A method for allelic replacement in Francisella tularensis
A method for allelic replacement in Francisella tularensis
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DOI：
10.1016/s0378-1097(03)00313-6
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发表时间：
2003-05-28
影响因子：
2.1
通讯作者：
Pavlov, V
中科院分区：
文献类型：
--
作者：
Golovliov, I;Sjöstedt, A;Pavlov, V
以pUC19为基础构建了图拉氏方济氏菌的诱变载体。通过插入枯草芽孢杆菌的sacb基因、Rp4的ORIT和福氏志贺氏菌的氯霉素抗性基因，获得了允许特异性诱变的载体pPV。开发了一种方案，允许通过接合将载体导入图拉氏丝虫活疫苗株LVS。作为原则的证明，我们的目标是开发一个在23 kDa蛋白(IglC)表达方面存在缺陷的特定突变体，我们之前已经证明，在F tularensis的细胞内生长过程中，该蛋白显著上调。一个命名为PPV-DeltaiglC的质粒被开发出来，它只包含编码基因iglC两侧的区域。通过双交叉事件，染色体iglC基因被缺失。然而，由此产生的菌株，标记为DeltaiglC1，仍然具有完整的iglC基因。Southern印迹分析证实，LVS含有iglC基因的两个拷贝。因此，重复诱变，获得了一个在两个iglC等位基因上都有缺陷的突变体，命名为DeltaiglC1+2。Deltaiglc1+2株与Deltaiglc1株相比，在小鼠体内显示出细胞内巨噬细胞生长受损，毒力减弱。所建立的遗传系统有可能为阐明图拉氏丝虫的毒力机制提供工具，并且该突变体说明了23-kDa蛋白iglC在图拉尔丝虫毒力中的关键作用。(C)2003年欧洲微生物学会联合会。爱思唯尔科学公司出版。版权所有。
A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F tularensis. A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained. The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F tularensis and the specific F tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F tularensis LVS. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.