Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis
Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis
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DOI：
10.1006/mpat.2000.0365
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发表时间：
2000-07-01
影响因子：
3.8
通讯作者：
Lowery, DE
中科院分区：
文献类型：
--
作者：
Fuller, TE;Kennedy, MJ;Lowery, DE
多杀性巴氏杆菌是全世界许多物种中发生的几种具有经济意义的兽医疾病的病原体。特征标记诱变 (STM) 是一种强大的遗传技术，用于同时筛选病原体的多个转座子突变体，以确定它们无法在体内存活。我们设计了一个基于微型 Tn10 转座子、化学发光检测和半定量分析的 STM 系统，并确定了转座子插入多杀性巴斯德氏菌基因中，从而减弱败血症小鼠模型中的毒力。使用含有强杂交标签的 96 个转座子库来创建 19 个多杀性巴氏杆菌转座子突变体池，每个池含有大约 70-90 个突变体。单独检查时，共有 62 个突变体被减毒，并从该组中鉴定出 25 个独特的单转座子插入突变。通过克隆或PCR扩增并对侧翼区域进行测序来确定每个减毒突变体的被破坏的ORF的序列。减毒突变体在编码生物合成酶、毒力因子、调节成分和未知功能的基因中含有转座子插入。这项研究应有助于了解多杀性巴氏杆菌和巴氏杆菌科其他病原体引起疾病的致病机制，并确定新型活疫苗候选物和新的潜在抗生素靶标。 (C) 2000 年学术出版社。
P. multocida is the causative agent of several economically significant veterinary diseases occurring in numerous species worldwide. Signature-tagged mutagenesis (STM) is a powerful genetic technique used to simultaneously screen multiple transposon mutants of a pathogen for their inability to survive in vivo. We have designed an STM system based on a mini-Tn10 transposon, chemiluminescent detection and semi-quantitative analysis and have identified transposon insertions into genes of Pasteurella multocida that attenuate virulence in a septicemic mouse model. A bank of 96 transposons containing strongly-hybridizing tags was used to create 19 pools of P. multocida transposon mutants containing approximately 70-90 mutants/pool. A total of 62 mutants were attenuated when checked individually, and 25 unique single transposon insertion mutations were identified from this group. The sequence of the disrupted ORF for each attenuated mutant was determined by either cloning or PCR-amplifying and sequencing the flanking regions. The attenuated mutants contained transposon insertions in genes encoding biosynthetic enzymes, virulence factors, regulatory components and unknown functions. This study should contribute to an understanding of the pathogenic mechanisms by which P. multocida and other pathogens in the Pasteurellaceae family cause disease and identify novel live vaccine candidates and new potential antibiotic targets. (C) 2000 Academic Press.