IDENTIFICATION AND CHARACTERIZATION OF VIRK, A VIRULENCE-ASSOCIATED LARGE PLASMID GENE ESSENTIAL FOR INTERCELLULAR SPREADING OF SHIGELLA-FLEXNERI
IDENTIFICATION AND CHARACTERIZATION OF VIRK, A VIRULENCE-ASSOCIATED LARGE PLASMID GENE ESSENTIAL FOR INTERCELLULAR SPREADING OF SHIGELLA-FLEXNERI
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DOI：
10.1111/j.1365-2958.1992.tb01413.x
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发表时间：
1992-08-01
影响因子：
3.6
通讯作者：
YOSHIKAWA, M
中科院分区：
文献类型：
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作者：
NAKATA, N;SASAKAWA, C;YOSHIKAWA, M
用Tn 5插入法对福氏2a志贺菌的230 kb质粒pMYSH 6000进行诱变，筛选出7个毒力位点。在这项研究中，我们使用Tn 10插入突变，并确定了一个新的毒力位点pMYSH 6000负责细菌传播。上皮细胞中Tn 10插入突变体的入侵细菌的表征揭示，细菌能够至少一些细胞内扩散，但不能细胞间扩散。用VirG特异性抗肽抗体对Tn 10插入突变体的裂解物进行免疫印迹分析，发现116 kDa VirG蛋白的水平降低。然而，突变体中的virG mRNA的表达水平与野生型相同。毒力表型所需的DNA区域定位于质粒上SalI-K片段中的1.6 kb DNA序列，因此该基因座被命名为virK。使用T7 RNA聚合酶依赖性启动子系统在大肠杆菌中表达virK产生36 kDa蛋白。测序结果表明，该基因全长1642 bp，编码316个氨基酸残基的开放阅读框（ORF）。virK区域在志贺氏菌和肠侵袭性大肠杆菌的大毒力质粒中高度保守。这些结果表明，VirK功能是一个重要的毒力决定因素，参与virG基因产物在转录后水平的表达。
Seven virulence loci have been identified by Tn5 insertion mutagenesis on the large 230 kb plasmid (pMYSH6000) of Shigella flexneri 2a. In this study, we used Tn10 insertion mutagenesis and identified a novel virulence locus on pMYSH6000 responsible for bacterial spread. Characterization of the invading bacteria of the Tn10 insertion mutants in the epithelial cells revealed that the bacteria were capable of at least some intracellular spreading but not intercellular spreading. Immunoblot analysis of lysates of the Tn10 insertion mutants with a VirG-specific antipeptide antibody revealed diminished levels of the 116 kDa VirG protein. The virG mRNA in the mutants, however, was expressed at the same level as that in the wild type. The DNA region required for the virulence phenotype was localized to a 1.6 kb DNA sequence in the SalI-K fragment on the plasmid, and thus the locus was designated virK. Expression of virK in Escherichia coli using a T7 RNA polymerase-dependent promoter system yielded a 36 kDa protein. The nucleotide sequence of 1642 bp encoding VirK function was determined, and an open reading frame encoding 316 amino acid residues was shown to encode the VirK protein. The virK region was highly conserved among the large virulence plasmids of shigellae and enteroinvasive Escherichia coli. These results suggest that VirK function is an essential virulence determinant for shigellae involved in the expression of virG gene product at post-transcriptional level.