Mahan M J, Tobias J W, Slauch J M, Hanna P C, Collier R J, Mekalanos J J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):669-73. doi: 10.1073/pnas.92.3.669.
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.
我们最近描述了一种称为体内表达技术（IVET）的遗传系统，该系统利用动物作为选择培养基来鉴定病原菌在感染宿主组织时特异性表达的基因。在此，随着转录融合载体pIVET8的开发，IVET方法的潜在用途得到了扩展，pIVET8以抗生素抗性作为在宿主组织中进行选择的基础。pIVET8包含无启动子的氯霉素乙酰转移酶（cat）和lacZY基因。用pIVET8产生的携带随机cat-lac转录融合的鼠伤寒沙门氏菌克隆库用于感染BALB/c小鼠，随后对其进行腹腔注射氯霉素处理。然后筛选那些在实验室培养基上低水平表达lacZY的、通过在动物体内表达cat基因而在选择中存活下来的菌株。这些菌株携带与在体内特异性诱导的基因（ivi基因）的操纵子融合。鉴定出的一个ivi基因（fadB）编码一种参与脂肪酸氧化的酶，这表明该酶可能有助于杀菌或促炎宿主脂肪酸的代谢。基于pIVET8的选择系统还用于鉴定在培养的巨噬细胞中诱导的鼠伤寒沙门氏菌基因。ivi基因产物的性质将使我们更全面地了解导致微生物病原体毒力的代谢、生理和遗传因素。
