Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host.
Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host.
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DOI：
10.1073/pnas.92.3.669
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发表时间：
1995-01
影响因子：
11.1
通讯作者：
M. Mahan;J. Tobias;J. Slauch;J. Slauch;P. Hanna;R. Collier;J. Mekalanos
中科院分区：
文献类型：
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作者：
M. Mahan;J. Tobias;J. Slauch;J. Slauch;P. Hanna;R. Collier;J. Mekalanos
我们最近描述了一种遗传系统，称为体内表达技术（IVET），它使用动物作为选择性培养基来鉴定病原菌感染宿主组织时特异性表达的基因。在这里，IVET方法的潜在效用已经随着转录融合载体pIVET 8的开发而扩展，该载体使用抗生素抗性作为在宿主组织中进行选择的基础。pIVET 8含有无启动子的氯霉素乙酰转移酶（cat）和lacZY基因。用pIVET 8产生的携带随机cat-lac转录融合物的鼠伤寒沙门氏菌克隆库用于感染BALB/c小鼠，随后用腹膜内注射氯霉素处理小鼠。通过在动物中表达cat基因而在选择中存活的菌株然后筛选那些在实验室培养基上具有低水平lacZY表达的菌株。这些菌株携带与体内特异性诱导的基因（ivi基因）融合的操纵子。其中一个ivi基因（fadB）编码一种参与脂肪酸氧化的酶，这表明这种酶可能有助于杀菌或促炎宿主脂肪酸的代谢。利用pIVET 8筛选系统对S.在培养的巨噬细胞中诱导的鼠伤寒杆菌基因。ivi基因产物的性质将提供一个更完整的了解代谢，生理和遗传因素，有助于微生物病原体的毒力。
We have recently described a genetic system, termed in vivo expression technology (IVET), that uses an animal as a selective medium to identify genes that pathogenic bacteria specifically express when infecting host tissues. Here, the potential utility of the IVET approach has been expanded with the development of a transcriptional-fusion vector, pIVET8, which uses antibiotics resistance as the basis for selection in host tissues. pIVET8 contains promoterless chloramphenicol acetyltransferase (cat) and lacZY genes. A pool of Salmonella typhimurium clones carrying random cat-lac transcriptional fusions, produced with pIVET8, was used to infect BALB/c mice that were subsequently treated with intraperitoneal injections of chloramphenicol. Strains that survived the selection by expressing the cat gene in the animal were then screened for those that had low-level lacZY expression on laboratory medium. These strains carry operon fusions to genes that are specifically induced in vivo (ivi genes). One of the ivi genes identified (fadB) encodes an enzyme involved in fatty acid oxidation, suggesting that this enzyme might contribute to the metabolism of bactericidal or proinflammatory host fatty acids. The pIVET8-based selection system was also used to identify S. typhimurium genes that are induced in cultured macrophages. The nature of ivi gene products will provide a more complete understanding of the metabolic, physiological, and genetic factors that contribute to the virulence of microbial pathogens.