Application of unstable Gfp variants to the kinetic study of Legionella pneumophila icm gene expression during infection.
An unstable type of green fluorescent protein (Gfp) tagged with a C-terminal extension, which is a target for tail-specific protease, was used as a reporter gene in Legionella pneumophila. To analyse Gfp expression in legionellae, transcriptional fusions of unstable gfp with the Legionella-specific icm (intracellular multiplication) promoters (P(icmS), P(icmT) and P(icmQ)) were constructed. Infection studies using J774.1 macrophages as the host, and L. pneumophila strains carrying P(icmS)-gfp, P(icmT)-gfp and P(icmQ)-gfp fusions, indicated that the icmS, icmT and icmQ genes could be expressed intracellularly. Expression of icmS, icmT and icmQ genes in infected cells was examined by flow cytometry. Furthermore, fluorescent intracellular legionellae were detected directly by confocal microscopy. Real-time quantitative RT-PCR revealed the differences in the gene expression of icmS, and that of icmT and icmQ, during infection. Expression of icmS was high in the late stage of infection, while that of icmT and icmQ was high in the early phase only. We show that unstable gfp is a useful reporter gene whose expression in legionellae can be followed in real-time, and that it allows analysis of promoter activities in legionellae and monitoring of the infection process.
Barysheva
Oksana V
OV
Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Fujii
Jun
J
Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Takaesu
Giichi
G
Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Yoshida
Shin-Ichi
SI
Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
D000818:Animals; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000096:biosynthesis*; D002460:Cell Line; D015169:Colony Count, Microbial; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016952:Legionella pneumophila / Q000502:physiology*; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D018832:Molecular Chaperones / Q000096:biosynthesis*; D013194:Staining and Labeling
D001426:Bacterial Proteins; C432955:ICMQ protein, Legionella pneumophila; C503175:IcmS protein, Legionella pneumophila; D018832:Molecular Chaperones; C443480:icmT protein, Legionella pneumophila; D049452:Green Fluorescent Proteins