Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells
Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells
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DOI：
10.1128/jb.175.18.5899-5906.1993
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发表时间：
1993-09
影响因子：
3.2
通讯作者：
R. Ménard;P. Sansonetti;C. Parsot
中科院分区：
文献类型：
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作者：
R. Ménard;P. Sansonetti;C. Parsot
福氏志贺菌大毒力质粒的一个31-kb片段是细菌体外进入上皮细胞所必需的。该片段的一个位点编码IpaA、-B、-C和-D蛋白，这些蛋白是志贺氏菌病期间体液免疫应答的优势抗原。为了解决ipa基因的作用，这些基因聚集在一个操纵子中，我们构建了一个不影响下游基因转录的可选择盒，并使用这个盒来表达ipaB、ipaC和iPad基因。这些非极性突变体中的每一个都在吞噬空泡的进入和溶解中有缺陷，但在与细胞的粘附中没有受损。我们发现，像IpaB和IpaC一样，iPad分泌到培养上清液中，并且这些蛋白质中没有一种是其他两种蛋白质分泌所必需的。该结果将指导进入过程的Ipa蛋白与指导Ipa蛋白分泌的Mxi和Spa蛋白区分开。此外，缺乏IpaB或iPad导致更大量的其他Ipa多肽释放到培养基中，这表明，除了它们在入侵中的作用之外，IpaB和iPad各自参与维持Ipa蛋白与细菌的结合。
A 31-kb fragment of the large virulence plasmid of Shigella flexneri is necessary for bacterial entry into epithelial cells in vitro. One locus of this fragment encodes the IpaA, -B, -C, and -D proteins, which are the dominant antigens of the humoral immune response during shigellosis. To address the role of the ipa genes, which are clustered in an operon, we constructed a selectable cassette that does not affect transcription of downstream genes and used this cassette to inactivate the ipaB, ipaC, and ipaD genes. Each of these nonpolar mutants was defective in entry and lysis of the phagocytic vacuole but was not impaired in adhesion to the cells. We showed that, like IpaB and IpaC, IpaD is secreted into the culture supernatant and that none of these proteins is necessary for secretion of the other two. This result differentiates the Ipa proteins, which direct the entry process, from the Mxi and Spa proteins, which direct secretion of the Ipa proteins. Moreover, lack of either IpaB or IpaD resulted in the release of larger amounts of the other Ipa polypeptides into the culture medium, which indicates that, in addition to their role in invasion, IpaB and IpaD are each involved in the maintenance of the association of the Ipa proteins with the bacterium.