containing rEppA treated with chaotrophic agents did not result in rEppA release. These findings are consistent with the idea that EppA is not peripherally associated with the outer membrane of E. coli but rather has an integral outer membrane association.
在本研究中，我们报告了对位于有毒力的伯氏疏螺旋体B31的一个9.0-kbp环形质粒上的基因（命名为eppA，即输出质粒蛋白A）进行的克隆、测序及分子分析。该基因编码一种由174个氨基酸组成的前体蛋白，包括一个20个氨基酸的信号肽和一个I型信号肽酶切割位点。由154个氨基酸组成的成熟EppA蛋白的计算分子量为17,972。几条证据表明，在体外培养期间，伯氏疏螺旋体B31不表达eppA。使用针对重组EppA（rEppA）的超免疫兔抗血清进行的免疫印迹分析未检测到体外培养的伯氏疏螺旋体B31中存在EppA。使用从体外培养的有毒力的伯氏疏螺旋体B31中分离的总RNA进行的Northern印迹分析未能检测到eppA转录本。在灵敏的抗原捕获酶联免疫吸附试验中，未在有毒力的伯氏疏螺旋体B31的培养上清液中检测到EppA。相比之下，感染期间eppA表达的证据基于以下观察结果：莱姆病患者以及经伯氏疏螺旋体B31实验感染的兔子产生了识别rEppA的抗体。由于脊椎动物感染中存在的生物体数量极少，无法在体内确定伯氏疏螺旋体中EppA的细胞定位，因此我们检查了在大肠杆菌中表达的rEppA的细胞定位。在大肠杆菌中，rEppA定位于外膜。此外，用离液剂处理的含有rEppA的纯化大肠杆菌外膜并未导致rEppA释放。这些发现与EppA并非外周结合于大肠杆菌外膜而是具有完整的外膜结合这一观点一致。
