IDENTIFICATION AND CLONING OF A NOVEL PLASMID-ENCODED ENTEROTOXIN OF ENTEROINVASIVE ESCHERICHIA-COLI AND SHIGELLA STRAINS
IDENTIFICATION AND CLONING OF A NOVEL PLASMID-ENCODED ENTEROTOXIN OF ENTEROINVASIVE ESCHERICHIA-COLI AND SHIGELLA STRAINS
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DOI：
10.1128/iai.63.12.4721-4728.1995
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发表时间：
1995-12-01
影响因子：
3.1
通讯作者：
MORRIS, JG
中科院分区：
文献类型：
--
作者：
NATARO, JP;SERIWATANA, J;MORRIS, JG
我们采用分子遗传学方法来表征肠侵袭性大肠杆菌 (EIEC) 肠毒性活性的性质，如先前在 Ussing 室中观察到的那样（A. Fasano、B. A. Kay、R. G. Russell、D. R. Maneval, Jr. 和 M. M. Levine, Infect, Immun. 58:3717-3723, 1990），TnphoA 的筛选EIEC突变体产生了单个插入突变体，其在尤斯室测定中显着降低了肠毒性活性水平，插入侧翼的DNA用作探针来筛选赋予促分泌活性的EIEC粘粒克隆，这种筛选导致鉴定出两个重叠的粘粒克隆，其引起粘膜短路电流（Isc）的显着变化。对粘粒克隆之一的 DNA 片段进行亚克隆和核苷酸序列分析，鉴定出赋予这种肠毒性活性的单一开放读码框。通过 DNA 杂交，在 75% 的 EIEC 菌株和 83% 的志贺氏菌菌株中发现了该基因（称为 sen 表示志贺氏菌肠毒素），并且定位于福氏志贺氏菌 2457T 的 inv 质粒，通过 PCR，发现 sen 基因具有从 2457T 克隆并测序了 99.7% 的核苷酸同一性，通过等位基因交换构建了 EIEC sen 基因中的缺失，导致 Isc 的升高明显低于野生型亲本引起的升高；然而，sen缺失突变体仍然具有显着的肠毒性活性。为了纯化Sen蛋白，将该基因克隆到表达载体pKK223-3的多克隆位点中。 sen基因产物的纯化产生了分子量为63 kDa的蛋白质，其引起Ussing室中Isc的升高。我们认为sen基因产物可能构成EIEC和志贺氏菌属中新型肠毒素的全部或部分。
We have employed a molecular genetic approach to characterize the nature of enteroinvasive Escherichia coli (EIEC) enterotoxic activity, as previously observed in Ussing chambers (A. Fasano, B. A. Kay, R. G. Russell, D. R. Maneval, Jr., and M. M. Levine, Infect, Immun. 58:3717-3723, 1990), The screening of TnphoA mutants of EIEC yielded a single insertion mutant which had significantly reduced levels of enterotoxic activity in the Ussing chamber assay, DNA flanking the insertion was used as a probe to screen for EIEC cosmid clones which conferred secretogenic activity, Such screening resulted in the identification of two overlapping cosmid clones which elicited significant changes in mucosal short-circuit current (Isc). Subcloning and nucleotide sequence analysis of a DNA fragment from one of the cosmid clones led to the identification of a single open reading frame which conferred this enterotoxic activity, By DNA hybridization, this gene (designated sen for shigella enterotoxin) was found in 75% of EIEC strains and 83% of Shigella strains and was localized to the inv plasmid of Shigella flexneri 2457T, By PCR, a sen gene with 99.7% nucleotide identity was cloned and sequenced from 2457T, A deletion in the EIEC sen gene was constructed by allelic exchange, resulting in significantly lower rises in Isc than were elicited by the wild-type parent; however, significant enterotoxic activity remained in the sen deletion mutant, To purify the Sen protein, the gene was cloned into the multiple cloning site of the expression vector pKK223-3. Purification of the sen gene product yielded a protein with a molecular mass of 63 kDa which elicited rises in Isc in the Ussing chamber, We believe that the sen gene product may constitute all or part of a novel enterotoxin in EIEC and Shigella spp.