Application of unstable Gfp variants to the kinetic study of Legionella pneumophila icm gene expression during infection

Abstract

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.

Quorum sensing modulates the formation of virulent Legionella persisters within infected cells

“…mCherry expression was improved using the T7 RBS amplified from pNT28 using the primers oNP45 forward/reverse, generating the plasmid pSN2. To increase GFPsfm2-LAA degradation rate 74 , the sequence encoding the peptide tag AANDENYAAAV at the carboxyl terminus was generated by site-directed mutagenesis of pSN2 using the primers oNP28 forward/reverse (QuikChange II site-directed mutagenesis; Agilent) yielding the plasmid pSN5. A hairpin transcriptional terminator followed by a multiple cloning site was synthesized and inserted upstream the gene encoding gfpsfm2-aav , yielding the plasmid pSN6.…”

Quantitative Analysis of Endocytosis with Cytoplasmic pHluorin Chimeras

“…Several variants of GFP have been generated that, in theory, could reduce the accumulation of detectable fluorescence in the vacuole. For example, protease‐sensitive variants, in which cleavage sites have been introduced into the sequence of GFP, are degraded upon delivery to sites of protease activity (3–5); however, their usefulness might be limited by the efficiency of cleavage. Instead, pH‐sensitive GFP variants, known as pHluorins, display changes in excitation and/or emission properties depending on the pH of their local environment (6).…”

Intracellular Growth and Morphological Characteristics of <i>Legionella pneumophila</i> during Invasion and Proliferation in Different Cells

“…26,27) However, our results were in line with Barysheva et al and Katagiri et al showed that the total number of bacteria increased over time in murine J774.1A and Raw264.7 cells. 25,28) The reasons for the deviation are not very clear now. Among the non-phagocytic cell lines, MCF-7 and Beas-2B cells were better hosts for up to 24 h post-infection, whereas 16HBE was a proper host between 48 and 72 h post-infection.…”