Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri

福氏志贺氏菌 生物 质粒 毒力 志贺氏菌 限制酶 微生物学 遗传学 分子生物学 大肠杆菌 DNA 基因
作者
Anthony T. Maurelli,Bernadette Baudry,Helend D'Hauteville,Thomas L. Hale,Philippe J. Sansonetti
出处
期刊:Infection and Immunity [American Society for Microbiology]
卷期号:49 (1): 164-171 被引量:293
标识
DOI:10.1128/iai.49.1.164-171.1985
摘要

A large plasmid is found in virulent isolates of Shigella sp. and encodes functions essential for invasion of mammalian cells. To identify plasmid sequences necessary for invasion, we isolated a series of Tn5 insertions in pWR100, the virulence plasmid of Shigella flexneri serotype 5. These insertions demonstrated that three separate EcoRI fragments of pWR100 were required for invasion of HeLa cells. However, the corresponding native EcoRI fragments, when cloned into pBR325, did not restore virulence to plasmidless strains. Construction of a lambda-sensitive, plasmidless Shigella recipient enabled us to shotgun clone plasmid DNA directly into S. flexneri by using the cosmid vector pJB8 and score for expression of invasive functions. In this fashion, we succeeded in isolating six independent recombinants which restored invasion of HeLa cells in plasmidless Shigella recipients. The cloned inserts all contained a common core of ca. 37 kilobases, thus defining a minimum sequence necessary for invasion of HeLa cells. Virulence-associated peptides produced by wild-type S. flexneri were also produced by the recombinants. Expression of these peptides and expression of invasiveness by the clones were regulated by growth temperature, as is expression of these traits in wild-type S. flexneri. A complete invasive phenotype was not expressed by the recombinants in that they failed to produce a positive Sereny test. Possible explanations for this behavior as it relates to the mechanism of bacterial invasion are discussed.

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