Identification of novel loci involved in entry by Legionella pneumophila
Identification of novel loci involved in entry by Legionella pneumophila
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DOI：
10.1099/00221287-146-6-1345
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发表时间：
2000-06-01
期刊：
影响因子：
2.8
通讯作者：
Cirillo, JD
中科院分区：
文献类型：
--
作者：
Cirillo, SLG;Lum, J;Cirillo, JD
嗜肺军团菌在感染过程中主要是一种细胞内的病原体；因此，进入宿主细胞的机制在发病机制中可能是重要的。通过选择以比野生型更高的频率进入宿主细胞的细菌，分离出几个表现增强进入(Enh)表型的嗜肺乳杆菌突变体。在对这些突变体之一C3的遗传基础进行表征的过程中，开发了一种从嗜肺乳杆菌L中分离实验室介质抑制的毒力决定簇的策略。使用来自C3的基因组文库筛选显性突变导致分离出三个赋予野生型嗜肺乳杆菌Enh表型的宇宙体，这些宇宙体的转座子突变允许鉴定三个影响进入的基因座，分析由这些基因座编码的可能的蛋白质，命名为rtxA和EnhC，分别与秀丽线虫结构毒素蛋白和分泌型Sei-1蛋白的重复序列相似。与野生型细菌相比，L、嗜肺菌rtxA和henC突变体显著减少了进入宿主细胞的能力，含有这些基因座的宇宙体所提供的表型很可能是由于它们在多拷贝载体上的存在而导致表达增加。利用增加的基因拷贝数来过度表达在实验室生长条件下通常被抑制的基因，通常适用于从嗜肺乳杆菌和其他细菌病原体中分离毒力决定因素。
Legionella pneumophila is primarily an intracellular pathogen during infection; thus, the mechanisms of entry into host cells are likely to be important for pathogenesis. Several L. pneumophila mutants that display an enhanced-entry (Enh) phenotype were isolated by selecting for bacteria that enter host cells at a higher frequency than wild-type, In the course of characterizing the genetic basis of one of these mutants, C3, a strategy was developed for the isolation of laboratory-media-repressed virulence determinants from L, pneumophila, Screens for dominant mutations using a genomic DNA library from C3 resulted in the isolation of three cosmids that confer an Enh phenotype to wild-type L. pneumophila, Transposon mutagenesis of these cosmids allowed identification of three loci that affect entry, Analysis of the putative proteins encoded by these loci, designated rtxA and enhC, demonstrated similarity to repeats in the structural toxin protein and the secreted SeI-1 protein from Caenorhabditis elegans, respectively. L, pneumophila rtxA and enhC mutants display significantly reduced entry into host cells, compared to wild-type bacteria, The phenotype that the cosmids containing these loci confer is most likely due to elevated expression resulting from their presence on multicopy vectors. The use of increased gene copy number to overexpress genes that are normally repressed under laboratory growth conditions is generally applicable to the isolation of virulence determinants from L. pneumophila and other bacterial pathogens.