CLONING OF PLASMID DNA-SEQUENCES INVOLVED IN INVASION OF HELA-CELLS BY SHIGELLA-FLEXNERI
CLONING OF PLASMID DNA-SEQUENCES INVOLVED IN INVASION OF HELA-CELLS BY SHIGELLA-FLEXNERI
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DOI：
10.1128/iai.49.1.164-171.1985
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发表时间：
1985-01-01
影响因子：
3.1
通讯作者：
SANSONETTI, PJ
中科院分区：
文献类型：
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作者：
MAURELLI, AT;BAUDRY, B;SANSONETTI, PJ
在志贺氏菌的毒力分离株中发现了一个大质粒，它编码了入侵哺乳动物细胞所必需的功能。为了确定侵染所需的质粒序列，我们在福氏志贺氏菌血清5型毒力质粒pWR100中分离了一系列Tn5插入。这些插入表明pWR100的三个独立的EcoRI片段是侵入HeLa细胞所必需的。然而，当将相应的原生EcoRI片段克隆到pBR325中时，并没有恢复对无质粒菌株的毒力。.lambda的构造。我们利用cosmid载体pJB8将质粒DNA直接克隆到S. flexneri中，并对其侵袭功能的表达进行评分。通过这种方式，我们成功地分离了6个独立的重组体，这些重组体在无质粒的志贺氏菌受体中恢复了HeLa细胞的侵袭。克隆的插入体都包含一个约37千碱基的共同核心，从而定义了侵入HeLa细胞所需的最小序列。该重组体还产生了野生型flexneri菌株的毒力相关肽。这些多肽的表达和侵染性的表达受生长温度的调控，这些性状在野生型flexneri中的表达也是如此。完整的侵袭性表型未被重组表达，因为它们未能产生阳性的Sereny测试。讨论了这种行为的可能解释，因为它与细菌入侵的机制有关。
A large plasmid is found in virulent isolates of Shigella sp. and encodes functions essential for invasion of mammalian cells. To identify plasmid sequence 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 functios. 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.